Monatomic nano-enzyme for targeting mitochondria and accelerating bone regeneration and preparation method of monatomic nano-enzyme
The nanozyme, which combines Fe/Cu single-atom nanozyme with TPP molecules, solves the problems of targeting and catalytic efficiency of existing nanozymes in bone regeneration, and achieves efficient bone regeneration and rapid repair.
Patent Information
- Application Number
- CN202511051486.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-10-31
AI Technical Summary
Existing nanozymes in the field of bone regeneration suffer from problems such as insufficient targeting, low catalytic efficiency, and limited functionality, resulting in poor repair effects and long repair cycles.
By employing Fe/Cu single-atom dual active sites, TPP molecules targeting mitochondria, and porous nanocarriers, specifically dendritic mesoporous silica nanoparticles, TPP-DMSN-Fe/Cu nanozymes were synthesized to target mitochondria and accelerate bone regeneration.
It significantly improves the efficiency of reactive oxygen species scavenging, reduces off-target effects, and simultaneously achieves antioxidant, metabolic regulation and osteogenic differentiation regulation, shortens the bone regeneration cycle, and has no heavy metal aggregation toxicity and excellent biocompatibility.
Smart Images
Figure CN120860059A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioactive components for bone regeneration, specifically to a single-atom nanozyme that targets mitochondria and accelerates bone regeneration, and its preparation method. Background Technology
[0002] Current mainstream technologies in bone regeneration include growth factor (e.g., BMP-2) delivery, bioactive scaffold implantation, and antioxidant therapy based on nanomaterials. While existing nanozymes (e.g., Fe3O4 nanoparticles, CeO2 nanoparticles) can scavenge reactive oxygen species (ROS), their catalytic efficiency is low, they lack targeting, and they cannot directly regulate mitochondrial function. Furthermore, traditional materials are prone to inducing inflammatory responses or off-target effects, leading to long bone repair cycles and unstable outcomes. The current technological bottlenecks can be summarized as follows:
[0003] 1. Insufficient targeting: Existing nanozymes cannot be precisely targeted to mitochondria, making it difficult to repair oxidative stress damage to bone cell mitochondria;
[0004] 2. Low catalytic efficiency: Multi-atom nanoparticles have few catalytic active sites, resulting in limited ROS scavenging ability;
[0005] 3. Limited functionality: Existing technologies only focus on antioxidant or osteogenic signal activation, lacking synergistic regulatory mechanisms.
[0006] Therefore, there is an urgent need for a novel single-atom nanozyme that targets mitochondria and accelerates bone regeneration, providing an effective solution to the shortcomings of existing technologies. Summary of the Invention
[0007] The purpose of this invention is to provide a single-atom nanozyme that targets mitochondria and accelerates bone regeneration, and a method for preparing the same, in order to solve the problems mentioned in the background art.
[0008] To achieve the above objectives, the present invention provides the following technical solution:
[0009] A single-atom nanozyme that targets mitochondria and accelerates bone regeneration comprises a Fe / Cu single-atom dual active site, a TPP molecule that targets mitochondria, and a porous nanocarrier.
[0010] Furthermore, the porous nanocarrier is dendritic mesoporous silica nanoparticles (DMSN).
[0011] Furthermore, the porous nanocarrier is a mesoporous titanium dioxide, zirconium dioxide, or polymer scaffold.
[0012] A method for preparing a single-atom nanozyme that targets mitochondria and accelerates bone regeneration includes the following steps:
[0013] S1 (DMSN Synthesis): First, 0.136 g TEA was added to 50 ml of water and gently stirred at 80 °C for 0.5 hours. Then, 760 mg CTAB and 336 mg NaSal were added to the solution, and stirring was continued at 80 °C for 1 hour. Next, 8 ml TEOS was mixed with 3.2 ml BTEE and stirred at 80 °C and 300 rpm for 6 hours. The resulting mixture was centrifuged at 10,000 rpm for 10 minutes and washed 1-2 times with pure ethanol. Finally, the mixture was refluxed at 80 °C with HCl (3 ml) and methanol (60 ml) solution for 2 hours, followed by centrifugation to obtain DMSN, and washed with ethanol.
