Intelligent hydrogen peroxide-driven nanorobot, its assembly method and application

Abstract

The application provides a kind of intelligent hydrogen peroxide driven nano robot, its assembly method and application, including asymmetric carbon polymer / metal-phenolic network nanomotor;Bovine serum albumin and cRGD peptide modified on the surface of asymmetric carbon polymer / metal-phenolic network nanomotor;And metal nuclide is directly marked on the metal-phenolic network layer of asymmetric carbon polymer / metal-phenolic network nanomotor without chelating agent[ 68 Ga]Ga, can respond to lesion microenvironment autonomous propulsion, and has multi-level active target recognition and intracellular endocytosis promotion capacity.

Description

Technical Field

[0001] This invention relates to the field of artificial nanorobots, specifically to an intelligent hydrogen peroxide-driven nanorobot, its assembly method, and its applications. Background Technology

[0002] Nanorobots are nanodevices that convert other forms of energy into mechanical motion. Due to their small size, autonomous movement, and high payload capacity, they hold great promise not only for applications in biosensing, microsurgery, medical imaging, and environmental remediation, but also for targeted drug delivery, enabling precise treatment of diseased cells. In recent years, extensive research has been conducted on chemically self-driven nanorobots. Inspired by biomolecules and self-driven microorganisms, artificial nanorobots can utilize catalytic reactions for energy to achieve autonomous movement. Currently, the precise motion control and multi-level targeted delivery of chemically self-driven nanorobots in complex pathological microenvironments are still in their early stages.

[0003] Rheumatoid arthritis (RA) is a systemic, chronic autoimmune disease characterized by an overabundance of highly reactive oxygen species (ROS, especially H2O2) and an imbalanced oxygen environment in its lesion microenvironment. Traditional chemically driven nanorobots primarily rely on a single H2O2 concentration gradient for propulsion. However, in the face of complex, systemic lesions like RA, single-factor driven motors are easily inactivated or passively cleared from the bloodstream. More importantly, existing nanomotors generally lack the ability to accurately and actively recognize the multi-level microenvironment of RA lesions. After entering the inflamed joint cavity, the motors struggle to accurately identify and actively bind to pathogenic M1 macrophages and inflammatory fibroblasts from the complex cell population, resulting in low endocytosis efficiency and local delivery and enrichment efficiency far below therapeutic expectations.

[0004] On the other hand, in order to assess the movement and multifocal accumulation of nanorobots in vivo in real time and achieve integrated diagnosis and treatment, it is usually necessary to perform radionuclide (such as […]) analysis. 68 [Ga]Ga) labeling is used for image monitoring. However, traditional radionuclide labeling methods heavily rely on exogenous grafting of bifunctional chelating agents (such as DOTA or NOTA). This complex chemical modification step is not only cumbersome and demanding, but it also severely masks or destroys the targeting ligand activity of nanorobot surface modifications, further reducing its recognition efficiency for lesion cells. As an emerging class of materials with good biocompatibility and coordination ability, metal-phenolic networks (MPNs) have attracted widespread attention in the biomedical field, and their inherent metal trapping ability makes chelate-free labeling possible. Summary of the Invention

[0005] The purpose of this invention is to provide an intelligent hydrogen peroxide-driven nanorobot, its assembly method and application. It constructs an intelligent nanosystem that synergistically integrates multi-level active recognition of lesion cells, endogenous H2O2 driving, simultaneous improvement of hypoxic microenvironment, and real-time tracking function of non-chelating direct radioactive labeling. It can also autonomously propel itself in response to the lesion microenvironment and has the ability to promote multi-level active targeting and cell endocytosis.

[0006] To achieve the above objectives, this technical solution provides an intelligent hydrogen peroxide-driven nanorobot, comprising:

[0007] Asymmetric carbon polymer / metal-phenolic network nanomotors; Bovine serum albumin and cRGD peptide modified on the surface of an asymmetric carbon polymer / metal-phenolic network nanomotor; And metal nuclides directly labeled on the metal-phenolic network layer of asymmetric carbon polymer / metal-phenolic network nanomotors without chelating agents [ 68 Ga]Ga.

[0008] The intelligent hydrogen peroxide-driven nanorobot of this scheme utilizes an asymmetric carbon polymer / metal-phenolic network nanomotor as its core framework, which possesses catalase-like activity. This allows for self-propulsion and treatment improvement through the catalytic decomposition of hydrogen peroxide, enhancing the hypoxic microenvironment. Bovine serum albumin and cRGD peptides modified on the nanomotor surface are used for precise targeting of rheumatoid arthritis lesions. Metal nuclides […]. 68 Ga]Ga is used to give it image monitoring function.

[0009] In the embodiments of this scheme, the asymmetric carbon polymer / metal-phenolic network nanomotor includes, from the inside out, a carbon polymer core and a metal-phenolic network coated on the carbon polymer core.

[0010] In some embodiments, the carbon polymer core includes a PmPD carbon polymer core and a mesoporous PmPD shell covering the PmPD carbon polymer core, wherein the PmPD core is a solid, smooth sphere without pores, and mesopores are formed on the mesoporous PmPD shell.

[0011] In some embodiments, the diameter of the PmPD carbon polymer core of the asymmetric carbon polymer / metal-phenolic network nanomotor of this scheme is 270~280nm, the diameter of the mesoporous PmPD shell is 380~390nm, and the thickness of the metal-phenolic network layer is 13~15nm.

[0012] Preferably, the diameter of the PmPD carbon polymer core of the asymmetric carbon polymer / metal-phenolic network nanomotor is 275 nm, the diameter of the mesoporous PmPD shell is 385 nm, and the thickness of the metal-phenolic network layer is 14 nm.

[0013] In some embodiments, the average pore size of the mesoporous PmPD shell is 12~14 nm.

