Preparation method of inorganic chiral nano-morphology-regulated extracellular vesicles and application of inorganic chiral nano-morphology-regulated extracellular vesicles in stem cell osteogenic differentiation

Abstract

The invention discloses a preparation method of inorganic chiral nano-morphology-regulated extracellular vesicles and application of the inorganic chiral nano-morphology-regulated extracellular vesicles in stem cell osteogenic differentiation, and relates to the technical field of EVs preparation, and the preparation method comprises the following steps: preparing a left-handed spiral nano-structure in a physical vapor deposition system by adopting a GLAD technology, and controlling deposition parameters to ensure that the structure is ordered; mSC is cultured on the surface of the left-handed spiral nanostructure, after directional culture is conducted through an EVs-free culture medium, supernate is collected, and EVs is obtained; and the obtained EVs are used for regulating and controlling the osteogenic differentiation of the MSC. According to the method, 99.99% high-purity silicon dioxide is taken as a raw material, a left-handed spiral nanostructure is accurately constructed through a GLAD technology, and the chiral regulation EVs is obtained by combining a multi-step purification and performance calibration process and is used for accurately regulating and controlling MSC osteogenic differentiation.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle (EV) preparation technology, specifically to an inorganic chiral nanomorphology-controlled EV preparation method and its application in osteogenic differentiation of MSCs. Background Technology

[0002] The regulation of osteogenic differentiation of mesenchymal stem cells (MSCs) is a core research area in bone tissue engineering. Endogenous vesicles (EVs), as the core carriers of MSC paracrine function, exhibit unique advantages in regulating MSC osteogenic differentiation due to their carried bioactive molecules such as miRNAs and proteins, and hold promise as novel biological agents for bone regeneration and repair. However, natural EVs suffer from insufficient bioactivity, complex regulation of active ingredients, and difficulty in precise design, limiting their clinical application and translation. Chiral nanomaterials, due to their ability to specifically interact with chiral molecules (such as proteins and nucleic acids) in living organisms, have gradually become ideal carriers for the regulation and delivery of extracellular energy (EVs). However, existing technologies for regulating EVs using chiral nanostructures still face several bottlenecks: First, the preparation process of chiral nanostructures is complex, often employing template methods or chemical synthesis, resulting in poor structural order and insufficient size uniformity, making it difficult to achieve precise regulation of EV activity. Second, EV isolation and purification methods are limited, often relying on centrifugation techniques, which can easily lead to low EV purity and impaired activity, failing to meet subsequent regulatory needs. Third, in terms of EV performance testing and comparison, there is a lack of systematic analysis and verification methods. Existing studies have not fully compared the differences in functional indicators such as microRNA expression profiles of EVs from different chiral nanostructure sources, leading to unclear regulatory mechanisms and difficulty in quantifying and verifying functional advantages. Fourth, in the application of osteogenic differentiation in MSCs, there is a lack of full-cycle monitoring and evaluation and targeted optimization strategies, making it difficult to ensure the stability and reliability of osteogenic effects.

[0003] Therefore, developing an inorganic chiral nanomorphology-controlled EV preparation method with controllable process, high purity, stable loading, and excellent osteogenic regulation effect has become a key technical problem that urgently needs to be solved in the field of bone tissue engineering. Summary of the Invention