[0014] S2 (DMSN-NH2 synthesis): 280 mg DMSN was dissolved in 80 ml ethanol and sonicated; then 7 ml APTES was added, and the solution was refluxed at 70 °C for 6 hours; after that, the solution was centrifuged to obtain DMSN-NH2;
[0015] S3 (DMSN-Fe / Cu Synthesis): First, a solution of 80 mg DMSN-NH2 dissolved in 20 ml MES buffer (pH=6) was prepared; then, 48 mg EDC and 12.8 mg NHS were activated for 30 minutes; subsequently, 320 mg L-Cys was added, and the mixture was kept at 37 °C for 6 hours. The resulting nanoparticles were centrifuged and then resuspended in 9 ml distilled water; finally, 1 ml of deionized water containing 41 mg Cu(NO3)2·3H2O and 100 mg Fe3O4·7H2O was added to obtain DMSN-Fe / Cu.
[0016] S4 (TPP-DMSN-Fe / Cu Synthesis): First, 20 mg DMSN / L-Cys / Fe / Cu was mixed with 24.552 mg NHS + 49.288 mg EDC and dispersed in 10 ml MES buffer at pH 6.00. The mixture was stirred at room temperature for 2 hours. Then, 80 mg TPP was dispersed in 8 ml deionized water and incubated at 37°C for 6 hours. The final product was centrifuged to obtain TPP-DMSN-Fe / Cu.
[0017] Furthermore, CTAB and NaSal were used as cationic surfactants in the synthesis of DMSN in S1.
[0018] Furthermore, in the synthesis process of DMSN in S1, the silicon sources are TEOS and BTEE, and the catalyst is TEA.
[0019] Furthermore, in S3, cobalt (Co) or vanadium (V) single atoms are used to replace Fe / Cu.
[0020] Furthermore, in S4, dequalinium (DQA) is used instead of TPP.
[0021] Compared with the prior art, the beneficial effects of the present invention are:
[0022] This invention utilizes single-atom catalytic active sites to significantly improve the efficiency of reactive oxygen species (ROS) scavenging compared to traditional nanozymes. Simultaneously, it reduces off-target effects by leveraging mitochondrial targeting ligands, thereby lowering the therapeutic dose. This invention simultaneously achieves antioxidant effects, metabolic regulation, osteogenic differentiation regulation, and accelerated bone regeneration, without heavy metal aggregation toxicity and exhibits excellent biocompatibility. This invention effectively addresses the shortcomings of existing bone regeneration technologies, such as insufficient mitochondrial targeting, low catalytic efficiency, and limited functionality, resulting in poor repair effects and long repair cycles. Attached Figure Description
[0023] Figure 1 Synthesis and characterization of TPP-DMSN-Fe / Cu nanozymes:
[0024] (1a) Schematic diagram of the synthesis process of TPP-DMSN-Fe / Cu nanozyme.
[0025] (1b, 1c) Transmission electron microscopy (TEM) images of synthesized nanozymes. Scale bars = 50 nm and 10 nm.
[0026] (1d) High-resolution HAADF image showing the dendritic mesoporous structure of the synthesized nanozyme. Scale bar = 50 nm.
[0027] (1e) Energy-dispersive X-ray spectroscopy (EDS) elemental mapping confirmed the uniform distribution of Cu, Fe, Si, and O in the TPP-DMSN-Fe / Cu nanozyme. Scale bar = 50 nm.
[0028] (1f) Zeta potential values of different nanoparticles. From left to right in the figure, they are Control group, DMSN group, DMSN-Fe / Cu group, and TPP-DMSN-Fe / Cu group.
[0029] (1g) Fourier transform infrared spectrum (FTIR).
[0030] (1h) N2 adsorption and desorption isotherms and corresponding pore size distribution of TPP-DMSN-Fe / Cu nanozymes.
[0031] (1i-1k) Antioxidant capacity test of different nanoparticles, comparing DPPH-(i), ·OH-(j) and H2O2 scavenging rate (k). From top to bottom in the figure, they are Control group, DMSN group, DMSN-Fe / Cu group, and TPP-DMSN-Fe / Cu group.
[0032] Figure 2 The experimental procedure and data were developed to verify the effect of TPP-DMSN-Fe / Cu nanozymes on bone tissue regeneration using a Sprague-Dowley (SD) rat tibial defect model. Detailed Implementation
[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0034] Example 1: A single-atom nanozyme that targets mitochondria and accelerates bone regeneration, comprising Fe / Cu single-atom dual active sites, a mitochondrial-targeting TPP molecule, and a porous nanocarrier.