[0014] Preferably, the average pore size of the mesoporous PmPD shell is 13 nm.

[0015] In some embodiments, the PmPD carbon polymer core is a solid poly(m-phenylenediamine) carbon polymer core, that is, the PmPD carbon polymer core is a solid, non-porous, smooth, spherical mesoporous PmPD shell covering 1 / 2 to 2 / 3 of the surface of the PmPD carbon polymer core.

[0016] In some embodiments, the metal-phenolic network layer is a tannic acid-iron ion metal-phenolic network layer, which is a mesh film formed by coordination of tannic acid polyphenol ligands and ferric ions.

[0017] The metal-phenolic network layer in this scheme can act as a hydrogen peroxide-like catalyst to provide the driving force for nanomotors, and the polyphenolic hydroxyl groups can capture inflammatory free radicals to alleviate oxidative stress in RA lesions.

[0018] As mentioned earlier, the asymmetric carbon polymer / metal-phenolic network nanomotor of this scheme relies on the stable catalase-like activity of the surface tannic acid-iron metal-phenolic network to catalyze the decomposition of endogenous hydrogen peroxide in rheumatoid arthritis lesions to produce oxygen. Thanks to the asymmetric structure of the solid poly(m-phenylenediamine) core with a mesoporous shell that is partially covered on one side, there is a difference in the catalytic reaction rate on both sides of the particle. The concentration gradient forms a diffusion propulsion force to achieve chemotactic autonomous movement of the lesion. At the same time, the oxygen generated by catalysis can improve the hypoxic microenvironment of the joint. In addition, the phenolic hydroxyl groups and iron ion cycling in the metal-phenolic network can efficiently remove excess reactive oxygen species in the lesion and regulate the polarization of pro-inflammatory M1 macrophages. It can simultaneously relieve oxidative stress and interrupt the vicious cycle of inflammation without additional drug loading, thus achieving catalytic self-driven and in-situ anti-inflammatory therapeutic functions in one integrated manner. Secondly, this solution provides a method for assembling an intelligent hydrogen peroxide-driven nanorobot, comprising the following steps: Step 1: Preparation of asymmetric carbon polymer / metal-phenolic network nanomotors; Step 2: Modify and link bovine serum albumin and cRGD peptide on the surface of the asymmetric carbon polymer / metal-phenolic network nanomotor to obtain a nanomotor with precise targeting function. Step 3: Combine the nanomotor with precise targeting function with a metal nuclide [ 68 A solution of Ga was mixed and heated to directly label the metal nuclide under conditions without chelating agents. 68 Ga]Ga, resulting in a smart hydrogen peroxide-driven nanorobot that combines therapeutic, targeted, and imaging monitoring functions.

[0019] The structure of the asymmetric carbon polymer / metal-phenolic network nanomotor is as described in the first aspect and will not be repeated here.

[0020] Regarding the preparation method: In step one, Pluronic P123 was dissolved in a mixed solution of water and ethanol. After stirring vigorously at room temperature for a period of time, 1,3,5-trimethylbenzene was added, and the mixture was ultrasonically dispersed at room temperature for a period of time to obtain an oil-water emulsion system. Then, m-phenylenediamine was added to the oil-water emulsion system, followed by ammonium persulfate to initiate polymerization, resulting in a poly(m-phenylenediamine) powder with an asymmetric structure. This asymmetric poly(m-phenylenediamine) powder was ultrasonically dispersed in deionized water, and then tannic acid and Fe were added. 3+ Tris buffer was added to maintain the pH of the system between 7.5 and 8.5. After reacting for a period of time, the precipitate was collected by centrifugation to obtain the asymmetric carbon polymer / metal-phenolic network nanomotor.

[0021] The first step of this scheme involves preparing poly(m-phenylenediamine) powder with an asymmetric structure using P123 / TMB emulsion template oxidative polymerization. Then, tannic acid and Fe are reacted in a weakly alkaline aqueous system. 3+ Asymmetric carbon polymer / metal-phenolic network nanomotors can be prepared by coordinating a self-assembled metal-phenolic network functional layer on the particle surface of poly(m-phenylenediamine) powder with an asymmetric structure in a two-step mild aqueous phase reaction.

[0022] In some embodiments, the poly(m-phenylenediamine) powder having an asymmetric structure includes a carbon polymer core and a mesoporous carbon polymer shell partially coated on one side of the carbon polymer core.

[0023] In some embodiments, Pluronic P123 is a PEO-PPO-PEO triblock amphiphilic copolymer, which is dissolved in a mixed solution of water and ethanol as a soft template agent and self-assembles to form micelles. It is combined with 1,3,5-trimethylbenzene pore expander to construct an oil-water emulsion system, which induces the oxidative polymerization of m-phenylenediamine in the oil-water emulsion system to directionally grow polym-phenylenediamine (PmPD) powder with an asymmetric structure. Subsequently, Pluronic P123 is completely removed by washing to form a through-pore structure in the shell.

[0024] In some embodiments, the mass ratio of ammonium persulfate to m-phenylenediamine is 2.5:1.

[0025] Further, Pluronic P123 was dissolved in a mixed solution of water and ethanol, and the mixture was stirred vigorously at room temperature for 1.5-2 hours. Then, 1,3,5-trimethylbenzene was added, and the mixture was ultrasonically dispersed at room temperature for 2-5 minutes to obtain an oil-water emulsion system. Then, m-phenylenediamine and ammonium persulfate were added to the oil-water emulsion system, and the mixture was stirred at room temperature for 10-12 hours to initiate polymerization and obtain polym-phenylenediamine powder with an asymmetric structure.