[0004] To address the aforementioned technical issues, this technical solution provides a method for preparing EVs with inorganic chiral nanomorphology regulation and its application in osteogenic differentiation of MSCs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An inorganic chiral nanostructure-controlled EV preparation method uses 99.99% pure silica as the chiral structure building material and MSCs as the EV donor. The method comprises three core steps: chiral nanostructure construction, EV separation and purification, and performance testing. The EV preparation method includes: S1. Construction of left-handed helical nanostructures: Left-handed helical nanostructures were prepared in a physical vapor deposition system using GLAD technology, and deposition parameters were controlled to ensure structural order. S2. EVs separation and purification: MSCs were cultured on the surface of left-handed helical nanostructures. After directional culture in EV-free medium, the supernatant was collected and separated by gradient centrifugation, membrane filtration, and size exclusion chromatography. After multi-index performance verification, left-handed nanomorphology-regulated EVs (L-EVs) were obtained. Performance testing of S3 and EVs: MicroRNA sequencing analysis was performed on L-EVs regulated by left-handed nanomorphology obtained from S2, as well as EVs obtained by right-handed nanomorphology, achiral nanomorphology, and conventional substrates (R-EVs, A-EVs, Control). By comparing the types and expression profiles of microRNAs in EVs from different sources, the results showed that L-EVs have a significant advantage in regulating the expression of osteogenic-related microRNAs, demonstrating their superior biological characteristics and functional potential.

[0006] Preferably, in step S1, the physical vapor deposition system pretreatment requires evacuation to 10~10 Torr, the electron beam evaporation parameters are set to 8.0 kV accelerating voltage and 83-87 mA emission current, silicon dioxide is deposited at an 85° oblique angle relative to the substrate normal direction, and the deposition rate is monitored in real time by a quartz crystal microbalance and stabilized at 0.3 nm / s.

[0007] Preferably, in step S1, the substrate is a silicon wafer or glass. During the deposition process, the substrate temperature is maintained at 0°C by a methanol / water cooling system. The deposition time is controlled by a pitch of 50-300 nm and a number of turns of 1-3. After deposition, the structural integrity and order are verified by scanning electron microscopy to ensure that there is no deformation or agglomeration.

[0008] Preferably, in S2, the MSC culture process is as follows: first, treat the inorganic chiral nanostructures (33 mm) that have been sterilized by high temperature and high pressure with 50 μg / mL fibronectin solution for 4 h; then, inoculate the MSCs (P5-P7) into low glucose DMEM medium containing 10% fetal bovine serum and culture them in a 37℃, 5% CO incubator until the cell density reaches 80%-90%; then replace the EVs-free basal medium and continue to culture for 2 weeks, collecting the supernatant every 72 h.

[0009] Preferably, in step S2, the gradient centrifugation and filtration steps are as follows: centrifugation at 300g and 4℃ for 10 min to remove suspended cells, centrifugation at 2000g and 4℃ for 20 min to remove cell debris, filtration through a 0.22μm polyethersulfone filter membrane, and then ultracentrifugation at 3000g and 4℃ for 30 min (twice) using Amicon Ultra-15 to obtain crude EVs. The crude EVs are gently resuspended in pre-cooled PBS to avoid violent shaking that could damage the EVs bilayer membrane structure.

[0010] Preferably, in S2, the SEC separation and purification parameters are as follows: using an ICO-35 column, PBS as the eluent, and collecting 1.5-2.5 mL of fractionated liquid; after purification, the size of EVs should be detected by a NanoSight NS300 system to be 30-150 nm, observed by transmission electron microscopy to be a bilayer membrane spherical structure, and the expression of markers CD63, CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5 and TSG101 should be positive.

[0011] Preferably, in step S3, EVs obtained from left-handed nanomorphic structures, right-handed nanomorphic structures, achiral nanomorphic structures, and conventional substrates are prepared and collected under the same conditions, and then high-throughput microRNA sequencing technology is used to systematically analyze the types and expression profiles of microRNAs in each group of EVs.

[0012] Preferably, in step S3, by comparing the microRNA sequencing results of EVs from different sources, the expression levels of osteogenic-related microRNAs (such as miR-27a, miR-218, etc.) are analyzed in detail. The detection results require that the expression levels of osteogenic-related microRNAs in L-EVs are significantly higher than those in right-handed, achiral, and conventional EVs, in order to verify their functional advantages in promoting osteogenic differentiation.

[0013] Preferably, in step S3, if the detection results fail to meet the above-mentioned microRNA expression enhancement standard, the preparation process of EVs needs to be optimized, including but not limited to adjusting cell culture conditions, optimizing nanostructure parameters or EV collection and purification methods, until high-quality L-EVs that meet performance requirements are obtained.

