Ultrasonic stimulation conversion treatment platform for improving mitochondrial membrane permeability, delaying senescence and promoting bone repair

Abstract

The invention relates to the field of nano treatment, in particular to an ultrasonic stimulation conversion treatment platform for improving mitochondrial membrane permeability, delaying senescence and promoting bone repair. The invention provides a hydrogel microsphere. The hydrogel microsphere comprises a PDA (Personal Digital Assistant) coated gold nanorod and GelMA hydrogel, the gold nanorod is loaded on the GelMA hydrogel. On the basis of reversing the potential of senescence stem cells for treating senescence bone defects through thermal stimulation, a treatment system based on a physical signal conversion strategy is designed, and potential pathways and targets of thermal stimulation therapy are further analyzed and analyzed in combination with multi-omics. And a brand new research platform is established for innovative development and application transformation of a new technology for treating senescence bone defects.

Description

Technical Field

[0001] The present invention relates to the field of nano-therapy, and particularly to an ultrasonic stimulation conversion therapy platform for improving mitochondrial membrane permeability, delaying aging and promoting bone repair. Background Art

[0002] At present, the global population aging situation is becoming increasingly severe. It is predicted that by 2050, the proportion of the global elderly population aged 65 and above will rise to 16%, and the incidence of age-related bone diseases is also increasing year by year. Aging is an important factor for the slow and poor bone healing in the elderly population, which has a profound impact on the quality of life of patients and social economy. The homeostasis and regeneration of the body's bone tissue depend on the intact function of mesenchymal stem cells (MSCs). However, with the increasing aging of individuals, the excessive accumulation of senescent MSCs damages the individual's bone repair and bone regeneration ability. Although the senescence of MSCs has been confirmed as a key factor hindering the repair of aging bones, there is still a lack of effective and precise treatment strategies to achieve targeted restoration of the function of senescent MSCs to promote the repair of aging bones. Mitochondria are important organelles for eukaryotes to regulate cell metabolism and maintain cell energy supply and homeostasis, and are also important hubs for determining cell fate. Therefore, intact mitochondrial function is crucial for maintaining the health of MSCs and realizing the physiological functions of MSCs. Mitochondrial dysfunction has been confirmed as one of the phenotypes of cell senescence, and the treatment strategy of targeted regulation of mitochondrial function provides a promising opportunity for the treatment of aging-related diseases. At present, the regulation of cell chemical signals using small molecule drugs is the most commonly used treatment plan for remodeling mitochondrial function, but it has defects such as diffuse in vivo targets and high cytotoxicity. For example, the classic mitochondrial function regulation drug, rapamycin, although it can maintain mitochondrial function by promoting mitophagy, has deficiencies such as immunosuppression and bone marrow cytotoxicity. The cytotoxicity of the new mitochondrial-targeted drug, resveratrol, has been significantly improved, but there is a risk of severe allergic reactions. Therefore, developing a strategy for remodeling mitochondrial function with high regulation efficiency and biocompatibility is a research hotspot in the current anti-aging field, but it is still challenging.

[0003] In recent years, in the field of biology, various physical signals such as temperature, ultrasound, and electricity have been widely used to regulate gene expression, gene editing, and manipulate cell behavior and function. Compared with traditional small molecule drugs, physical signals have many advantages, such as non-invasiveness, spatiotemporal precision, real-time monitoring, and modularity. In recent years, thermal signals have been reported to stimulate changes in tissue protein conformation, improve cell metabolism and immune function, and promote rejuvenation, thereby achieving the effect of tissue and organ repair. Lin Feng et al., by utilizing the heat conduction effect of acupuncture to activate the endogenous heat shock protein network in aging chondrocytes, remodel the mitochondrial function of aging cells to treat osteoarthritis. This suggests that thermal stimulation has great potential in remodeling mitochondria and is one of the strong candidates for anti-aging strategies, but the specific mechanism needs further exploration.

[0004] Therefore, restoring the function of aging MSCs and promoting repair in the aging body based on in vitro thermal signals holds great potential. However, achieving stable and sustainable thermal stimulation signals in deep bone tissue through noninvasive therapeutic techniques remains a significant challenge. While acupuncture and other therapeutic techniques can effectively transmit thermal stimulation to deep tissues, they can damage the patient's skin and mucosal barrier, significantly increasing the risk of infection and even osteomyelitis. Furthermore, previous studies have shown that under high-temperature conditions (46°C), the effective heat exchange depth at the skin surface is only 5 mm, making heat transfer through soft tissue inefficient. In recent years, ultrasound-assisted thermal conversion strategies have been applied to deep tissue heat transfer, a strategy that circumvents these challenges. Furthermore, ultrasound-assisted thermal conversion strategies offer controllability and precision, allowing for adjustment of energy output and area of action according to treatment needs, providing an innovative therapeutic approach for achieving stable and sustainable thermal stimulation signals. Exogenous injectable implants may offer a new avenue for the application of ultrasound-assisted thermal conversion strategies. Summary of the Invention

[0005] In view of this, the present invention provides an ultrasonic stimulation conversion therapy platform for improving mitochondrial membrane permeability, delaying aging, and promoting bone repair. First, through in vitro experimental studies, the present invention discovers the potential of heat stimulation to reverse the treatment of aging bone repair with senescent stem cells. Secondly, PDA@GNRs and GelMA microspheres with ultrasonic-heat signal response are organically integrated to build a new research and application platform for ultrasonic signal and heat effect signal conversion therapy of aging bone repair. This treatment system not only effectively avoids side effects such as skin damage and energy loss caused by direct heat stimulation, but also the efficient physical signal conversion effect enhances the osteogenic differentiation ability at multiple levels by delaying the senescence of BMSCs and restoring the disordered glucose metabolism of BMSCs. This is related to its ability to restore the expression of HSP70, which can inhibit the activation of BAX in the senescent mitochondrial membrane and reduce mitochondrial membrane permeability. On the one hand, it reduces the release of ROS in mitochondria and alleviates oxidative stress in senescent BMSCs. On the other hand, it reduces the leakage of mtDNA, thereby inhibiting the polarization of the NF-κB pathway triggered by cGAS-STING and reducing the SASP phenotype, reversing the senescent chronic inflammatory state. In addition, the in vivo experimental results further confirm the efficacy of this treatment platform in restoring mitochondrial function, improving the senescent phenotype, and effectively promoting bone regeneration. In summary, based on the potential of heat stimulation to reverse the treatment of aging bone defects with senescent stem cells, we designed a treatment system based on a physical signal conversion strategy, and further combined multi-omics analysis to analyze the potential pathways and targets of heat stimulation therapy, thus building a new research platform for the innovative development and application transformation of new technologies for treating aging bone defects.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0007] The present invention provides hydrogel microspheres, including: gold nanorods wrapped with PDA and GelMA hydrogel; the gold nanorods are loaded in the GelMA hydrogel.

[0008] In some embodiments of the present invention, in the above-mentioned hydrogel microspheres, the concentration ratio of the gold nanorods to the GelMA hydrogel is: 400:1.

[0009] The present invention also provides a preparation method of the hydrogel microspheres, including the following steps: in the presence of a photoinitiator, crosslink and wrap the surface of the gold nanorods wrapped with PDA with the GelMA hydrogel to obtain the hydrogel microspheres.

[0010] In some embodiments of the present invention, in the above-mentioned preparation method, the mass ratio of the concentration of the gold nanorods wrapped with PDA to the concentration of the GelMA hydrogel is 5:4.

[0011] In some embodiments of the present invention, in the above-mentioned preparation method, the preparation method of the hydrogel microspheres includes the following steps: adding 0.4 g of GelMA solid to 2 mL of pure water, then adding a 0.5 mg / mL concentration of PDA@GNRs (PDA-wrapped gold nanorods) solution, dissolving in a 55°C water bath, and then adding 10 mg of I2595 photoinitiator, and mixing thoroughly to obtain three aqueous phases; adding 5 mL of Span80 to 100 mL of isopropyl myristate, and mixing thoroughly to obtain an oil phase; using a coaxial needle (21G / 30G) as a device, and setting the injection speed of the aqueous phase and the oil phase to 1:50; collecting the generated microspheres in a beaker and cleaning them with 75% ethanol solution to obtain hydrogel microspheres (GMPG).