[0026] Furthermore, after ultrasonically dispersing poly(m-phenylenediamine) powder with an asymmetric structure in deionized water, tannic acid and FeCl₂ were added sequentially. 3 The reaction was stirred separately, and Tris buffer was added to maintain the pH of the system between 7.5 and 8.5. After reacting at room temperature for 15 min, the precipitate was collected by centrifugation to obtain the asymmetric carbon polymer / metal-phenolic network nanomotor.

[0027] This method involves preparing an asymmetric carbon polymer / metal-phenolic network nanomotor with inherent catalase-like activity and autonomous propulsion performance in step one. This nanomotor responds to the endogenous hydrogen peroxide highly expressed in the lesion area of ​​rheumatoid arthritis (RA), catalyzing the decomposition of hydrogen peroxide into nanoscale autonomous propulsion power, and simultaneously regulating and improving the severely hypoxic microenvironment in the joint cavity during movement.

[0028] In step two, asymmetric carbon polymer / metal-phenolic network nanomotors were dispersed in PBS buffer, BAS powder was added and incubated at room temperature for a period of time, then the free BAS was removed, washed with PBS buffer to obtain BSA-modified particles; the BAS-modified particles were dispersed in PBS buffer, EDC and NHS were added, activated at room temperature, then cRGD peptides were added, reacted at room temperature, centrifuged, and unreacted cRGD peptides were removed with PBS buffer to obtain nanomotors with precise targeting function.

[0029] Furthermore, the mass ratio of bovine serum albumin to asymmetric carbon polymer / metal-phenolic network nanomotor is 1:2, and the mass ratio of the cRGD peptide to the asymmetric carbon polymer / metal-phenolic network nanomotor is 1:10.

[0030] In some embodiments, asymmetric carbon polymer / metal-phenolic network nanomotors are dispersed in PBS buffer, BAS powder is added and incubated at room temperature for 3-5 hours, then the free BAS is removed by centrifugation, and the mixture is washed 1-3 times with PBS buffer.

[0031] In some embodiments, the molar ratio of added EDC to NHS is 1:1, and activation is performed at room temperature for 0.5 to 1.5 hours.

[0032] In some embodiments, after adding the cRGD peptide, the mixture is placed at room temperature in the dark for 3-5 hours. After the reaction is completed, the mixture is centrifuged and washed 2-4 times with PBS.

[0033] This approach involves multi-level modification of the surface of an asymmetric carbon polymer / metal-phenolic network nanomotor with bovine serum albumin and cRGD peptide in step two. Since the microenvironment of rheumatoid arthritis lesions highly specifically expresses SPARC protein, the specific binding of bovine serum albumin to SPARC protein guides the nanorobots to more precisely accumulate in the arthritis lesions. This effectively avoids non-specific mistargeting caused by the high expression of hydrogen peroxide in other conventional inflammatory areas, greatly improving targeting accuracy. Furthermore, the cRGD peptide further targets pathogenic M1 macrophages and inflammatory fibroblasts in the lesions, significantly promoting the endocytosis efficiency of the robots by pathogenic cells.

[0034] In step three, HCl solution is used to [ 68 Ga]Ga is obtained by elution from the generator. 68 Ga]GaCl3 solution was neutralized with ammonia and immediately added to a nanomotor solution containing a nanomotor with precise targeting function. The mixture was incubated at 50°C with low-speed oscillation, purified by high-speed centrifugation, and then redispersed in PBS to obtain an intelligent hydrogen peroxide-driven nanorobot with therapeutic, targeting, and imaging monitoring functions.

[0035] In some embodiments, the mixture is incubated at 50°C with shaking at 400-600 rpm for 30-50 min, purified by centrifugation at 9,000-11,000 rpm for 3-8 min, and then redispersed in PBS.

[0036] This scheme involves step three, which involves the non-chelating direct labeling of metal nuclides on a metal-phenolic network layer. 68 Ga]Ga can provide highly sensitive real-time tracking signals.

[0037] In summary, the intelligent hydrogen peroxide-driven nanorobot prepared by this invention successfully integrates autonomous driving, hypoxia regulation therapy, highly precise targeting, and in vivo monitoring functions. Compared with traditional drug carriers that passively diffuse and lack active recognition capabilities, this invention has the advantages of high delivery efficiency, good lesion targeting and enrichment effect, and integrated diagnosis and treatment.

[0038] Thirdly, this solution provides the application of this intelligent hydrogen peroxide-driven nanorobot in the preparation of targeted therapeutic agents for rheumatoid arthritis with real-time image monitoring capabilities.

[0039] Specifically, the real-time imaging monitoring and treatment process is as follows: intelligent hydrogen peroxide-driven nanorobots are made into injectable agents and injected into an animal model of rheumatoid arthritis. Micro-PET / CT is used to perform real-time imaging at predetermined time points to monitor the distribution of intelligent hydrogen peroxide-driven nanorobots in mice and their targeted enrichment in the lesion area of ​​rheumatoid arthritis.

[0040] At this point, the intelligent hydrogen peroxide-driven nanorobot of this scheme utilizes the catalase-like activity inherent in the asymmetric carbon polymer / metal-phenolic network nanomotor of step one to catalyze the decomposition of intra-regional hydrogen peroxide in the lesion area to generate power for self-propulsion, and simultaneously improves the hypoxic microenvironment of the lesion to exert therapeutic function; through the modified bovine serum albumin of step two, it precisely targets the highly expressed SPARC protein in the rheumatoid arthritis lesion, and through the cRGD peptide, it targets the pathogenic M1 macrophages and inflammatory fibroblasts in the lesion to promote endocytosis and achieve precise targeting; through the labeled metal nuclide of step three... 68 Ga]Ga provides Micro-PET / CT tracer signals to enable real-time image monitoring.