[0014] An application of chiral nanomorphology-modulated EVs in osteogenic differentiation of MSCs is as follows: Step 1, Sample Pretreatment and Inoculation: The L-EVs (left-handed nanomorphology-regulated extracellular vesicles) obtained in Step S2 were added to the MSC osteogenic induction culture system at a concentration of 1 × 10 EVs / mL. MSC suspension was inoculated into wells containing L-EVs at a volume of 2 mL per well in 6-well plates and 0.5 mL per well in 24-well plates, and cultured at 37℃ and 5% CO2 for 7–14 days. The culture was initially performed using basal medium. After reaching a aggregation degree of 70–80, the medium was replaced with osteogenic differentiation medium containing 100 nmol dexamethasone, 10 mmol β-glycerophosphate, and 50 μg / mL L-ascorbic acid. The medium was changed every 2–3 days.

[0015] Step 2, Osteogenic Differentiation Detection and Evaluation: On day 7 of culture, alkaline phosphatase (ALP) activity was detected, and the mRNA expression level of osteogenic-related genes was detected by real-time quantitative PCR; on day 7 of culture, the nuclear localization of Runx2 protein was observed by immunofluorescence staining; on day 14 of culture, Alizarin Red S (ARS) staining was used to detect the formation of mineralized nodules, and the absorbance value was quantified by microplate reader. At the same time, the expression levels of osteogenic-related genes and proteins such as OCN and COL1A1 were detected.

[0016] Optimization measures when indicators are not met: If the above osteogenic differentiation indicators are not met, the concentration of L-EVs will be increased to 2 × 10 particles / EVs, and the osteogenic induction culture period will be extended to 14-21 days; in addition, the pitch and number of turns of the chiral nanostructure can be optimized, EVs can be collected again, and differentiation detection and full-cycle evaluation can be carried out again after optimization until the relevant osteogenic indicators meet the requirements for bone regeneration applications.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention proposes to use 99.99% high-purity silica as raw material to accurately construct left-handed helical nanostructures through GLAD technology, and obtain chiral regulated EVs by combining multi-step purification and performance calibration processes, which can be used to precisely regulate osteogenic differentiation of MSCs. Moreover, the process is controllable and easy to industrialize. 2. This invention proposes a novel chiral nanostructure—a left-handed helical nanostructure—for regulating the secretion of extracellular energy (EVs) from mesenchymal stem cells (MSCs) growing on it. By using GLAD technology to precisely control the deposition parameters of the nanostructure, the high degree of order of the nanostructure is ensured, thereby providing an ideal surface for the stable loading of EVs. This regulation of chiral nanomorphology enables the prepared EVs to play a better role in the osteogenic differentiation of MSCs. 3. This invention also designs several optimization strategies for situations where performance does not meet standards. For example, if osteogenic differentiation indicators do not meet standards, the concentration of L-EVs is increased to 2 × 10⁻⁶ particles / EVs, and the osteogenic induction culture period is extended to 14-21 days. Alternatively, the pitch and number of turns of the chiral nanostructure can be optimized, EVs can be collected again, and differentiation testing and full-cycle evaluation can be performed again after optimization until the relevant osteogenic indicators meet the requirements for bone regeneration applications. These optimization measures ensure that the performance of the final product meets the requirements for MSC osteogenic differentiation applications, improving overall efficiency and operability. 4. This invention ensures the stability and efficiency of the isolated and purified EVs in terms of morphology, function and bioactivity by precisely controlling the collection and quality inspection of chiral inorganic nanostructures and EVs, and by combining multiple performance verification methods. This comprehensive verification system effectively improves the application performance of EVs in biomedicine. 5. In the osteogenic differentiation process of MSCs, this invention uses left-handed nanomorphology-regulated EVs to induce MSCs. By optimizing the culture medium composition and the staged culture strategy, the osteogenic differentiation effect of MSCs is significantly improved, and it will be used for the treatment of bone defects. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of the present invention.