[0012] In some embodiments of the present invention, in the above preparation method, the preparation method of the PDA-wrapped gold nanorods comprises the following steps:

[0013] S1: Mix HAuCl4 solution, sodium citrate solution and NaBH4 solution to obtain a seed solution;

[0014] S2: Mixing CTAB solution, HAuCl4 solution and AA solution to obtain a stock solution;

[0015] S3: mixing the seed solution in S1 and the stock solution in S2 to obtain solution A;

[0016] S4: mixing the solution A described in S3 with the stock solution described in S2 to obtain solution B;

[0017] S5: mixing the solution B described in S4 and the stock solution described in S2, reacting and centrifuging to obtain gold nanorods;

[0018] S6: mixing the Tris-HCl solution with dopamine hydrochloride to obtain a PDA layer;

[0019] S7: Mixing the gold nanorods described in S5 and the PDA layer described in S6, initiating a reaction, centrifuging, collecting the precipitate, and obtaining the PDA-wrapped gold nanorods.

[0020] In some embodiments of the present invention, in the above preparation method, the concentration ratio of the HAuCl4 solution to the sodium citrate solution is 1:1.

[0021] In some embodiments of the present invention, in the above preparation method, the concentration of the HAuCl4 solution is 2.5×10 -4 M and the concentration of the sodium citrate solution was 2.5×10 -4 M.

[0022] In some embodiments of the present invention, in the above preparation method, the concentration of the NaBH4 solution is 0.1M.

[0023] In some embodiments of the present invention, in the above preparation method, the concentration of the CTAB solution is 0.1M; the concentration of the AA solution is 0.1M.

[0024] In some embodiments of the present invention, in the above preparation method, the preparation method of the PDA-wrapped gold nanorods comprises the following steps: -4 M HAuCl4 solution and 2.5×10 -4 M sodium citrate solution was fully mixed, and then 0.6 mL of 0.1 M NaBH4 solution was added and stirred for 2 h to obtain a brown seed solution; 0.1 M CTAB solution and 2.5 × 10 -4 A 0.1M HAuCl₄ solution was thoroughly mixed, and 0.1M AA solution was added until the solution turned from yellow to colorless, resulting in a transparent stock solution. 1mL of the seed solution was added to 9mL of the stock solution to obtain Solution A. 1mL of Solution A was then added to 9mL of the stock solution to obtain Solution B. 10mL of Solution B was then added to 90mL of the stock solution to obtain the final solution. The solution was incubated in a 30°C incubator overnight, and the final solution was centrifuged and purified to obtain gold nanorods. Dopamine hydrochloride was then dissolved in Tris-HCl with gentle stirring to prepare a PDA coating. After 5 minutes, the nanoparticles were added to the dopamine solution to initiate the reaction. The product was then collected by centrifugation and purified three times with water to obtain PDA-coated gold nanorods (PDA@GNRs).

[0025] The present invention also provides a bioreceptor comprising: the above-mentioned hydrogel microspheres and / or the hydrogel microspheres obtained by the above-mentioned preparation method.

[0026] In some embodiments of the present invention, the above-mentioned bioreceptor includes: an ultrasonic signal receptor.

[0027] The present invention also provides the use of the hydrogel microspheres, the hydrogel microspheres obtained by the above preparation method and / or the above bioreceptors in the preparation of products for treating aging bone defects.

[0028] In some embodiments of the present invention, in the above application, the treatment of aging bone defects includes: restoring mitochondrial function, improving aging phenotype and promoting bone regeneration or one or more thereof.

[0029] In some embodiments of the present invention, in the above application, the restoration of mitochondrial function comprises: inhibiting one or more of BAX activation of aged mitochondrial membrane, reducing mitochondrial membrane permeability and reducing the release of ROS in mitochondria.

[0030] In some embodiments of the present invention, in the above application, the improvement of the aging phenotype includes: reducing the SASP phenotype.

[0031] The present invention also provides a product, including: the above hydrogel microspheres, the hydrogel microspheres obtained by the above preparation method and / or the above bioreceptor, and acceptable auxiliaries, excipients, carriers and / or platforms.

[0032] In some embodiments of the present invention, the above product includes: one or more of a drug delivery system, a product for diagnosis and / or detection, a product for tissue engineering, a drug, a dressing, a product for growth factor delivery, and a cosmetic.

[0033] The "ultrasonic signal bioreceptor" provided by the present invention is integrated into the therapeutic platform of hydrogel microspheres, which can provide a promising therapeutic strategy for aging bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0035] Figure 1 Characterization of GNRs and PDA@GNRs; wherein: A shows the SEM images of GNRs and PDA@GNRs; B shows the TEM images of GNRs and PDA@GNRs; C shows the EDS elemental mapping of PDA@GNRs; D shows the aspect ratio analysis of the gnr particle size; E shows the Zeta potential of GNRs and PDA@GNRs; F shows the UV-visible absorption spectra of GNRs and PDA@GNRs; G shows the DLS images of GNRs and PDA@GNRs; H shows the schematic diagram of the acoustic temperature conversion ability induced by GNRs; I shows the temperature heat map of gnr and PDA@GNRs over time under ultrasonic stimulation; J shows the temperature curve of PDA@GNRs for 15 minutes under ultrasonic stimulation; K shows the temperature curves of PDA@GNRs under different ultrasonic intensities; L shows the temperature curves of PDA@GNRs under ultrasonic stimulation at different concentrations;

[0036] Figure 2 Characterization of GM, GMG and GMPG; A shows the optical microscope images of GM, GMG and GMPG; B shows the SEM images of GM, GMG and GMPG; C shows the surface roughness analysis of GM, GMG and GMPG by AFM; D shows the particle size analysis of GMPG; E shows the pore size analysis of GMPG; F shows the EDS analysis of GM and GMPG; G shows the temperature heat map of GM, GMG, GMPG for 15 minutes per week; H shows the temperature curves of GM, GMG, GMPG under ultrasonic stimulation per week; I shows the in vitro release curves of GNRs in GMG and GMPG; J shows the in vitro degradation curves of GM, GMG, GMPG;

[0037] Figure 3 To show the ability of GMPG / US to improve the phenotype of senescent bone marrow mesenchymal stem cells; wherein: A shows the β-gal staining and immunofluorescence images of P21 and P53 in microsphere cells; B shows the quantitative analysis of β-gal staining in bone marrow mesenchymal stem cells (n = 10); C shows the semi-quantitative analysis of the immunofluorescence staining intensity of P21 in microsphere cells (n = 10); D shows the semi-quantitative analysis of the immunofluorescence staining intensity of P53 in microsphere cells (n = 10); E shows the flow cytometry analysis of Ki67; F shows the flow cytometry analysis of Ki67 (n = 3); G shows the qPCR analysis of SASP in BMSCs; H shows the protein analysis of P16, P21, P53, IL-6 and IL-1β.

[0038] Figure 4 To show the evaluation of the ability of GMPG / US to improve the metabolism and osteogenic function of senescent bone marrow mesenchymal stem cells; wherein: A shows the heat map of metabolites related to glycolysis and the TCA cycle; B shows the quantitative analysis of metabolites related to glycolysis and the TCA cycle (n = 6); C shows the schematic diagram of the changes in target metabolites involved in glycolysis and the TCA cycle; D shows the real-time ecar of bone marrow mesenchymal stem cells in the glycolysis stress test; E shows the semi-quantitative analysis of glycolysis, glycolytic capacity and glycolytic reserve (n = 3); F shows the real-time ocr of bone marrow mesenchymal stem cells in the cellular mitochondrial stress test; G shows the semi-quantitative analysis of basal respiration, maximal respiration and ATP production (n = 3); H shows the protein analysis of glycolysis and OXPHOS marker proteins; I shows the ALP staining and RUNX2 immunofluorescence images of microsphere cells 7 days after osteogenic induction, and alizarin red staining and OPN immunofluorescence images 14 days after induction; (scale bar = 20 μm); J shows the quantitative analysis of ALP staining in microsphere cells; K shows the calcium nodule content (n = 10); L shows RUNX2 in microsphere cells; M shows the semi-quantitative analysis of the immunofluorescence staining intensity of OPN (n = 10); N shows the protein analysis of RUNX2, OPN and OCN.