[0041] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects:

[0042] The assembly method of the intelligent hydrogen peroxide-driven nanorobot according to the present invention achieves stable and controllable preparation process because the asymmetric core-shell morphology and pore structure of poly(m-phenylene diamine) nanoparticles are precisely controlled through an interfacial superassembly strategy; more importantly, the present invention utilizes the inherent metal coordination properties of the metal-phenolic network to achieve the […] of metal nuclides under mild oscillating heating conditions. 68 Ga]Ga's direct and efficient radiolabeling without chelators completely avoids the cumbersome chemical modification steps introduced by grafting complex bifunctional chelators (such as DOTA and NOTA) in traditional labeling methods, thus maximally preserving the biotargeting ligand activity of bovine serum albumin and cRGD peptides modified on the nanorobot surface. Furthermore, this assembly method requires no complex post-purification, is simple to perform, highly operable, and exhibits extremely high reproducibility and biocompatibility.

[0043] The application of the intelligent hydrogen peroxide-driven nanorobots of this invention in the preparation of targeted therapeutic agents for rheumatoid arthritis with real-time imaging monitoring is significant. Due to the extremely complex microenvironment of rheumatoid arthritis lesions, the agents prepared by this invention, when used, can, on the one hand, monitor the biodistribution of nanorobots in vivo and their active enrichment efficiency in the rheumatoid arthritis lesion area in real-time and with high precision using Micro-PET / CT in vivo tracking technology. This solves the technical bottleneck of traditional nano-preparations being unable to accurately and non-invasively assess their distribution in deep tissues in real-time. On the other hand, thanks to their highly precise active targeting capabilities, the nanorobots, after being enriched in the lesion and efficiently internalized by pathogenic cells, can continuously catalyze the decomposition of local hydrogen peroxide to generate oxygen, fundamentally alleviating the hypoxic state of the lesion and exerting therapeutic functions. This achieves precise targeted therapy for rheumatoid arthritis lesions under real-time imaging monitoring, opening up new avenues for the clinical translation of next-generation intelligent nanomedicine agents. Attached Figure Description

[0044] Figure 1 This is a transmission electron microscope image of the PmPD powder prepared in Example 1 of the present invention; Figure 2 This is a transmission electron microscope image of the asymmetric carbon polymer / metal-phenolic network nanomotor in Embodiment 1 of the present invention. Figure 3 This is a histogram of the motion velocity of the nanorobot in different concentrations of hydrogen peroxide in Embodiment 2 of the present invention; Figure 4 This is a histogram of the diffusion coefficient of the nanorobot in different concentrations of hydrogen peroxide in Embodiment 2 of the present invention; Figure 5 The figure shows the CCK-8 cytotoxicity test results of the nanorobots on L929 cells and Raw264.7 cells in Embodiment 3 of the present invention. Figure 6 This is a laser confocal microscope image of cells after LPS stimulation and FITC-labeled nanomotors were co-cultured for 24 hours in Example 4 of the present invention. Figure 7 This is a fluorescence microscopy comparison image of intracellular ROS level detection using the DHE probe in Embodiment 4 of the present invention; Figure 8 In Example 5 of this invention, AIA model mice were injected via the tail vein. 68 Micro-PET / CT images of Ga]Ga-labeled nanorobots; Figure 9 This is a histogram showing the radiobiological distribution (%ID / g) of the main tissues and organs at different time points after drug administration in Example 5 of the present invention. Detailed Implementation

[0045] To make the technical means, creative features, objectives and effects of this invention easy to understand, the following embodiments, in conjunction with the accompanying drawings, specifically illustrate the intelligent hydrogen peroxide-driven nanorobot, its assembly method and application.

[0046] Example 1: This embodiment provides a specific assembly method for an intelligent hydrogen peroxide-driven nanorobot, including the following steps: Step 1: Preparation of asymmetric carbon polymer / metal-phenolic network nanomotors with catalase-like activity and autonomous driving function: Weigh 22-24 mg of Pluronic P123 and dissolve it in a mixed solution of 3.5 mL deionized water and 1.5 mL ethanol at a ratio of 1 mg: 145-160 μL. Stir vigorously at room temperature for 1.5-2 h. Add 130 μL of 1,3,5-trimethylbenzene (TMB) and ultrasonically disperse at room temperature for 2-5 min to form a stable oil / water emulsion system. Add 40 mg of m-phenylenediamine (mPD) and stir at room temperature for 1 h. Then add 100 mg of ammonium persulfate (APS) and stir at room temperature for 11 h. Collect the precipitate by centrifugation, wash it, and vacuum dry it at 55±5 ℃ for 12 h to obtain poly-m-phenylenediamine powder (PmPD powder) with an asymmetric structure. Its core diameter is about 275 nm, the shell diameter is about 385 nm, and the average pore size of the mesopores on the shell surface is about 13 nm.

[0047] 10 mg of the obtained PmPD powder was ultrasonically dispersed in 10 mL of deionized water; 20 μL of 20 mM tannic acid (TA) solution was added and stirred for 5 min, followed by 40 μL of 20 mM FeCl3 solution and stirring for 15 min; finally, 10–12 mL of 10 mM Tris buffer (pH 8) was added to maintain the pH of the mixed solution between 7.5 and 8.5, and the mixture was stirred at room temperature for 15 min; the precipitate was collected by centrifugation, washed, and dried, and a metal-phenolic network layer with a thickness of approximately 14 nm was assembled in situ on the surface of the asymmetric carbon polymer, thus obtaining an asymmetric carbon polymer / metal-phenolic network nanomotor. The morphologies of the obtained PmPD powder and the asymmetric carbon polymer / metal-phenolic network nanomotor are shown in the figures below. Figure 1 and Figure 2 As shown.