[0019] Figure 2 Characterization of silica nanohelices (SiO2_NHs). Scanning electron microscopy (SEM) cross-sectional image (scale bar: 150 nm) of SiO2_NHs with a 150 nm pitch (P) and 2 turns; (top left) SEM top view of SiO2_NHs (scale bar: 300 nm), (a) left-handed nanostructure, (b) right-handed nanostructure, (c) achiral nanostructure. (d) Circular dichroism (CD) spectrum (solid line) and extinction curve (dashed line) of SiO2_NHs.

[0020] Figure 3 Characterization of EVs. (a) Schematic diagram of cell culture in left-handed nanostructures. (b) NTA results. (c) Specific protein expression results of EVs. (d) Transmission electron microscopy image of EVs.

[0021] Figure 4 Mechanisms of osteogenic induction of EVs by different substrates. (a) Heatmap of differentially expressed miRNAs between the L-EVs and R-EVs groups. (b) Heatmap of differentially expressed miRNAs between the L-EVs and A-EVs groups. (c) Heatmap of differentially expressed miRNAs between the L-EVs and f-EVs (Control) groups.

[0022] Figure 5 L-EVs most effectively enhanced osteogenic differentiation of MSCs. (a) mRNA levels of osteogenic marker genes in cells after different nanoparticle treatments (n=4). (b) RUNX2 immunofluorescence staining. Scale bar = 100 µm. (c) Alkaline phosphatase (ALP) staining and Alizarin Red (ARS) staining of MSCs after nanoparticle treatment. Scale bar = 2 mm (inset) and 500 µm (magnified view). (d) Quantitative analysis of ALP and ARS staining (n=4). (e) Representative Western blot bands of RUNX2, ALP, and OCN and corresponding quantitative analysis (n=3). Detailed Implementation

[0023] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0024] Reference Figure 1 As shown, an inorganic chiral nanostructure-controlled EV preparation method uses 99.99% pure silica as the chiral structure construction material and MSCs as the EV donor. The method is achieved through three core steps: chiral nanostructure construction, EV separation and purification, and performance testing. The EV preparation method includes: S1. Construction of left-handed helical nanostructures: Left-handed helical nanostructures were prepared in a physical vapor deposition system using GLAD technology, and deposition parameters were controlled to ensure structural order. S2. EVs separation and purification: MSCs were cultured on the surface of left-handed helical nanostructures. After directional culture in EV-free medium, the supernatant was collected and separated by gradient centrifugation, membrane filtration, and size exclusion chromatography. After multi-index performance verification, left-handed nanomorphology-regulated EVs (L-EVs) were obtained. Performance testing of S3 and EVs: MicroRNA sequencing analysis was performed on L-EVs obtained from S2, as well as EVs obtained from right-handed nanomorphology, non-chiral nanomorphology, and conventional substrates (R-EVs, A-EVs, Control). By comparing the types and expression profiles of microRNAs in EVs from different sources, the results showed that L-EVs have a significant advantage in regulating the expression of osteogenic-related microRNAs, demonstrating their superior biological characteristics and functional potential.

[0025] In S1, the physical vapor deposition system pretreatment requires evacuation to 10~10 Torr, the electron beam evaporation parameters are set to 8.0kV accelerating voltage and 83-87mA emission current, and silicon dioxide is deposited at an 85° oblique angle relative to the substrate normal direction. The deposition rate is monitored in real time by a quartz crystal microbalance and stabilized at 0.3nm / s. By precisely setting key parameters such as vacuum degree and acceleration voltage, and combining real-time speed control with a quartz crystal microbalance, the problem of structural disorder caused by parameter ambiguity and deposition rate fluctuation in the preparation of traditional chiral structures has been solved, ensuring the uniformity of left-handed helical nanostructures.