[0039] Figure 5Figure 3 shows the changes in mitochondrial membrane permeability and the analysis of mitochondrial content leakage; A shows representative high-resolution SIM microscopy images of Cyt c (green) and TOM20 (red); B shows the co-localization analysis of TOM20-Cyt-c complex in bone marrow mesenchymal stem cells; C shows the mitochondrial membrane potential detection, DCFH-DA fluorescence and MitoSOX fluorescence detection of bone marrow mesenchymal stem cells JC-1; D shows the semi-quantitative analysis of mitochondrial membrane potential, DCFH-DA fluorescence and MitoSOX fluorescence detection of bone marrow mesenchymal stem cells JC-1 (n=10); E shows the Bio-TEM detection of bone marrow mesenchymal stem cell mitochondria; F shows the N Protein analysis of rf2 and HO-1; G shows representative high-resolution SIM microscopy images of DNA (red), TFAM (green), and mitosis tracker (gray) in bone marrow mesenchymal stem cells; (Scale bar = 5 μm); H shows colocalization analysis of DNA-TFAM complex and mitosis tracker in bone marrow mesenchymal stem cells. The magnified image shows that TFAM colocalizes with DNA outside the mitochondrial network, indicating mtDNA leakage; I shows protein analysis of proteins related to the cGAS-STING signaling pathway and the NF-κB signaling pathway;

[0040] Figure 6 Figure 3 shows the analysis of channel proteins related to membrane permeability; A shows the immunofluorescence image of mPTP in bone marrow mesenchymal stem cells; B shows the semi-quantitative analysis of the immunofluorescence staining intensity of mPTP in bone marrow mesenchymal stem cells (n=10); C shows the immunofluorescence image of VDAC in bone marrow mesenchymal stem cells; D shows the semi-quantitative analysis of the immunofluorescence staining intensity of VDAC (n=10); E shows the immunofluorescence image of BAX6A7 in bone marrow mesenchymal stem cells; F shows the semi-quantitative analysis of the immunofluorescence staining intensity of BAX6A7 (n=10); G shows the protein analysis of VDAC and BAX6A7; H shows the semi-quantitative analysis of VDAC and BAX6A7 in BMSCs; I shows Representative high-resolution microscopy images of TFAM (red), BAX6A7 (green), and mitochondria (gray) in bone marrow mesenchymal stem cells; J shows colocalization analysis of the BAX6A7-TFAM complex and mitotracker in bone marrow mesenchymal stem cells; K shows a single mitochondria undergoing nucleoid externalization, as shown by overlaying a 3D surface reconstruction on the original data using Imaris software; L shows an image of microsphere cells showing HSP70 immunofluorescence; M shows semi-quantitative analysis of HSP70 immunofluorescence staining intensity in microsphere cells (n=10); N shows protein analysis of HSP70 in young and aged bone marrow mesenchymal stem cells under heat stimulation (n=3);

[0041] Figure 7Identification of mitochondrial DNA leakage; wherein: A shows representative electron microscopy images of DNA immunogold in BMSCs among different groups; B shows the percentage of mtDNA outside mitochondria; C shows the mtDNA image after 1% agarose gel electrophoresis; D shows qPCR analysis of D-loop, Cytb, and Nd4 in BMSCs; E shows Western blotting of Cyt c and TFAM.

[0042] Figure 8 Verification of the effect of HSP70 on the channel protein BAX; wherein: A shows representative high-resolution SIM microscope images of BAX6A7 (green) and TOM20 (red); (scale bar = 5 μm); B shows the co-localization analysis of the TOM20-BAX complex in BMSCs; C shows representative high-resolution SIM microscope images of DNA (green) and TOM20 (red); scale bar = 5 μm, and the enlarged image shows DNA outside the mitochondrial network; D shows the co-localization analysis of the TOM20-DNA complex in BMSCs; E shows the JC-1 mitochondrial membrane potential detection, DCFH-DA fluorescence, and MitoSOX fluorescence detection in BMSCs; scale bar = 10 μm; F shows the semi-quantitative analysis of the JC-1 mitochondrial membrane potential in BMSCs (n = 10); G shows the semi-quantitative analysis of DCFH-DA fluorescence in BMSCs (n = 10); H shows the semi-quantitative analysis of MitoSOX fluorescence in BMSCs (n = 10); I shows Western blotting of BMSCs after adding agonists and inhibitors;

[0043] Figure 9 Radiological and thermal effect evaluation of in vivo osteogenesis; wherein: A shows the flowchart of the animal experiment; B shows the temperature heat maps of the GM, GMG, and GMPG groups over time under ultrasound stimulation; C shows the temperature heat maps of the GM, GMG, and GMPG groups over time after ultrasound stimulation; D shows the temperature change curves of the GM, GMG, and GMPG groups over time under ultrasound stimulation; E shows the temperature curves of the GM, GMG, and GMPG groups after ultrasound stimulation; F shows the Micro-CT evaluation of bone regeneration in the femoral condyle defects of different groups at 2 and 4 weeks after surgery; scale bar = 1.5 mm;

[0044] Figure 10 Evaluation of in vivo osteogenesis by tissue immunostaining and immunofluorescence staining; wherein: A shows the macroscopic and magnified images of the femoral condyle at 2 weeks after surgery, H&E staining and Masson staining; B shows the macroscopic view and magnified images of the femoral condyle at 4 weeks after surgery, H&E staining and Masson staining; C shows the immunofluorescence staining of BAX6A7 (green) and TOM20 (red) in the postoperative tissue; D shows the immunofluorescence staining of STING (green) in the postoperative tissue; E shows the immunofluorescence staining of P16 (red) in the postoperative tissue; F shows the immunofluorescence staining of OCN (green) in the postoperative tissue; (scale bar = 50 μm);

[0045] Figure 11 Show the in vivo imaging of the femoral condyles of rats; among them: A shows the fluorescence imaging of the femoral condyle defect in rats after injecting the ROS probe; B shows the semi-quantitative analysis of the fluorescence intensity in in vivo imaging (n = 10).

[0046] Figure 12 Show the live / dead cell staining at 4 days and 7 days (scale bar = 25 mm). Detailed implementation manners

[0047] The present invention discloses an ultrasonic stimulation conversion treatment platform for improving mitochondrial membrane permeability, delaying aging and promoting bone repair.

[0048] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0049] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0050] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0051] The use of any and all examples or exemplary language herein, such as "for example" or "including", is only intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0052] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0053] The present invention explores the potential of heat stimulation to reverse aging and constructs a signal transducer-therapy platform based on the crosstalk amplification mode of ultrasound and thermodynamic signals. The platform uses composite nanoparticles (PDA@GNRs) with efficient ultrasound-thermal conversion effects as the core and couples them into GelMA hydrogel microsphere carriers (GMPG) through microfluidic technology; among them, PDA@GNRs are composed of gold nanorods (GNRs) with acoustic-thermal coupling effects and a PDA outer layer with enhanced thermal effects. The discontinuous characteristics of GMPG enable it to widely accommodate the filling and bone grafting of open surgery and the local injection requirements of minimally invasive surgery. The PDA@GNRs uniformly distributed inside the GelMA microspheres act as sensitive "ultrasound signal sensors", efficiently converting ultrasound signals into in-situ thermal signals at the lesion site, achieving non-invasive and enriched conduction of local thermal signals. In in vitro studies, we found that GMPG / US can effectively improve the state of senescent cells and enhance the repair of senescent bone defects. Based on metabolomics and transcriptomics, we deeply explored the metabolic mode and function of this novel signal conversion therapy platform to restore mitochondrial disorders in senescent BMSCs and the mechanism of improving cell senescence in vitro. In addition, in vivo studies showed that the platform has efficient ultrasound-thermal conversion effects, targets the restoration of mitochondrial function, effectively reshapes the bone regeneration potential of senescent BMSCs, and promotes the repair of senescent bones. In summary, our research results show that this therapy platform integrating "ultrasound signal sensors" into hydrogel microspheres can provide a promising therapeutic strategy for senescent bone regeneration.