[0048] Step 2: 10 mg of the prepared asymmetric carbon polymer / metal-phenolic network nanomotor was dispersed in 10 mL of PBS (pH 7.4), and 5 mg of BSA was added. The mixture was incubated at room temperature for 4 h. Unbound BSA was removed by centrifugation, and the particles were washed twice with PBS to obtain BSA-modified particles. Another 10 mg of the powder was dispersed in 10 mL of PBS buffer (0.1 M, pH 7.4) and sonicated until homogeneous. EDC and NHS (molar ratio 1:1) were added, and the mixture was activated at room temperature for 1 h. Then, 1 mg of cRGD(fK) peptide was added, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction, the mixture was centrifuged (12000 rpm, 10 min), washed three times with PBS to remove unreacted peptides, and then freeze-dried for 12 h.

[0049] Step 3: Directly heat-label the metal nuclide under chelating agent-free conditions. 68 Ga: Use 2 mL of 0.1 M HCl solution to mix the metal nuclide [ 68 Ga]Ga from 68 Ge / 68 Eluting in a Ga generator yields [ 68 Ga]GaCl3 solution; neutralized with ammonia water. 68 After processing the Ga]GaCl3 solution, it was immediately added to a nanomotor solution with a concentration of 2 mg / mL; the mixture was incubated at 50 °C with shaking at 500 rpm for 40 min on a thermostatic mixer; after the reaction was completed, the nanoparticles were purified by centrifugation (10,000 rpm, 5 min) to remove unbound free [ 68 Ga]Ga; Discard the supernatant and redisperse the labeled particles in PBS.

[0050] Figure 1 The image shown is a transmission electron microscope (TEM) image of the poly(m-phenylenediamine) core framework prepared in Example 1 of this invention. Figure 1 As shown, the prepared poly(m-phenylenediamine) powder with asymmetric structure exhibits a distinct asymmetric core-shell structure, in which the core is a solid sphere and is surrounded by a shell with mesopores distributed on the surface. Statistical results show that the core diameter is about 275 nm, the shell diameter is about 385 nm, and the average pore size of the mesopores on the shell surface is about 13 nm.

[0051] Figure 2 This is a transmission electron microscope (TEM) image of the asymmetric carbon polymer / metal-phenolic network nanomotor coated in Embodiment 1 of the present invention. Figure 2As shown, a uniform metal-phenolic network (MPN) layer was successfully assembled in situ on the surface of the poly(m-phenylenediamine) core framework. This MPN layer is continuous and dense, with a thickness of approximately 14 nm, and does not disrupt the original asymmetric core-shell mesoporous structure. This MPN layer not only endows the nanomotor with excellent metal coordination ability, providing sites for subsequent non-chelating radioactive labeling, but also enhances the catalase-like catalytic activity of the material.

[0052] Example 2 This embodiment describes the motion performance of the intelligent hydrogen peroxide-driven nanorobot prepared in Example 1.

[0053] To objectively evaluate the motion performance of the intelligent hydrogen peroxide-driven nanorobot prepared in Example 1, this example tested its autonomous motion behavior in a hydrogen peroxide environment. The real-time X and Y coordinate trajectories of each nanorobot particle in different concentrations of hydrogen peroxide fuel were observed and recorded using a fluorescence microscope, and trajectory analysis was performed using ImageJ software. The mean square displacement (MSD) curves of the particles were further plotted using the obtained trajectory data, and the particle velocity and diffusion coefficient of the nanorobot in various hydrogen peroxide environments were calculated using the obtained MSD values.

[0054] Figure 3 This is a histogram of the motion velocity of the nanorobot in different concentrations of hydrogen peroxide in Embodiment 2 of the present invention, as shown below. Figure 3 As shown, when no hydrogen peroxide is added to the system, the nanorobots exhibit only weak random Brownian motion with low speed. As the hydrogen peroxide concentration increases from 1% to 3% and then to 5%, the movement speed of the nanorobots shows a significant dose-dependent increase. These results demonstrate that the nanorobots constructed in this invention possess excellent hydrogen peroxide-responsive self-propulsion properties, enabling autonomous chemotactic movement within the microenvironment of rheumatoid arthritis lesions with high hydrogen peroxide concentrations.

[0055] Figure 4 This is a histogram of the diffusion coefficient of the nanorobot in Embodiment 2 of the present invention under different concentrations of hydrogen peroxide. Figure 4 As shown, consistent with the trend of motion speed, the diffusion coefficient of the nanorobots increases significantly with increasing hydrogen peroxide concentration. Under 5% hydrogen peroxide conditions, the diffusion coefficient is approximately 7 times higher than under fuel-free conditions. This result further quantitatively confirms that nanorobots can acquire enhanced diffusion capabilities through the catalytic decomposition of endogenous hydrogen peroxide, providing strong support for their autonomous penetration and active accumulation in lesions within complex pathological microenvironments.

[0056] Example 3 This embodiment is a cytotoxicity test of the intelligent hydrogen peroxide-driven nanorobot prepared in Example 1.

[0057] This embodiment used a CCK-8 assay to detect the effect of different concentrations of the material on the survival rate of L929 normal cells and Raw264.7 macrophages after 24 h of treatment, in order to evaluate its biosafety at the in vitro cellular level. The specific steps are as follows: First, L929 cells and Raw264.7 cells in good growth condition were digested, and the cell pellet was collected. Then, the cells were resuspended in complete culture medium to prepare a single-cell suspension, which was seeded into 96-well plates (100 μL per well, containing 5 × 10³ cells) and incubated overnight in a cell culture incubator to ensure complete adhesion. A cell-free control group and a drug-free cell control group were reserved in the experiment, with 3 parallel wells in each group. After adhesion, solutions of nanorobot materials at different concentration gradients were added to the corresponding wells of the 96-well plates containing L929 cells and Raw264.7 cells, respectively, and the cells were incubated for another 24 h. After 24 h of incubation with the drug, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for another 2 h. After incubation, the optical density (OD) value of each well was measured using a microplate reader at an excitation wavelength of 450 nm. The cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank control) / (OD value of control group - OD value of blank control) × 100%.