[0026] In S1, the substrate is selected as a silicon wafer or glass. During the deposition process, the substrate temperature is maintained at 0°C by a methanol / water cooling system. The deposition time is controlled by a pitch of 50-300nm and a number of turns of 1-3. After deposition, the structural integrity and order are verified by scanning electron microscopy to ensure no deformation and no agglomeration. By clearly defining the substrate compatibility type and temperature control method, quantifying structural parameters such as pitch and number of turns, and verifying with scanning electron microscopy to form a closed loop, the problems of poor substrate compatibility, lack of quantitative structural standards, and lack of integrity verification in traditional technologies are solved.

[0027] In S2, the MSC culture process is as follows: First, treat the inorganic chiral nanostructures (33 mm) that have been sterilized by high temperature and high pressure with 50 μg / mL fibronectin solution for 4 h. Then, inoculate the MSCs (P5-P7) into low glucose DMEM medium containing 10% fetal bovine serum and culture them in a 37℃, 5% CO incubator until the cell density reaches 80%-90%. Then replace the basal medium without EVs and continue to culture for 2 weeks. Collect the supernatant every 72 h. The three-step method of "fibronectin coating + high-activity MSC amplification + directional secretion of EVs-free basal medium" is adopted, combined with high temperature and high pressure sterilization and standardized medium change process, which effectively improves cell adhesion and amplification efficiency, avoids interference from exogenous EVs impurities, and solves the pain points of uneven cell attachment, insufficient EVs purity and poor batch stability in traditional culture.

[0028] In step S2, the gradient centrifugation and filtration steps are as follows: centrifuge at 300g and 4℃ for 10 min to remove suspended cells, centrifuge at 2000g and 4℃ for 20 min to remove cell debris, filter through a 0.22μm polyethersulfone filter membrane, and then use Amicon Ultra-15 to centrifuge at 3000g and 4℃ for 30 min (twice) to obtain crude EVs. The crude EVs are gently resuspended in pre-cooled PBS to avoid violent shaking that could damage the EVs bilayer membrane structure. The design incorporates multi-gradient centrifugation for graded impurity removal, combined with fine filtration using a specific pore size filter membrane and a gentle resuspension method. This solves the problems of incomplete impurity removal and easy damage to the double-layer membrane structure of EVs caused by single centrifugation technology.

[0029] In S2, the SEC separation and purification parameters are as follows: an ICO-35 column is used, PBS is used as the eluent, and 1.5-2.5 mL of fraction is collected; after purification, the size of EVs is detected by a NanoSight NS300 system and is between 30 nm and 150 nm. Transmission electron microscopy shows a bilayer membrane spherical structure, and Exo-Check antibody array detection shows positive expression of markers CD63, CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5, and TSG101. By setting exclusive SEC separation parameters and establishing a multi-dimensional verification system of "concentration + speciation + biomarkers", the problem of lack of quantitative standards for the purity of EVs after traditional purification and the inability to guarantee activity have been solved, thus improving the reliability of EV quality.

[0030] In S3, EVs obtained from left-handed nanomorphology, right-handed nanomorphology, achiral nanomorphology and conventional substrate were prepared and collected under the same conditions, and high-throughput microRNA sequencing technology was used to systematically analyze the types and expression profiles of microRNAs in each group of EVs. The two-step method of "simultaneous preparation of EVs from multiple substrates + analysis by high-throughput microRNA sequencing system" is adopted. By combining standardized preparation and strict control, it can comprehensively and quantitatively compare the functional molecular differences of EVs with different chiralities and conventional substrates. It avoids the limitations of single detection methods and solves the pain points of the one-sided evaluation of EV function and the difficulty in systematically quantifying the advantages and disadvantages in traditional methods.

[0031] In section S3, by comparing the microRNA sequencing results of EVs from different sources, the expression levels of osteogenic-related microRNAs (such as miR-29a and miR-34a) were analyzed in detail. The detection results required that the expression levels of osteogenic-related microRNAs in L-EVs were significantly higher than those in right-handed, achiral, and conventional EVs, in order to verify their functional advantages in promoting osteogenic differentiation. The "quantitative comparison of osteogenic-related microRNAs" method was used to accurately verify the osteogenic functional advantages of L-EVs, solving the pain point of unclear functional evaluation of traditional EVs.