[0054] In Examples 1 to 6 of the present invention, the raw materials and reagents used can be purchased from the market.

[0055] The present invention is further described below in conjunction with examples:

[0056] Example 1 Preparation of GNRs and Preparation of PDA@GNRs

[0057] Mix 2.5×10 -4 M HAuCl4 solution (Aladdin, China) and 2.5×10 -4 M sodium citrate solution (Aladdin, China) thoroughly, and then add 0.6 mL of 0.1 M NaBH4 solution (Aladdin, China) and stir for 2 h to obtain a pale yellow seed solution.

[0058] Mix 0.1 M CTAB solution (Aladdin, China) and 2.5×10 -4 M HAuCl4 solution thoroughly, and add 0.1 M AA solution (Aladdin, China) until the solution changes from yellow to colorless to obtain a transparent stock solution.

[0059] Add 1 mL of the seed solution to 9 mL of the stock solution to obtain Solution A. Then, take 1 mL of Solution A and add it to 9 mL of the stock solution to obtain Solution B. Next, take 10 mL of Solution B and add it to 90 mL of the stock solution to obtain the final solution. Incubate the final solution in an incubator at 30 °C overnight. Take out the final solution and centrifuge it for purification to obtain gold nanorods.

[0060] Dissolve dopamine hydrochloride (Aladdin, China) in Tris-HCl (Aladdin, China) under gentle stirring to prepare the PDA coating. After 5 min, add the nanoparticles to the dopamine solution to initiate the reaction. Then, centrifuge to collect the product and purify it three times with water to obtain PDA@GNRs.

[0061] Preparation of Examples 2 GM, GMG, and GMPG

[0062] Add 0.4 g of GelMA solid (EngineeringForLife, China) to 2 mL of pure water, and then add 2 mL of pure water or a 0.5 mg / mL GNRs solution or a 0.5 mg / mL PDA@GNRs solution. Dissolve it in a water bath at 55 °C, and then add 10 mg of the I2595 photoinitiator (EngineeringForLife, China). After thorough mixing, three aqueous phases are obtained. Add 5 mL of Span80 (Aladdin, China) to 100 mL of isopropyl myristate (Aladdin, China) and mix well to obtain the oil phase. Use a coaxial needle (21G / 30G) as the device, and set the injection speed of the aqueous phase and the oil phase to 1:50. Collect the generated microspheres in a beaker and wash them clean with a 75% ethanol solution to obtain GelMA microspheres (GM), GelMA microspheres loaded with gold nanorods (GMG), and GelMA microspheres loaded with PDA-coated gold nanorods (GMPG).

[0063] Example 3

[0064] 1. Material Characterization

[0065] The microspheres prepared in Example 2 were spread on a conductive adhesive, and a scanning electron microscope (SEM, Hitachi, Japan) was used. At an acceleration voltage of 10 kV, the morphology and surface condition of each microsphere were observed. Elemental analysis of the microspheres was performed by X-ray energy dispersive spectroscopy (EDS) and mapping analysis. Two hundred microspheres were selected for particle size analysis, and the average size and variability of the microspheres were calculated. The surface roughness of different groups of microspheres was compared by atomic force microscopy (AFM, Bruker, USA). The chemical bond composition of different microsphere groups was compared by Fourier transform infrared spectroscopy (FTIR, Thermo Scientific, USA). The heat differences of the microspheres in each group were compared by differential scanning calorimetry, and the optimal ultrasonic intensity and GNRs concentration were explored.

[0066] 2. Cell culture and intervention

[0067] The cells used in this experiment were BMSCs. BMSCs were extracted from the primary bone marrow of 4-week-old rats. Under sterile conditions, BMSCs were separated into small cell clusters by mechanical shearing. The cells were cultured in DMEM-α medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% double antibody in a 37 °C, 5% CO2 incubator and passaged after the cells reached 70% confluence. For in vitro experiments, 24-well plates were used, and 100 microspheres, 2×10 4 cells were added to each well for co-culture with 1 mL of DMEM-α medium. The microspheres were co-cultured with the cells cultured to the third generation after being disinfected by alcohol immersion. The culture medium was changed every 3 days and continued to be cultured until the cells grew on the microspheres. Then, 50 mg / mL D-galactose was used to intervene in BMSCs for 2 hours to induce senescence. The groups that required ultrasonic intervention were stimulated at an intensity of 1 W / cm 2 for 15 min.

[0068] Regarding the thermal stimulation required to improve senescence, current in vitro methods of continuous infrared and other heat transfer may cause skin burns or deep soft tissue burns. Moreover, bone is located deep within the tissue, and there are adverse consequences such as energy loss in the external thermal effect. Therefore, there is an urgent need to construct a biomaterial for deep tissue heat transfer. Gold nanorods can respond to ultrasonic stimulation in vitro and convert it into a thermal effect, which provides an idea for us to design a biomaterial that can respond to external physical signals in deep bone tissue and improve individual bone senescence through local effects.

[0069] We successfully prepared GNRs with uniform size and good dispersibility through the seed growth method. Further, taking advantage of the property that dopamine can spontaneously deposit on GNRs under alkaline conditions, we constructed a composite nanomaterial coated with a PDA layer. This is related to the fact that electrons in GNRs are excited to high-energy states by ultrasonic stimulation and undergo electron transitions. The system tends to release excess energy (such as heat) in a non-radiative manner to restore stability. At the same time, the resulting lattice vibrations also intensify the intermolecular interactions and enhance the heat release. The recombination process of electrons and holes in GNRs after excitation can also generate a certain amount of heat. Compared with GNRs, the thermal effect of PDA@GNRs under ultrasonic stimulation is further enhanced, which is attributed to the fact that the acoustic properties of the PDA layer improve the energy conversion efficiency of the GNRs core. Further experiments show that the signal amplification effect of the PDA layer can ensure that PDA@GNRs can effectively respond to external ultrasonic stimulation in deep tissues and efficiently convert it into the thermal effect required by organisms. In addition, parameters such as ultrasonic intensity and PDA@GNRs concentration are crucial for the control of ultrasonic thermal effects. By comparing the effects of different ultrasonic intensities and nanoparticle concentrations on temperature rise, we determined that the optimal ultrasonic intensity is 1W / cm 2 and the optimal concentration of gold nanorods is 0.25mg / mL, and these are used as parameters for subsequent experiments.

[0070] We further successfully prepared stable and uniformly sized composite GelMA microspheres (GMPG) loaded with PDA@GNRs using microfluidic technology. The particle sizes and pore sizes of the three types of microspheres are similar, indicating that the addition of PDA@GNRs does not significantly affect the particle size and pore size of the microspheres. Compared with GelMA microspheres (GMG) loaded with GNRs, GMPG exhibits better thermal effect retention ability, which may be due to the formation of hydrogen bonds between PDA and the side chains of GelMA, thereby increasing the heat retention effect of the composite microspheres. Release and degradation experiments confirmed that the PDA layer can improve the long-term residence of PDA@GNRs in GMPG to meet the long-term physical signal stimulation requirements for bone tissue healing.