[0035] The porous nanocarrier is DMSN.
[0036] This embodiment utilizes DMSN for one-step synthesis, and transmission electron microscopy (TEM) confirms its uniform spherical morphology and well-defined mesoporous structure. Subsequent functionalization steps include sequential loading of Fe / Cu and covalent coupling of mitochondrial-targeting TPP via amidation, ultimately preparing the TPP-DMSN-Fe / Cu nanozyme. Figure 1 High-angle annular dark-field (HAADF) imaging reveals a dendritic structure with radially arranged mesopores. Figure 1 d), while energy dispersive spectroscopy (EDS) elemental mapping confirmed the uniform distribution of Cu, Fe, Si, and O in the composite structure. Figure 1 e).
[0037] The physicochemical properties of the single-atom nanozyme in this embodiment were systematically characterized. Zeta potential measurements showed that the surface charge changed from negative to positive after TPP coupling. Figure 1 f) The average surface charge value is 34.7 mV, indicating colloidal stability (absolute value > 30 mV). Furthermore, Fourier transform infrared (FTIR) spectroscopy further confirmed the successful synthesis of the nanozyme, observing characteristic Si–O–Si stretching vibration peaks (410, 755, and 1019 cm⁻¹). -1 The C=O absorption peak (1607 cm⁻¹) generated by L-Cys-mediated amidation reaction. -1 ()( Figure 1 g). Meanwhile, the nitrogen adsorption-desorption isotherm shows that the TPP-DMSN-Fe / Cu nanozyme has a high specific surface area (592.8 m²). 2 g-1 ) and nanoporous structures ( Figure 1 h), which is beneficial to Fe / Cu loading and catalytic activity.
[0038] Since Fe / Cu co-catalysis can increase ATP production, the synergistic catalysis of Fe / Cu single-atom dual active sites in this embodiment simultaneously enhances fatty acid oxidation and scavenging reactive oxygen species. Replacing either metal would disrupt the metabolic balance crucial for osteogenic formation. Furthermore, the positive charge of TPP drives accumulation through mitochondrial membrane potential. In this embodiment, TPP targeting mitochondria can directly regulate fatty acid oxidases (such as CPT1A) and the electron transport chain (ETC) complex.
[0039] The antioxidant capacity of different nanoparticles was verified by testing and comparing the scavenging rates of DPPH-(i), ·OH-(j), and H2O2 (k). ROS accumulation has been shown to disrupt mitochondrial function and hinder tissue regeneration. To evaluate the ROS scavenging potential of nanozymes, free radical scavenging experiments were conducted. The results showed that, compared with unmodified DMSN, the Fe / Cu loading in the nanoparticles significantly improved the scavenging rates of DPPH radicals, hydroxyl radicals (·OH), and hydrogen peroxide (H2O2). Figure 1 (ik). The potent antioxidant activity in this embodiment makes TPP-DMSN-Fe / Cu an effective therapeutic agent for alleviating oxidative stress and restoring the redox balance of stem cells, thereby supporting mitochondrial health during bone formation.
[0040] This embodiment utilizes single-atom catalytic active sites, significantly improving ROS scavenging efficiency compared to traditional nanozymes. Simultaneously, it leverages mitochondrial-targeting ligands to reduce off-target effects, thereby lowering the therapeutic dose. This embodiment simultaneously achieves antioxidant, metabolic regulation, osteogenic differentiation regulation, and accelerated bone regeneration, without heavy metal aggregation toxicity and exhibiting excellent biocompatibility. This embodiment effectively addresses the shortcomings of existing bone regeneration technologies, such as insufficient mitochondrial targeting, low catalytic efficiency, and limited functionality, resulting in poor repair effects and long repair cycles.
[0041] Example 1: The in vivo evaluation of the effect of TPP-DMSN-Fe / Cu nanozyme on bone tissue regeneration in Example 1 was conducted using an SD rat tibial critical bone defect model. Figure 2 a)
[0042] Pure methacrylamide gelatin [GelMA, 10% (wt / v)] scaffolds and GelMA scaffolds loaded with 500 μg / mL DMSN, DMSN-Fe / Cu, and TPP-DMSN-Fe / Cu (labeled GelMA / DMSN scaffold, GelMA / DMSN-Fe / Cu scaffold, and GelMA / TPP-DMSN-Fe / Cu scaffold, respectively) were implanted into the defect sites. Bone defect specimens were collected from the tibias of SD rats at 4 and 8 weeks after treatment and evaluated using micro-CT.