[0032] In S3, if the detection results fail to meet the above-mentioned microRNA expression enhancement standards, the preparation process of EVs needs to be optimized, including but not limited to adjusting cell culture conditions, optimizing nanostructure parameters or EV collection and purification methods, until high-quality L-EVs that meet performance requirements are obtained. Targeted optimization schemes were developed for different performance defects, and optimization parameters and activity limits were defined to form a closed loop of "detection-optimization-re-verification", which solved the problem of no effective solution for the substandard performance of traditional technologies.

[0033] An application of chiral nanomorphology-modulating EVs in osteogenic differentiation of MSCs, the application steps include MSC seeding and culture and osteogenic induction, as detailed below: Step 1, Sample Pretreatment and Inoculation: The L-EVs (left-handed nanomorphology-regulated extracellular vesicles) obtained in Step S2 were added to the MSC osteogenic induction culture system at a concentration of 1 × 10 EVs / mL. MSC suspension was inoculated into wells containing L-EVs at a volume of 2 mL per well in 6-well plates and 0.5 mL per well in 24-well plates, and cultured at 37℃ and 5% CO2 for 7–14 days. The culture was initially performed using basal medium. After reaching a aggregation degree of 70–80, the medium was replaced with osteogenic differentiation medium containing 100 nmol dexamethasone, 10 mmol β-glycerophosphate, and 50 μg / mL L-ascorbic acid. The medium was changed every 2–3 days.

[0034] Step 2, Osteogenic Differentiation Detection and Evaluation: On day 7 of culture, alkaline phosphatase (ALP) activity was detected, and the mRNA expression level of osteogenic-related genes was detected by real-time quantitative PCR; on day 7 of culture, the nuclear localization of Runx2 protein was observed by immunofluorescence staining; on day 14 of culture, Alizarin Red S (ARS) staining was used to detect the formation of mineralized nodules, and the absorbance value was quantified by microplate reader. At the same time, the expression levels of osteogenic-related genes and proteins such as OCN and COL1A1 were detected.

[0035] Optimization measures when indicators are not met: If the above osteogenic differentiation indicators are not met, the concentration of L-EVs will be increased to 2 × 10 particles / EVs, and the osteogenic induction culture period will be extended to 14-21 days; in addition, the pitch and number of turns of the chiral nanostructure can be optimized, EVs can be collected again, and differentiation detection and full-cycle evaluation can be carried out again after optimization until the relevant osteogenic indicators meet the requirements for bone regeneration applications.

[0036] By establishing standardized application procedures and designing a full-cycle monitoring system covering early, middle, and late osteogenesis, coupled with optimization strategies such as load density adjustment and extended culture time, the problems of non-standard procedures, large fluctuations in osteogenesis effects, and lack of effective control methods in traditional applications have been solved, thus improving the reliability of clinical translation.