[0058] Figure 5 The image shows the CCK-8 cytotoxicity test results of the nanorobots on L929 cells and Raw264.7 cells in Embodiment 3 of this invention. Figure 5 As shown, Figure 5 (a) is a histogram of cell viability of L929 cells; Figure 5 (b) is a histogram of cell viability for Raw264.7 cells. From... Figure 5 It can be seen that after 24 h of treatment with different drug concentration gradients, the survival rates of L929 cells and Raw264.7 cells remained within the safe range (cell survival rate >80%), and no obvious cytotoxicity was observed, indicating that the nanorobot has good cellular biosafety.

[0059] Example 4 This embodiment is an evaluation experiment on the cellular uptake and intracellular ROS elimination of the intelligent hydrogen peroxide-driven nanorobot prepared in Example 1.

[0060] The specific process is as follows: First, FITC fluorescent dye was dissolved in DMSO. Then, 50 μL of dye solution (10 mg / mL) was added dropwise to 1 mL of nanomaterial solution (10 mg / mL), and the mixture was stirred and crosslinked overnight at 4 ℃ in the dark. Subsequently, the reaction solution was centrifuged at 4 ℃ and 12000 rpm for 10 min, and the centrifugation and washing were repeated three times to completely remove unbound free fluorescent molecules, yielding FITC-labeled nanomaterials. L929 cells and Raw264.7 cells (2 × 10⁻⁶ cells) were then used. 4 Cells were seeded in 20 mm confocal dishes and cultured for 24 h. 24 h before drug administration, 1 μg / mL LPS was added to the culture medium to simulate cellular inflammation. After stimulation, the initial culture medium was replaced with serum-free medium containing 100 μg / mL FITC-labeled nanomaterials and incubated for 24 h. After drug administration, cells were washed three times with PBS, and 500 μL of 4% paraformaldehyde fixative was added to each dish for 10 min at room temperature to fix the cells. The fixative was discarded, and cells were washed three times with PBS. 200 μL of Triton X-100 permeabilization buffer was added for 10 min at room temperature to permeate the cell membrane, followed by three washes with PBS. Actin-Tracker Red staining solution was diluted 1:200, and 200 μL of the staining working solution was added to each dish for 60 min at room temperature in the dark to label the cytoskeleton, followed by three washes with PBS. Finally, 200 μL of DAPI staining solution was added for 15 min at room temperature in the dark. The cell nuclei were labeled with 0 min; the cells were then washed three times with PBS, and cell uptake was observed using a laser confocal microscope. The results are as follows: Figure 6 As shown. Meanwhile, in the reactive oxygen species (ROS) elimination experiment, L929 cells (1×10⁻⁶) were used... 4 / well) and Raw264.7 cells (1×10 5 DHE probes were seeded in 96-well plates (n / well) and two control groups (untreated negative control and H2O2 treated positive control) were set up. The DHE probe was diluted to 10 μM with serum-free medium and the original medium was replaced. 100 μL of working solution was added to each well and incubated at 37 °C in the dark for 30 min. After washing three times with PBS, the color development was observed directly using a fluorescence microscope. The results are as follows: Figure 7 As shown.

[0061] Figure 6 This is a laser confocal microscopy image of cells after LPS stimulation and co-culturing with FITC-labeled nanomotors for 24 hours, as described in Example 4 of this invention. Figure 6 As shown, Figure 6 (a) is a cellular uptake image of L929 cells; Figure 6 (b) Cell uptake image of Raw264.7 cells. From Figure 6It can be seen that the nanorobots are diffusely distributed in the cytoplasm of both cell types. This indicates that H2O2-driven autonomous movement endows the nanorobots with active chemotaxis, while BSA / RGD dual-targeting modification enhances cell recognition and endocytosis.

[0062] Figure 7 This is a fluorescence microscopy comparison image of intracellular ROS levels detected using a DHE probe in Embodiment 4 of the present invention. Figure 7 As shown, Figure 7 (a) is an image of the DHE probe in L929 cells; Figure 7 (b) Image of the DHE probe in Raw264.7 cells. From Figure 7 It can be seen that the DHE fluorescence intensity of both cell types was significantly lower than that of the untreated negative control group. This is attributed to the Fe³⁺ in the MPN structure. + / Fe² + The study revealed a Fenton-like mediated reaction and the efficient free radical scavenging effect of phenolic hydroxyl groups. This indicates that nanorobots can be efficiently endocytosed into inflammatory cells and scavenge excess intracellular ROS, thereby alleviating oxidative stress levels.

[0063] Example 5 This embodiment is an evaluation experiment on the monitoring and targeted enrichment performance of the intelligent hydrogen peroxide-driven nanorobot prepared in Example 1 in a mouse model of rheumatoid arthritis.

[0064] The specific procedure was as follows: Healthy female BALB / c mice (8 weeks old) were selected for in vivo experiments. Aphrodine solution was used for intraperitoneal injection anesthesia (dose was 10 mL / kg). After the anesthesia took effect, 100 μL of complete Freund's adjuvant (CFA) was injected subcutaneously into the right hind paw of the mouse using a microsyringe to establish a mouse model of unilateral arthritis (AIA).

[0065] In a Micro-PET / CT in vivo imaging experiment, 150 μCi of [a specific drug / method] was injected into the tail vein of mice that had successfully developed the model. 68 Ga]Ga-nanorobots; after drug administration and injection, mice were placed in a warm environment to wait for the drug to complete targeted distribution and metabolism in the body for 40 minutes; then the mice were fixed on a scanning bed, and the mice under anesthesia were scanned using a small animal PET / CT system, and the imaging results were reconstructed.