[0071] Example 4

[0072] 1. Cell adhesion and proliferation

[0073] Bone marrow mesenchymal stem cells (BMSCs) were seeded at a density of 2.0×10 3The density of cells per well was co-cultured with 50 GelMA microspheres in a 96-well culture plate. The culture plate was placed in an incubator at 37 °C, 95% relative humidity and 5% CO₂ partial pressure, and the culture medium was changed daily. Cell proliferation was measured. After 1, 3, and 5 days respectively, the reagent in the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was mixed and diluted 10-fold with complete culture medium, and then 100 μL of the culture medium in each well of the well plate was added to the above solution. After reacting in the incubator for 2 h, 100 μL of the mixed solution in each well was taken and placed in a new 96-well plate. After incubation for 4 h, the absorbance value was read at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader.

[0074] 2. Cell viability and cytotoxicity staining

[0075] The Live / Dead Cell Staining Kit (Introvegen, USA) was used in the experiment. Under light-proof conditions, 5 μL of calcein and 20 μL of ethidium homodimer-1 were mixed evenly in 10 mL of PBS to prepare the cell viability and cytotoxicity staining solution. After washing the co-cultured cells on the 1st and 3rd days with PBS, 200 μL of the staining solution was added to each well. After incubation at room temperature in the dark for 30 min, the cells were washed 3 times with PBS and observed using a confocal fluorescence microscope. Live cells showed green fluorescence and dead cells showed red fluorescence under the microscope. Photos were taken and counted for analysis.

[0076] 3. Cell immunofluorescence staining

[0077] Cell immunofluorescence was used to detect the expression of BMSCs-related proteins. The cells in different microsphere groups were fixed with 4% paraformaldehyde at room temperature for 20 min (for experiments requiring mitochondrial staining, mitotracker was used to stain at 37 °C for 40 min and then fixed with paraformaldehyde). Subsequently, 0.5% Triton X-100 was added for perforation, and the cells were incubated at room temperature for 15 min. Then, 5% BSA was used to block overnight at 4 °C. Subsequently, the BSA was aspirated and the primary antibody was added, and the cells were incubated overnight at 4 °C. Finally, the corresponding secondary antibody, rhodamine phalloidin, and DAPI were added and incubated at 37 °C for 2 h. Before each operation, the cells were washed three times with PBS for 10 min each time. Observation and photography were performed under a fluorescence microscope (Carl Zeiss, USA), and finally, semi-quantitative analysis and fluorescence co-localization analysis were performed using Image J.

[0078] 4. β-gal staining

[0079] In this study, a β-gal staining kit (Solarbio, China) was used. The cells to be tested were inoculated in 6-well plates or culture dishes and cultured to an appropriate density. The cells were fixed with β-gal fixative and treated at room temperature for 15 min to ensure the integrity of cell morphology and the maintenance of β-gal enzyme activity. The staining working solution was prepared in an appropriate ratio, added to the cells, sealed with plastic wrap and incubated overnight at 37 °C. Observation and photographs were taken under a light microscope.

[0080] 5. Detection of alkaline phosphatase (ALP) and calcium deposition levels

[0081] After 7 days of cell culture, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and then 500 μL of the staining solution in the ALP staining kit was added for half an hour of staining. The staining situation was observed and photographed under a microscope. After each step of operation, the cells were washed 3 times with PBS for 10 minutes each time. Subsequently, quantitative analysis was performed. The cells were lysed with RIAP cell lysate, and after centrifugation of the lysate to obtain the supernatant, the AKP / ALP kit (Beyotime, China) was used to quantify the ALP activity.

[0082] After 14 days of cell culture, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and then 500 μL of alizarin red staining solution was added for 5 min of staining. The staining situation was observed and photographed under a microscope. After each step of operation, the cells were washed 3 times with PBS for 10 minutes each time. Subsequently, quantitative analysis was performed. 1 mL of 10% perchloric acid solution was added to each well of the stained well plate, and the reaction was carried out at room temperature in the dark until the calcium nodules were completely dissolved. The absorbance was measured at a wavelength of 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0083] 6. Flow cytometry analysis

[0084] The cells were transferred to 1.5 mL epppendorf (EP) tubes and centrifuged at 2000 rpm for 5 minutes at 4 °C. The collected cells were analyzed with Ki67 (ab16667, Abclonal, USA) antibody, and the analysis was performed using FlowJo software.

[0085] 7. Hippocampal analysis

[0086] The Seahorse Extracellular Flux XFe 24 analyzer (Agilent, USA) was used to study the mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The cells were inoculated in Seahorse XF-24 plates and material intervention was added. Before analysis, the cell plates were incubated in a CO2-free incubator for 1 hour. For ECAR analysis, glucose (10×10 -3 m), Oligo (1×10 -6 m) and 2-DG (50×10-3 m). Glycolysis was evaluated as the maximum ECAR value before Oligo intervention minus the last ECAR value before glucose injection, glycolytic capacity was evaluated as the maximum ECAR value after Oligo injection minus the last ECAR value before glucose injection, and glycolytic reserve was glycolytic capacity minus glycolysis. For OCR analysis, Oligo (1×10 -6 m), FCCP (1.5×10 -6 m), and rotenone / antimycin (R / A) (2×10 -6 m) were sequentially injected. Basal respiration was the last OCR value before Oligo injection minus non-mitochondrial respiration, maximum respiration was the maximum OCR value after FCCP injection minus the minimum OCR value after R / A injection, and ATP production was the final OCR value before Oligo injection minus the minimum OCR value after Oligo injection.

[0087] 8. Western Blot

[0088] After co-culturing the cells with microspheres, total proteins were extracted using RIPA lysis buffer (Beyotime, China) containing protease inhibitors (Beyotime, China) and phosphatase inhibitors (Beyotime, China). Protein concentration was confirmed using a BCA kit (Solarbio, China). Subsequently, equal amounts of proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane for target proteins. Then, blocking was performed for 1 hour, and the membrane was incubated with the primary antibody overnight. Next, the membrane was incubated with a horseradish peroxidase (HRP)-labeled secondary antibody. Finally, protein bands were detected using an enhanced chemiluminescence (ECL) kit (ThermoFisher, USA) and visualized using a gel imaging system. Finally, semi-quantitative analysis was performed using ImageJ.

[0089] 9. qRT-PCR

[0090] Cell RNA was extracted using an RNA extraction kit (Vazyme, China) according to the steps, the RNA concentration and purity were measured, EDTA was added to make the RNA concentrations of each group consistent. An RNA reverse transcription reaction system was prepared by volume, incubated in a 70°C metal bath for 5 min for reverse transcription, and 100 μL of enzyme-free water was added after the reaction to obtain the reverse transcription product cDNA stock solution. Subsequently, a qRT-PCR reaction system was prepared, and after denaturation, annealing, and extension reactions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference control to analyze the relevant target genes. All primer sequences are shown in Table 1.

[0091] Table 1

[0092]

[0093] Good biocompatibility is the basis for maintaining the viability of BMSCs co-cultured with microspheres and is also a key factor for the ultrasound transducer to exert its effect. Live / dead staining showed that all microsphere groups exhibited good biocompatibility, and the GMPG group showed the most favorable cell proliferation. This may be due to the crosslinking of PDA and GelMA in PDA@GNRs, which further enhanced the biocompatibility of the side chains and made it more conducive to cell adhesion and proliferation.

[0094] Cell senescence is a process of cellular physiological changes induced by internal and external factors, usually manifested as a decline in proliferative capacity, changes in metabolic function, and an increase in the expression of typical senescence markers. The designed signal transducer aims to precisely intervene in the senescence phenotype of cells and provide new strategies for delaying or reversing aging. The staining results of this example showed that the signal transducer could restore the metabolism of senescent BMSCs cells and improve senescence. The Ki67 flow cytometry results revealed that the cells in the GMPG / US group had stronger cell proliferation ability and were more youthful. Senescence is often accompanied by the production of senescence-associated secretory phenotype (SASP), which can exacerbate the inflammatory response and further promote the aging process. Through the analysis of the qRT-PCR results of SASP-related markers, it was further clarified that the ultrasound thermal effect could improve the level of cell senescence and reduce the SASP secretion of senescent BMSCs. In summary, the GMPG / US group could improve the cell senescence phenotype and restore cell proliferation ability, and reduce the secretion of SASP, delaying the process of cell senescence at multiple levels.