[0037] In summary, the advantages of this invention are as follows: Using GLAD technology combined with a physical vapor deposition system, and 99.99% high-purity silica as raw material, highly ordered left-handed helical nanostructures were prepared by precisely controlling parameters such as vacuum degree, deposition angle, rate, pitch and number of turns. This solved the problems of disordered chiral structures and uneven size in traditional methods, provided a stable chiral regulation microenvironment for EVs, and laid the structural foundation for precise regulation of osteogenic differentiation. Innovatively, MSCs were cultured on the surface of a left-handed helical nanostructure, and combined with a multi-step purification process including gradient centrifugation, membrane filtration, and size exclusion chromatography, which significantly improved the purity and bioactivity of EVs, removed impurities to the greatest extent and reduced activity loss, and met the subsequent application requirements of high-quality EVs. A systematic system for detecting and comparing the performance of EVs was established. By performing microRNA sequencing analysis on EVs of different chiralities and conventional substrate sources, their performance in functional indicators such as osteogenic-related microRNA expression was comprehensively evaluated. For the first time, the significant advantages of left-handed nanomorphology-regulated EVs in terms of biological characteristics and functional potential were verified, providing a theoretical and technical basis for the precise regulation and functional optimization of EVs. The chiral nanomorphology-regulating EVs prepared in this invention can precisely regulate osteogenic differentiation of MSCs. A full-cycle monitoring and evaluation system is set up during the application process, covering key indicators in the early stage of osteogenic differentiation (ALP activity), the middle stage (Runx2 expression), and the late stage (mineralized nodules, OCN / COL1A1 expression). Optimization strategies such as adjusting the loading density and extending the culture time are formulated to ensure that the osteogenic indicators meet the standards (ALP activity increased by 168% and mineralization absorbance increased by 134%), which significantly improves the stability and reliability of the application effect and provides an efficient and feasible technical solution for bone tissue regeneration and repair. The GLAD technology, gradient centrifugation, SEC separation and other processes used in this invention are all mature biomaterial preparation technologies. The equipment is readily available and the operation process is standardized. The parameters of each step are clear and highly controllable. Moreover, they do not require complex chemical modifications, are green and environmentally friendly, and are easy to scale up for industrial production, with broad prospects for clinical translation.

[0038] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for preparing inorganic chiral nanostructured EVs, characterized in that, Methods for preparing EVs include: S1. Construction of left-handed helical nanostructures: Left-handed helical nanostructures were prepared in a physical vapor deposition system using GLAD technology, and deposition parameters were controlled to ensure structural order. S2. EVs separation and purification: MSCs were cultured on the surface of left-handed helical nanostructures. After directional culture in EV-free medium, the supernatant was collected and separated by gradient centrifugation, membrane filtration, and size exclusion chromatography. After multi-index performance verification, left-handed nanomorphology-regulated EVs (L-EVs) were obtained. Performance testing of S3 and EVs: MicroRNA sequencing analysis was performed on L-EVs obtained from S2 with left-handed nanomorphology regulation, as well as EVs obtained from right-handed nanomorphology, achiral nanomorphology, and conventional substrates without nanomorphology (R-EVs, A-EVs, Control). By comparing the types and expression profiles of microRNAs in EVs from different sources, the results showed that L-EVs have a significant advantage in regulating the expression of osteogenic-related microRNAs, demonstrating their superior biological characteristics and functional potential.

2. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In S1, the physical vapor deposition system pretreatment requires evacuation to 10~10 Torr, the electron beam evaporation parameters are set to 8.0 kV accelerating voltage and 83-87 mA emission current, and silicon dioxide is deposited at an 85° oblique angle relative to the substrate normal direction. The deposition rate is monitored in real time by a quartz crystal microbalance and stabilized at 0.3 nm / s.

3. The method for preparing EVs with inorganic chiral nanostructure control according to claim 1, characterized in that, In S1, the substrate is selected as silicon wafer or glass. During the deposition process, the substrate temperature is maintained at 0°C by a methanol / water cooling system. The deposition time is controlled by a pitch of 50-300 nm and a number of turns of 1-3. After deposition, the structural integrity and order are verified by scanning electron microscopy to ensure that there is no deformation or agglomeration.

4. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In S2, the MSC culture process is as follows: First, treat the inorganic chiral nanostructures (base diameter of 33 mm) that have been sterilized by high temperature and high pressure with 50 μg / mL fibronectin solution for 4 h. Then, inoculate the MSCs (P5-P7) into low glucose DMEM medium containing 10% fetal bovine serum and culture them in a 37℃, 5% CO incubator until the cell density reaches 80%-90%. Then, replace the basal medium without EVs and continue to culture for 2 weeks. Collect the supernatant every 72 h.

5. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In step S2, the gradient centrifugation and filtration steps are as follows: centrifuge at 300g and 4℃ for 10 min to remove suspended cells, centrifuge at 2000g and 4℃ for 20 min to remove cell debris, filter through a 0.22μm polyethersulfone filter membrane, and then use Amicon Ultra-15 to centrifuge at 3000g and 4℃ for 30 min (twice) to obtain crude EVs. The crude EVs are gently resuspended in pre-cooled PBS to avoid violent shaking that could damage the EVs bilayer membrane structure.

6. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In S2, the SEC separation and purification parameters are as follows: using an ICO-35 column, PBS as the eluent, and collecting 1.5–2.5 mL of fractionated liquid; after purification, the size of EVs is detected by a NanoSight NS300 system to be between 30 nm and 150 nm, and a bilayer membrane spherical structure is observed by transmission electron microscopy. The Exo-Check antibody array detects positive expression of markers CD63, CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5, and TSG101.

7. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In step S3, EVs obtained from left-handed nanomorphic structures, right-handed nanomorphic structures, achiral nanomorphic structures, and conventional substrates were prepared and collected under the same conditions. Then, high-throughput microRNA sequencing technology was used to systematically analyze the types and expression profiles of microRNAs in each group of EVs.

8. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In step S3, the expression levels of osteogenic-related microRNAs (such as miR-29a and miR-34a) were analyzed by comparing the microRNA sequencing results of EVs from different sources. The detection results required that the expression levels of osteogenic-related microRNAs in L-EVs were significantly higher than those in right-handed, achiral, and conventional EVs, in order to verify their functional advantages in promoting osteogenic differentiation.

9. The method for preparing EVs with inorganic chiral nanomorphology control according to claim 1, characterized in that, In step S3, if the detection results fail to meet the above-mentioned microRNA expression enhancement standards, the preparation process of EVs needs to be optimized, including but not limited to adjusting cell culture conditions, optimizing nanostructure parameters or EV collection and purification methods, until high-quality L-EVs that meet performance requirements are obtained.

10. An application of chiral nanomorphology-regulated EVs in osteogenic differentiation of mesenchymal stem cells (MSCs), applicable to the method for preparing inorganic chiral nanomorphology-regulated EVs according to any one of claims 1-9, characterized in that, The specific application steps are as follows: Sample pretreatment and inoculation: L-EVs (left-handed nanomorphology-regulated extracellular vesicles) obtained in step S2 were added to the MSC osteogenic induction culture system at a concentration of 1 × 10 EVs / mL. MSC suspension was inoculated into wells containing L-EVs at a volume of 2 mL per well of 6-well plates and 0.5 mL per well of 24-well plates, and cultured at 37℃ and 5% CO for 7–14 days. The culture was initially carried out using basal medium. After reaching a aggregation degree of 70–80, it was replaced with osteogenic differentiation medium containing 100 nmol dexamethasone, 10 mmol β-glycerophosphate, and 50 μg / mL L-ascorbic acid. The medium was changed every 2–3 days. Osteogenic differentiation detection and evaluation: On day 7 of culture, alkaline phosphatase (ALP) activity was detected, and the mRNA expression level of osteogenic-related genes was detected by real-time quantitative PCR; on day 7 of culture, the nuclear localization of Runx2 protein was observed by immunofluorescence staining; on day 14 of culture, Alizarin Red S (ARS) staining was used to detect the formation of mineralized nodules, and the absorbance value was quantified by microplate reader. At the same time, the expression levels of osteogenic-related genes and proteins such as OCN and COL1A1 were detected. Optimization measures when indicators fail to meet the standards: If the above osteogenic differentiation indicators fail to meet the standards, the concentration of L-EVs will be increased to 2 × 10 particles / EVs, and the osteogenic induction culture period will be extended to 14-21 days; in addition, the pitch and number of turns of the chiral nanostructure can be optimized, EVs can be collected again, and differentiation detection and full-cycle evaluation can be carried out again after optimization until the relevant osteogenic indicators meet the requirements for bone regeneration applications.

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