[0066] In a radiobiological distribution experiment, mice were injected with 150 μCi of [a specific drug] via the tail vein. 68Ga-Ga nanorobots were administered. One hour after injection, mice were euthanized using an overdose anesthesia method. Blood, heart, liver, spleen, lungs, kidneys, stomach, muscles, and inflamed right hind paw tissue were rapidly dissected and separated. The wet weight (g) of each tissue block was measured using an electronic balance, and then the radioactivity count (cpm) of each tissue block was measured using a gamma counter. The percentage dose rate (%ID / g) injected per gram of tissue was calculated. The results further confirmed that the radioactivity enrichment of the nanorobot at the inflamed right hind paw lesion site was significantly higher than that of the normal control tissue, successfully achieving in vivo imaging monitoring of the active enrichment of the nanorobot into RA lesions.

[0067] Figure 8 In Example 5 of this invention, AIA model mice were injected via the tail vein. 68 Micro-PET / CT images of Ga-labeled nanorobots. (Example) Figure 8 As shown, the lesion area in the right hind paw exhibits a clear and highly specific concentration of radioactive signals. This indicates that H2O2-driven autonomous movement and BSA / RGD dual-targeting modification endow the nanorobot with precise lesion targeting capabilities under in vivo conditions, and this targeting result can be monitored in real time using Micro-PET / CT.

[0068] Figure 9 This is a histogram showing the radiobiodistribution (%ID / g) of major tissues and organs 1 hour after drug administration in Example 5 of the present invention. Figure 9 As shown, the radioactive enrichment of nanorobots at the inflamed right hind paw lesion site was significantly higher than that of normal control tissue, further verifying the targeting behavior of nanorobots actively enriching at the RA lesion in vivo.

[0069] The role and effect of the embodiments The intelligent hydrogen peroxide-driven nanorobot described in this embodiment possesses a unique asymmetric structure and excellent catalase-like catalytic activity in its core framework. This allows it to respond to the high expression of endogenous hydrogen peroxide in lesions and convert it into autonomous propulsion. Simultaneously, through multi-level surface modification with bovine serum albumin and cRGD peptide, the binding of bovine serum albumin to SPARC protein, which is specifically highly expressed in rheumatoid arthritis lesions, guides the motor to precisely target the arthritis lesion area. This effectively avoids non-specific mistargeting caused by the similarly high expression of hydrogen peroxide in other conventional inflammatory sites in the body. Furthermore, the cRGD peptide further enhances the recognition and endocytosis efficiency of pathogenic inflammatory cells on the robot. In addition, its metal-phenolic network layer is directly labeled with metal nuclides without chelation. 68 Ga]Ga can provide a strong radioactive monitoring signal. Therefore, the nanorobots of this invention not only exhibit excellent biosafety at the cellular level (cell viability greater than 80%), but also can utilize Fe in their structure.3+ / Fe 2+ The Fenton-like reaction mediated by the phenolic hydroxyl group and its efficient capture of free radicals demonstrate outstanding performance in scavenging intracellular reactive oxygen species (ROS) and alleviating oxidative stress, successfully achieving the synergistic integration of autonomous propulsion, highly specific active targeting, and in vivo imaging monitoring functions.

[0070] According to the assembly method of the intelligent hydrogen peroxide-driven nanorobot of the present invention, a morphology-controllable asymmetric mesoporous poly(m-phenylene diamine) core framework is first precisely constructed through emulsion template and oxidative polymerization reaction, and then a uniform metal-phenolic network coating layer is constructed in situ on the surface using the coordination effect of tannic acid and iron ions; more importantly, the present invention utilizes the inherent metal-capturing activity of the metal-phenolic network to achieve the capture of metal nuclides under mild oscillating heating conditions. 68 The direct radiolabeling of Ga]Ga without chelators completely avoids the cumbersome chemical modification steps introduced by grafting complex bifunctional chelating agents in traditional labeling methods. Therefore, this assembly method is not only simple to operate, highly reproducible, and has extremely high biocompatibility, but also maximizes the preservation of the biotargeting activity of bovine serum albumin and cRGD peptides modified on the nanorobot surface, significantly improving labeling efficiency and radiochemical purity.

[0071] The application of the intelligent hydrogen peroxide-driven nanorobot of the present invention in the preparation of a targeted therapeutic agent for rheumatoid arthritis with real-time imaging monitoring function is discussed. Because the microenvironment of rheumatoid arthritis lesions is extremely complex, the agent prepared by the present invention, when used, can utilize unchelated metal nuclides... 68 The high-sensitivity signal provided by Ga]Ga, through Micro-PET / CT in vivo tracking technology, enables real-time and high-precision monitoring of the distribution of nanorobots in vivo and their active enrichment efficiency in rheumatoid arthritis lesions. High-specificity radioactive signal concentration in the right hind paw lesion area was clearly observed, effectively solving the technical bottleneck of the inability to non-invasively and accurately assess the in vivo trajectory of traditional nano-formulations. On the other hand, thanks to its excellent hydrogen peroxide-responsive self-propulsion performance and dual-target active recognition capability, the nanorobots can efficiently accumulate at arthritis lesions and be efficiently internalized by pathogenic inflammatory cells, continuously catalyzing the decomposition of hydrogen peroxide within the cells. This achieves highly precise targeted control and treatment of rheumatoid arthritis lesions under real-time imaging monitoring, opening new avenues for the clinical diagnosis and treatment translation of next-generation intelligent nanomedicine formulations.