[0095] Significant changes also occur in the energy metabolism mode of senescent cells, which are often manifested as continuous metabolic activity and the secretion of more SASP, affecting the surrounding microenvironment. As the center of cell energy metabolism, mitochondrial function changes will secondary to the changes in cell metabolic status. Previous studies have shown that compared with normal cells, senescent cells have increased glucose consumption and lactate levels. Our research results showed that GMPG / US could reprogram the abnormal metabolism of senescent BMSCs, improve cell metabolic function, relieve the metabolic stress state, reshape the mitochondrial homeostasis of senescent BMSCs, and maintain a stable energy supply for cells.

[0096] The decline in the osteogenic differentiation ability of BMSCs in the senescent state impairs the regenerative ability of bone tissue. To verify whether GMPG / US can delay the senescence of BMSCs and improve its bone regeneration function, we further comprehensively analyzed its osteogenic ability. Alkaline phosphatase (ALP) and alizarin red (ARS) staining, as early and late osteogenic mineralization markers, were used to evaluate the osteogenic ability of cells. It is worth noting that in this example, the color of ALP staining in the GMPG / US treatment group was the darkest, and it had the best mineral matrix formation ability, indicating that GMPG / US can improve the osteogenic differentiation function of senescent BMSCs. To further evaluate the osteogenic potential of BMSCs after ultrasound hyperthermia intervention, we detected the key transcription factor RUNX2 involved in osteogenic differentiation and osteocalcin (OPN), a key regulator of calcium metabolism, by immunofluorescence to evaluate the osteogenic potential of BMSCs after ultrasound hyperthermia effect intervention. The research results show that GMPG / US, as an effective signal converter, can effectively enhance the osteogenic differentiation of BMSCs in vitro and has great potential in promoting bone regeneration in senescent individuals.

[0097] This example proves that the designed GMPG / US can delay the senescent phenotype of BMSCs, reduce the secretion of SASP, increase the cell proliferation ability, and thus delay the senescent state of cells from multiple levels. Moreover, GMPG / US can relieve the cellular glucose metabolism stress state and restore the metabolic function of senescent BMSCs by inhibiting glycolysis and oxidative phosphorylation. In addition, GMPG / US can also improve the osteogenic differentiation function of senescent BMSCs.

[0098] Example 5

[0099] 1. Transcriptomics and metabolomics

[0100] BMSCs cultured with microspheres were collected for transcriptomics and metabolomics analysis. Total RNA was extracted using an RNA purification kit (ThermoFisher, USA) for transcriptomics, and the cells were quickly frozen in liquid nitrogen for 5 min to extract metabolites for metabolomics. For the comparison between each two groups, genes or metabolites with a false discovery rate (FDR) < 0.05 were considered significant, and the log 2-fold change (logFC) in expression between conditions was reported. Subsequently, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used to determine the biological functions or pathways mainly affected by the differential transcripts.

[0101] 2. Mitochondrial permeability transition pore (mPTP) detection

[0102] Cells were detected using a mitochondrial permeability transition pore (mPTP) detection kit (Beyotime, China). They were incubated with Calcein AM staining solution or Calcein-AM staining solution supplemented with CoCl2 at 37 °C for 20 min, and then washed three times with PBS for 10 min each time. Observation and photography were carried out under a fluorescence microscope (Carl Zeiss, USA), and finally semi-quantitative analysis and fluorescence co-localization analysis were performed using Image J.

[0103] 3. Measurement of mitochondrial function

[0104] They were incubated with the JC-1 matrix metalloproteinase detection kit staining working solution (MedChemExpress, USA) at 37 °C for 20 min, and then washed twice with PBS. Observation and photography were carried out under a fluorescence microscope. Semi-quantitative analysis was performed using ImageJ software, and MMP (the ratio of red / green fluorescence intensity) was calculated.

[0105] Cells in different microsphere groups were incubated with DCFH-DA working solution (MedChemExpress, USA) and MitoSOX working solution (MedChemExpress, USA) at 37 °C for 30 min. Observation and photography were carried out under a fluorescence microscope. Semi-quantitative analysis was performed using ImageJ software.

[0106] 4. Cytosol separation

[0107] The cytosol of BMSC cells was extracted using a mitochondrial isolation kit (Beyotime, China). The cytosolic supernatant without mitochondria was collected for the extraction of cytosolic proteins and cytosolic mtDNA. The process of extracting cytosolic proteins was the same as step 8 in Example 4. Genomic DNA micro-preparation kit (Axygen, USA) was used to extract cytosolic mtDNA.

[0108] 5. Immunogold electron microscopy

[0109] Cells in each group were fixed with paraformaldehyde, dehydrated step by step with acetone, and embedded and polymerized with resin. Cryosectioning was performed using a cryostat (Leica, Germany) with a section thickness of 60 nm. Subsequently, the sections were blocked with 1% BSA for 30 min, incubated with the primary antibody DNA (CBL186, Merck, Germany) overnight at 4 °C, and then incubated with the anti-mouse IgG gold secondary antibody (G7652, Merck, Germany) for 2 hours at room temperature. Finally, high-resolution TEM images were taken (FEI Tecnai T12, USA), and semi-quantitative analysis was performed.

[0110] 6. Agarose gel electrophoresis

[0111] Add 0.3 g of agarose (ThermoFisher, USA) to 30 mL of 1× TAE and heat it in a microwave oven until the solution is clear and free of particles. Wait for it to cool to 55 °C, add 6 μL of nucleic acid staining reagent (G814, Solarbio, China), and mix well. Add the agarose solution to the electrophoresis mold and wait for it to cool and solidify into a gel. Mix the extracted mtDNA in the cytosol and loading buffer evenly at a ratio of 1:5, and add them to each well of the agarose gel according to the grouping. Place the loaded gel into the electrophoresis apparatus and perform electrophoresis at 90 V for 90 min. Finally, place the gel on an ultraviolet light imager to observe and take pictures.

[0112] To explore the potential mechanism of GMPG / US in delaying cell senescence, we performed transcriptome analysis on senescent BMSCs to investigate the potential mechanism of GMPG / US in delaying the process of cell senescence. Interestingly, the MAPK and NF-κB signaling pathways are closely related to the specific therapeutic mechanism of GMPG / US in BMSCs. Specifically, the MAPK signaling pathway reflects the biological response of BMSCs to heat stimulation; the NF-κB signaling pathway represents the regulatory effect of GMPG / US on the senescence-inflammation network hub in BMSCs. Gene Ontology enrichment analysis (GO) showed that multiple cellular biological processes and functions such as positive regulation of cell population proliferation, senescence, and inflammatory responses were affected after heat stimulation intervention. The enrichment of functional changes in the response to heat suggested the response of cells to heat stimulation, while the significant change in the negative role of mitochondrial membrane permeability suggested the change in mitochondrial membrane permeability of cells. GSEA analysis showed that after GMPG / US treatment, the downregulation of cell cycle and DNA replication pathways closely related to senescence verified that GMPG / US could regulate senescence-related signaling pathways to improve the cell senescence state. Therefore, the ultrasonic thermal effect plays an important role in dealing with senescence, inflammation, and the increased mitochondrial membrane permeability caused by the cascade.

[0113] Previous studies have shown that mtDNA leakage can be captured by the cytosolic DNA sensor (cGAS) to generate the cyclic dinucleotide GMP-AMP (cGAMP), which activates the STING (stimulator of interferon genes) dimer. On the one hand, the activation of STING promotes the autophosphorylation of TBK1; on the other hand, it can promote the activation of NF-κB through the compensation of TAK1 and the IKK complex. The activated IRF3 and NF-κB can jointly regulate the cell-inflammatory response, prompting senescent cells to produce SASP. GMPG / US can negatively regulate the NF-κB signaling pathway by reducing the activation of the cGAS-STING pathway caused by mtDNA leakage, inhibit the occurrence of SASP, and relieve the inflammatory response, which is consistent with the results of transcriptomics analysis.