[0043] like Figure 2 As shown in b, after 4 weeks of treatment, varying degrees of new bone formation were observed in the defect areas of all experimental groups. After another 4 weeks, the amount of new bone formation in all groups further increased. Furthermore, we analyzed the bone volume fraction (BV / TV) and bone mineral density (BMD) of the new bone tissue. Compared with the control group, the BV / TV values of the GelMA / TPP-DMSN-Fe / Cu group were significantly higher at both 4 and 8 weeks. At week 4, the mean BV / TV value of the GelMA / TPP-DMSN-Fe / Cu group was 2.77 times, 2.05 times, 1.50 times, and 1.49 times that of the control group, GelMA group, GelMA / DMSN group, and GelMA / TPP-DMSN-Fe / Cu group, respectively. Figure 2 c). By week 8, these BV / TV multiples had further increased to 4.10, 1.67, 1.45, and 1.28 ( Figure 2 d).
[0044] Example 2: A method for preparing a single-atom nanozyme that targets mitochondria and accelerates bone regeneration, the difference from Example 1 is that the porous nanocarrier is mesoporous titanium dioxide, zirconium dioxide or polymer scaffold.
[0045] In this embodiment, mesoporous titanium dioxide, zirconium dioxide, or polymer scaffolds are used to replace DMSN as the single-atom nanozyme carrier. Although mesoporous titanium dioxide, zirconium dioxide, or polymer scaffolds alter degradation kinetics / biocompatibility, they retain the core function, and their surface chemistry is suitable for TPP grafting.
[0046] Example 3: See Figure 1 a. A method for preparing single-atom nanozymes that target mitochondria and accelerate bone regeneration, comprising the following steps:
[0047] S1 (DMSN Synthesis): First, 0.136 g TEA was added to 50 ml of water and gently stirred at 80 °C for 0.5 hours. Then, 760 mg CTAB and 336 mg NaSal were added to the solution, and stirring was continued at 80 °C for 1 hour. Next, 8 ml TEOS was mixed with 3.2 ml BTEE and stirred at 80 °C and 300 rpm for 6 hours. The resulting mixture was centrifuged at 10,000 rpm for 10 minutes and washed 1-2 times with pure ethanol. Finally, the mixture was refluxed at 80 °C with HCl (3 ml) and methanol (60 ml) solution for 2 hours, followed by centrifugation to obtain DMSN, and washed with ethanol.
[0048] S2 (DMSN-NH2 synthesis): 280 mg DMSN was dissolved in 80 ml ethanol and sonicated; then 7 ml APTES was added, and the solution was refluxed at 70 °C for 6 hours; after that, the solution was centrifuged to obtain DMSN-NH2;
[0049] S3 (DMSN-Fe / Cu Synthesis): First, a solution of 80 mg DMSN-NH2 dissolved in 20 ml MES buffer (pH=6) was prepared; then, 48 mg EDC and 12.8 mg NHS were activated for 30 minutes; subsequently, 320 mg L-Cys was added, and the mixture was kept at 37 °C for 6 hours. The resulting nanoparticles were centrifuged and then resuspended in 9 ml distilled water; finally, 1 ml of deionized water containing 41 mg Cu(NO3)2·3H2O and 100 mg Fe3O4·7H2O was added to obtain DMSN-Fe / Cu.
[0050] S4 (TPP-DMSN-Fe / Cu Synthesis): First, 20 mg DMSN / L-Cys / Fe / Cu was mixed with 24.552 mg NHS + 49.288 mg EDC and dispersed in 10 ml MES buffer at pH 6.00. The mixture was stirred at room temperature for 2 hours. Then, 80 mg TPP was dispersed in 8 ml deionized water and incubated at 37°C for 6 hours. The final product was centrifuged to obtain TPP-DMSN-Fe / Cu.
[0051] The synthesis of DMSN in S1 uses CTAB and NaSal as cationic surfactants.
[0052] In the synthesis of DMSN in S1, the silicon sources are TEOS and BTEE, and the catalyst is TEA.
[0053] In this embodiment.
[0054] Example 4: A method for preparing a single-atom nanozyme that targets mitochondria and accelerates bone regeneration. The difference from Example 3 is that cobalt (Co) or vanadium (V) single atoms are used instead of Fe / Cu in S3.