[0072] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A smart hydrogen peroxide-driven nanorobot, characterized in that, include: An asymmetric carbon polymer / metal-phenolic network nanomotor, comprising, from the inside out, a carbon polymer core and a metal-phenolic network coating the carbon polymer core. The carbon polymer core includes a PmPD carbon polymer core and a mesoporous PmPD shell covering the PmPD carbon polymer core. The PmPD core is a solid, smooth sphere without pores, and mesopores are formed on the mesoporous PmPD shell, which covers 1 / 2 to 2 / 3 of the surface area of ​​the PmPD carbon polymer core. The metal-phenolic network layer is a tannic acid-iron ion metal-phenolic network layer, which is a mesh film formed by the coordination of tannic acid polyphenol ligands and ferric ions. Bovine serum albumin and cRGD peptide modified on the surface of an asymmetric carbon polymer / metal-phenolic network nanomotor; And metal nuclides directly labeled on the metal-phenolic network layer of asymmetric carbon polymer / metal-phenolic network nanomotors without chelating agents [ 68 Ga]Ga.

2. The intelligent hydrogen peroxide-driven nanorobot according to claim 2, characterized in that, The diameter of the PmPD carbon polymer core is 270~280nm, the diameter of the mesoporous PmPD shell is 380~390nm, and the thickness of the metal-phenolic network layer is 13~15nm.

3. A method for assembling an intelligent hydrogen peroxide-driven nanorobot, characterized in that, Includes the following steps: Step 1: Prepare an asymmetric carbon polymer / metal-phenolic network nanomotor. The asymmetric carbon polymer / metal-phenolic network nanomotor, from the inside out, includes a carbon polymer core and a metal-phenolic network coated on the carbon polymer core. The carbon polymer core includes a PmPD carbon polymer core and a mesoporous PmPD shell covering the PmPD carbon polymer core. The PmPD core is a solid, smooth sphere without pores. Mesoporous pores are formed on the mesoporous PmPD shell, which covers 1 / 2 to 2 / 3 of the surface area of ​​the PmPD carbon polymer core. The metal-phenolic network layer is a tannic acid-iron ion metal-phenolic network layer, which is a mesh film formed by the coordination of tannic acid polyphenol ligands and trivalent iron ions. Step 2: Modify and link bovine serum albumin and cRGD peptide on the surface of the asymmetric carbon polymer / metal-phenolic network nanomotor to obtain a nanomotor with precise targeting function. Step 3: Combine the nanomotor with precise targeting function with a metal nuclide [ 68 A solution of Ga was mixed and heated to directly label the metal nuclide under conditions without chelating agents. 68 Ga]Ga, resulting in a smart hydrogen peroxide-driven nanorobot that combines therapeutic, targeted, and imaging monitoring functions.

4. The assembly method of the intelligent hydrogen peroxide-driven nanorobot according to claim 3, characterized in that, In step one, Pluronic P123 was dissolved in a mixed solution of water and ethanol. After stirring vigorously at room temperature for a period of time, 1,3,5-trimethylbenzene was added, and the mixture was ultrasonically dispersed at room temperature for a period of time to obtain an oil-water emulsion system. Then, m-phenylenediamine was added to the oil-water emulsion system, followed by ammonium persulfate to initiate polymerization, resulting in a poly(m-phenylenediamine) powder with an asymmetric structure. This asymmetric poly(m-phenylenediamine) powder was ultrasonically dispersed in deionized water, and then tannic acid and Fe were added. 3+ Tris buffer was added to maintain the pH of the system between 7.5 and 8.

5. After reacting for a period of time, the precipitate was collected by centrifugation to obtain asymmetric carbon polymer / metal-phenolic network nanomotors. In step two, asymmetric carbon polymer / metal-phenolic network nanomotors were dispersed in PBS buffer, BAS powder was added and incubated at room temperature for a period of time, then the free BAS was removed, and the particles were washed with PBS buffer to obtain BSA-modified particles; the BAS-modified particles were dispersed in PBS buffer, EDC and NHS were added, and after activation at room temperature, cRGD peptides were added, reacted at room temperature, centrifuged, and unreacted cRGD peptides were removed with PBS buffer to obtain nanomotors with precise targeting function; In step three, HCl solution is used to [ 68 Ga]Ga is obtained by elution from the generator. 68 Ga]GaCl3 solution was neutralized with ammonia and immediately added to a nanomotor solution containing a nanomotor with precise targeting function. The mixture was incubated at 50°C with low-speed oscillation, purified by high-speed centrifugation, and then redispersed in PBS to obtain an intelligent hydrogen peroxide-driven nanorobot with therapeutic, targeting, and imaging monitoring functions.

5. The assembly method of the intelligent hydrogen peroxide-driven nanorobot according to claim 3, characterized in that, The mass ratio of bovine serum albumin to asymmetric carbon polymer / metal-phenolic network nanomotor was 1:2, and the mass ratio of cRGD peptide to asymmetric carbon polymer / metal-phenolic network nanomotor was 1:

10.

6. The assembly method of the intelligent hydrogen peroxide-driven nanorobot according to claim 4, characterized in that, The mass ratio of ammonium persulfate to m-phenylenediamine is 2.5:

1.

7. The assembly method of the intelligent hydrogen peroxide-driven nanorobot according to claim 4, characterized in that, In step three, the mixture is incubated at 50°C with shaking at 400-600 rpm for 30-50 min, purified by centrifugation at 9,000-11,000 rpm for 3-8 min, and then redispersed in PBS.

8. The application of a smart hydrogen peroxide-driven nanorobot as described in any one of claims 1 to 2 in the preparation of a targeted formulation for rheumatoid arthritis with real-time image monitoring function.

9. The application of the intelligent hydrogen peroxide-driven nanorobot according to claim 8 in the preparation of a targeted formulation for rheumatoid arthritis with real-time image monitoring function, characterized in that, Intelligent hydrogen peroxide-driven nanorobots were made into injectable formulations and injected into an animal model of rheumatoid arthritis. Micro-PET / CT was used to perform real-time imaging at predetermined time points to monitor the distribution of intelligent hydrogen peroxide-driven nanorobots in mice and their targeted enrichment in the lesion areas of rheumatoid arthritis.

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