[0114] To further explore the effect of GMPG on the mitochondrial membrane permeability of senescent BMSCs after in vitro exposure to ultrasound stimulation, we first used high-resolution structured illumination microscopy (HIS-SIM) to observe the effects of changes in the mitochondrial membrane permeability of senescent BMSCs on mitochondrial contents and mitochondrial function. The fluorescence co-localization results of Cyt c and TOM20 showed that the GMPG / US group effectively maintained the intact network structure of the mitochondria in senescent BMSCs and significantly reduced the efflux of Cyt c inside the mitochondria, which also verified the increased mitochondrial membrane permeability in senescent cells.

[0115] The results of JC-1, Mitosox, DCFH-DA fluorescence probes and mitochondrial electron microscopy showed that the mitochondrial MMP level of senescent BMSCs in the GMPG / US group was improved and the mitochondrial function was restored. Previous studies have shown that the abnormal accumulation of intracellular ROS is a key factor inducing senescence, and the increased mitochondrial membrane permeability in senescent cells will trigger the explosive release of mitochondrial ROS, further disrupting mitochondrial homeostasis. WB analysis also revealed that GMPG / US enhanced the antioxidant capacity of senescent BMSCs. The above results all revealed that ultrasound hyperthermia could improve the mitochondrial dysfunction of senescent BMSCs, stabilize the mitochondrial membrane permeability, and reshape its redox homeostasis.

[0116] Recent studies have shown that the aging process may be accompanied by the leakage of another key mitochondrial content - mitochondrial DNA. In this application, we found that mtDNA was present in the cytoplasm of the GMPG group, while no obvious mtDNA leakage occurred in the GMPG / US group. These results demonstrated that GMPG / US significantly reduced the leakage of mtDNA from senescent BMSCs into the cytoplasm, which may be attributed to the decreased mitochondrial membrane permeability.

[0117] To deeply explore the specific mechanism by which GMPG / US inhibits the increase in mitochondrial membrane permeability induced by senescence leading to mtDNA leakage, the activities of channels related to mitochondrial membrane permeability under ultrasound hyperthermia stimulation were detected, especially the mitochondrial permeability transition pore (mPTP), voltage-dependent anion channel (VDAC), and pro-apoptotic protein BAX and other megachannels that can leak large molecular mitochondrial contents. Using CalceinAM staining to verify the opening of the mPTP channel, we found that the presence of CoCl2 was associated with a significant quenching of the cytoplasmic calcein signal, while the mitochondrial signal remained unchanged. This indicated that mtDNA did not leak through the mPTP and ultrasound hyperthermia did not affect the permeability of the mPTP channel. As the main anion channel in the outer mitochondrial membrane, the change in its permeability under ultrasound hyperthermia stimulation was also monitored. The absence of significant change in the VDAC fluorescence intensity indicated that its openness remained in a steady state and its permeability and functional state were not affected by ultrasound treatment.

[0118] BAX is an important pro-apoptotic protein in the mitochondrial pathway, and it usually promotes the transmission of apoptotic signals by forming pores on the membrane. The results of fluorescence staining with a conventional confocal microscope suggest that there are significant differences in BAX after ultrasound stimulation. Subsequently, we performed double immunostaining for BAX6A7 and TFAM, labeled mitochondria with Mitotracker, and used super-resolution structured illumination microscopy to explore whether mtDNA leakage depends on the pores formed on the outer mitochondrial membrane after BAX oligomerization. Different from the stable performance of mPTP and VDAC, the visualization image results show that BAX is localized to the annular pores formed by mitochondria, and mtDNA leaks from the pores from inside the mitochondria into the cytoplasm. These results all indicate that GMPG / US maintains the stability of mitochondrial membrane permeability by inhibiting BAX activation.

[0119] Previous transcriptome sequencing suggested that ultrasound thermal stimulation significantly upregulated the pathways responsive to heat. The cellular stress response mechanism is partly composed of heat shock proteins (HSPs), and these molecular chaperones play an indispensable role in maintaining intracellular homeostasis by assisting protein folding. The HSP70 protein is the most strongly heat shock-induced target and the most well-characterized HSP. Both immunofluorescence and WB experiments showed that the expression level of HSP70 in BMSCs co-cultured with composite microspheres under ultrasound stimulation was significantly higher than that in the composite microsphere group without ultrasound stimulation. Therefore, this signal transducer can help cells sense ultrasound thermal stimulation and convert it into a biochemical signal with high expression of HSP70. Therefore, we proposed the hypothesis that HSP70 may be associated with mitochondrial membrane permeability, the NF-κB signaling pathway, and the Cell cycle pathway.

[0120] Next, we investigated the specific mechanism by which GMPG / US inhibits BAX activation and reduces mtDNA leakage. Previous results showed that GMPG / US could promote the high expression of HSP70 in senescent BMSCs. Therefore, we hypothesized that HSP70 could protect cells from apoptosis by inhibiting the activation of BAX. VER-155008 and ML346 were used as a specific inhibitor and agonist of HSP70, respectively, to explore the effect of HSP70 on mitochondrial membrane permeability. The results showed that GMPG / US could alleviate the oligomerization of BAX induced by senescence in BMSCs. The addition of ML346 promoted the therapeutic effect, while the addition of the VER-155008 inhibitor reversed this change, suggesting that HSP70 could inhibit BAX activation. This might depend on the HSP70 chaperone proteins HSP40, NEFs, and the ATP hydrolysis domain. HSP40 and NEFs enhanced the binding of HSP70 to BAX by regulating the ATP hydrolysis and nucleotide exchange of HSP70, thereby preventing its conformational change and keeping it stable in the cytoplasm without oligomerization. The fluorescence co-localization results of DNA and TOM20 further revealed that HSP70 could reduce the leakage of mtDNA. The JC-1 immunofluorescence results confirmed that ML346 promoted the stabilizing effect of GMPG / US on mitochondrial membrane potential, while VER-155008 reversed the therapeutic effect. In addition, the DCFH-DA and Mitosox staining results demonstrated that HSP70 had a protective effect on the release of ROS in mitochondria. The above results all indicated that HSP70 could inhibit BAX activation, prevent the formation of mitochondrial membrane channels, reduce mitochondrial membrane permeability, and thus reduce the release of ROS and the leakage of mtDNA.

[0121] In addition, we verified that the activation of HSP70 further enhanced the anti-aging effect of GMPG / US on BMSCs, and the inhibition of HSP70 weakened the inhibitory effect of ultrasound hyperthermia on the cGAS-STING pathway and its downstream NF-κB pathway, thereby increasing the expression of SASP. We also found that the expression levels of osteogenic differentiation-related proteins RUNX2, OPN, and OCN in the GMPG / US group were significantly decreased after inhibiting HSP70, which also proved that this treatment platform could promote the high expression of HSP70 in cells to improve BMSCs senescence and restore osteogenic differentiation function. These all indicated that the signal transducer based on the ultrasound response and thermal conversion effect of PDA@GNRs in GelMA microspheres improved the senescent state of cells by stimulating the high expression of HSP70 in senescent BMSCs.