[0055] Replacing Fe / Cu with cobalt (Co) or vanadium (V) as a metal substitute may reduce catalytic activity.
[0056] Example 5: A method for preparing a single-atom nanozyme that targets mitochondria and accelerates bone regeneration. The difference from Example 3 is that in S4, dequalinium (DQA) is used instead of TPP.
[0057] Using dequalinium (DQA) as a targeted alternative has certain cytotoxic effects; at high concentrations, it can disrupt mitochondrial membrane integrity, and its safety profile is lower than that of TPP.
Claims
1. A single-atom nanozyme that targets mitochondria and accelerates bone regeneration, characterized in that, It includes Fe / Cu single-atom dual active sites, triphenylphosphine (TPP) targeting mitochondria, and porous nanocarriers.
2. The single-atom nanozyme targeting mitochondria and accelerating bone regeneration according to claim 1, characterized in that, The porous nanocarrier is dendritic mesoporous silica nanoparticles (DMSN).
3. The single-atom nanozyme targeting mitochondria and accelerating bone regeneration according to claim 1, characterized in that, The porous nanocarrier is a mesoporous titanium dioxide, zirconium dioxide, or polymer scaffold.
4. A method for preparing a single-atom nanozyme that targets mitochondria and accelerates bone regeneration, characterized in that, Includes the following steps: S1 (DMSN Synthesis): First, 0.136 g TEA was added to 50 ml of water and gently stirred at 80 °C for 0.5 hours. Then, 760 mg CTAB and 336 mg NaSal were added to the solution, and stirring was continued at 80 °C for 1 hour. Next, 8 ml TEOS was mixed with 3.2 ml BTEE and stirred at 80 °C and 300 rpm for 6 hours. The resulting mixture was centrifuged at 10,000 rpm for 10 minutes and washed 1-2 times with pure ethanol. Finally, the mixture was refluxed at 80 °C with HCl (3 ml) and methanol (60 ml) solution for 2 hours, followed by centrifugation to obtain DMSN, and washed with ethanol. S2 (DMSN-NH2 synthesis): 280 mg DMSN was dissolved in 80 ml ethanol and sonicated; then 7 ml APTES was added, and the solution was refluxed at 70 °C for 6 hours; after that, the solution was centrifuged to obtain DMSN-NH2; S3 (DMSN-Fe / Cu Synthesis): First, a solution of 80 mg DMSN-NH2 dissolved in 20 ml MES buffer (pH=6) was prepared; then, 48 mg EDC and 12.8 mg NHS were activated for 30 minutes; subsequently, 320 mg L-Cys was added, and the mixture was kept at 37 °C for 6 hours. The resulting nanoparticles were centrifuged and then resuspended in 9 ml distilled water; finally, 1 ml of deionized water containing 41 mg Cu(NO3)2·3H2O and 100 mg Fe3O4·7H2O was added to obtain DMSN-Fe / Cu. S4 (TPP-DMSN-Fe / Cu Synthesis): First, 20 mg DMSN / L-Cys / Fe / Cu was mixed with 24.552 mg NHS + 49.288 mg EDC and dispersed in 10 ml MES buffer at pH 6.
00. The mixture was stirred at room temperature for 2 hours. Then, 80 mg TPP was dispersed in 8 ml deionized water and incubated at 37°C for 6 hours. The final product was centrifuged to obtain TPP-DMSN-Fe / Cu.
5. The method for preparing a single-atom nanozyme targeting mitochondria and accelerating bone regeneration according to claim 4, characterized in that: The synthesis of DMSN in S1 uses CTAB and NaSal as cationic surfactants.
6. The method for preparing a single-atom nanozyme targeting mitochondria and accelerating bone regeneration according to claim 4, characterized in that: In the synthesis of DMSN in S1, the silicon sources are TEOS and BTEE, and the catalyst is TEA.
7. The method for preparing a single-atom nanozyme targeting mitochondria and accelerating bone regeneration according to claim 4, characterized in that: In S3, cobalt (Co) or vanadium (V) single atoms are used to replace Fe / Cu.
8. The method for preparing a single-atom nanozyme targeting mitochondria and accelerating bone regeneration according to claim 4, characterized in that: Dequalinium (DQA) is used instead of TPP in S4.