[0122] Example 6

[0123] 1. Preparation for animal experiment

[0124] The experimental animals were 18-week-old female rats, with an average weight of about 250 - 350 g. They were all provided by the Experimental Animal Center of Soochow University. During the experiment, all surgical operations and animal handling during the perioperative period were approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. (SUDA 20240326 A02)

[0125] 2. Establishment and treatment of animal models

[0126] First, prepare a 2% sodium pentobarbital solution and inject it intraperitoneally for anesthesia at a standard of 0.1 mL / 100 g. After successful anesthesia, prepare the skin around the knee joint within a 5-cm radius. Then, make a longitudinal incision along the lateral margin of the patella, about 2 cm long, and expose the skin, fascia, muscle layer by layer, strip the muscle, push aside the patella, and expose the femoral condyle. Subsequently, locate the attachment point of the lateral collateral ligament on the femoral condyle, and use the intersection of the horizontal line at this attachment point and the perpendicular bisector of the midpoint between the femoral condyles as the drilling point. Use a drill bit with a diameter of 3.0 mm to drill a hole with a depth of 4.0 mm, reset the patella, and suture the muscle, fascia, subcutaneous tissue, and skin layer by layer. Finally, disinfect the wound and inject the disinfected microsphere material into the bone defect. Intramuscularly inject 400,000 units of sodium penicillin once a day for three consecutive days. Each group was stimulated with an intensity of 1 W / cm 2 for 60 min, and stimulated once every two days.

[0127] 3. Sample collection and micro-CT analysis

[0128] At 2 weeks and 4 weeks after surgery, the rats were euthanized, and the femoral specimens were taken out and immersed in 10% formalin solution for fixation. Subsequently, Micro-CT (SkyScan 1276, Belgium) was used to take pictures at parameters of 18 μm, 65 kV, and 385 mA. The analysis software was used to perform 3D reconstruction of the entire femur and the defect site, and analyze the bone volume / total volume (BV / TV), bone mineral density (BMD), trabecular thickness (TP.th), and trabecular separation (TP.sp).

[0129] 4. Section preparation, H&E staining, and MASSON staining

[0130] The specimens were decalcified in EDTA solution for 2 weeks, dehydrated successively in 70%, 80%, 90%, and 100% ethanol. Then, they were cleared with xylene, immersed in paraffin, embedded, cooled, and fixed. Sections were made at a thickness of 5 μm, and the femoral sections were dried. They were placed in xylene solution again, soaked in absolute ethanol twice, then placed in ethanol solutions with decreasing concentration gradients for 3 min each, and finally rinsed with distilled water.

[0131] The sections were stained with hematoxylin for 5 min, then treated with 1% hydrochloric acid and 70% alcohol for 1 min, soaked in distilled water containing ammonia (pH 8.7) for 5 min; soaked in 0.5% eosin aqueous solution for 5 min, dehydrated with 70%, 80% and 90% ethanol for 1 min each, dehydrated with 100% ethanol for 10 min, treated with xylene for 30 min, and finally sealed with resin and cover glass. Observation and photography were carried out under a microscope.

[0132] The sections were stained with Masson's trichrome stain for 5 min, then washed with 0.2% acetic acid solution, treated with 5% phosphotungstic acid solution for 10 min, washed with 0.2% acetic acid solution twice, stained with aniline blue for 5 min, washed with 0.2% acetic acid solution twice, and finally dehydrated with absolute ethanol twice, cleared and sealed with xylene. Observation and photography were carried out under a microscope.

[0133] 5. Immunofluorescence staining of sections

[0134] Fixation and dehydration were the same as in 4 of Example 6. 100 μL of non-immune animal sheep serum was added dropwise and incubated at 37 °C for 20 min. Then 100 μL of primary antibodies BAX6A7 (NBP1-28566, Novus, USA), TOM20 (CL647-11802, Proteintech, USA), STING (A21051, Abclonal, USA), P16 (A23882, Abclonal, USA), P65 (A22331, Abclonal, USA), IL-6 (A21264, Abclonal, USA), and OCN (23418-1-AP, Proteintech, USA) were added and incubated overnight at 4 °C. The next day, the wet box was taken out, placed at 37 °C for 30 min, PBS was removed, 100 μL of labeled fluorescent secondary antibody was added dropwise, incubated at 37 °C for 1 h, washed 3 times with PBS, and finally sealed with Fluoroshield Mounting Medium With DAPI.

[0135] 6. Detection of in vivo ROS level

[0136] ROS Brite 700 (AAT Bioquest, USA) was used to detect the in vivo ROS level in the femoral supracondylar fractures of adult and aged rats in each group 7 days after surgery. First, the rats were anesthetized with isoflurane, and then ROS Brite 700 (in vivo imaging solution, containing 20×10 -3 m in Hanks with mHHBS of 100×10 -6 m) was injected into the lesion site. A bioluminescence image was obtained using an in vivo imaging system (Spectral Instruments Imaging, USA).

[0137] In this section, by establishing a femoral condyle bone defect model in aging rats, we demonstrated the anti-aging effect of the prepared signal conversion therapy platform in vivo. The prepared signal conversion therapy platform showed a faster temperature increase at the bone defect site, reaching the optimal temperature for high expression of HSP70, while reducing the ROS level and alleviating oxidative damage in the bone defect area. Imaging results demonstrated that the prepared signal conversion therapy platform significantly promoted new bone formation, with a better effect than other groups. The results of H&E and Masson staining confirmed that the prepared signal conversion therapy platform had the most excellent bone tissue calcification and remodeling ability. Immunofluorescence staining results showed that the prepared signal conversion therapy platform promoted the high expression of HSP70, inhibited the activation of BAX in aging rats, improved mitochondrial function, reduced ROS and mtDNA leakage, thereby alleviating the inflammatory response and promoting the regeneration of aging bone.

[0138] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Hydrogel microspheres, characterized in that, Comprising: PDA-coated gold nanorods and GelMA hydrogel; The gold nanorods are loaded in the GelMA hydrogel.

2. The hydrogel microspheres according to claim 1, wherein The concentration ratio of the gold nanorods to the GelMA hydrogel is: 400:

1.

3. The preparation method of the hydrogel microspheres according to claim 1 or 2, characterized in that, Comprising the following steps: in the presence of a photoinitiator, crosslink and coat the surface of the PDA-coated gold nanorods with the GelMA hydrogel to obtain the hydrogel microspheres.

4. The preparation method according to claim 3, characterized in that, The preparation method of the PDA-coated gold nanorods comprises the following steps: S1: Mix an HAuCl4 solution, a sodium citrate solution and a NaBH4 solution to obtain a seed solution; S2: Mix a CTAB solution, an HAuCl4 solution and an AA solution to obtain a stock solution; S3: Mix the seed solution in S1 and the stock solution in S2 to obtain a solution A; S4: Mix the solution A in S3 and the stock solution in S2 to obtain a solution B; S5: Mix the solution B in S4 and the stock solution in S2, react and centrifuge to obtain gold nanorods; S6: Mix a Tris-HCl solution and dopamine hydrochloride to obtain a PDA layer; S7: Mix the gold nanorods in S5 and the PDA layer in S6, initiate the reaction, centrifuge, and collect the precipitate to obtain the PDA-coated gold nanorods.

5. A bioreceptor, characterized in that, Comprising: The hydrogel microspheres as described in claim 1 or 2 and / or the hydrogel microspheres obtained by the preparation method as described in claim 3 or 4.

6. Use of the hydrogel microspheres as described in claim 1 or 2, the hydrogel microspheres obtained by the preparation method as described in claim 3 or 4 and / or the bioreceptor as described in claim 5 in the preparation of a product for treating senile bone defects.

7. The application according to claim 6, characterized in that, The treatment of senile bone defects includes: restoring mitochondrial function, improving the senile phenotype and promoting bone regeneration, one or more of them.

8. The application according to claim 7, wherein The restoration of mitochondrial function includes: inhibiting the activation of BAX in the senile mitochondrial membrane, reducing the mitochondrial membrane permeability and reducing the release of ROS in the mitochondria, one or more of them.

9. The application according to claim 7 or 8, characterized in that, The improvement of the senile phenotype includes: reducing the SASP phenotype.

10. Product, characterized in that, Comprising: The hydrogel microspheres as described in claim 1 or 2, the hydrogel microspheres obtained by the preparation method as described in claim 3 or 4 and / or the bioreceptor as described in claim 5, and acceptable adjuvants, excipients, carriers and / or platforms.

Images