Nano-robot capable of efficient and targeted enrichment in inflammatory lesion tissues and use thereof

Abstract

The present disclosure relates to the field of micro-nano device technology, and in particular, to a nano-robot capable of efficient and targeted enrichment in inflammatory lesion tissues and use thereof. The nano-robot comprises a nanoparticle and at least two enzymes connected to the nanoparticle. At least one enzyme is used for driving the nano-robot in a physiological medium, and at least one other enzyme is used for sensing an inflammation lesion and controlling the motion direction of the nano-robot.

Description

A nanorobot capable of efficiently targeting and enriching inflammatory disease tissues and its application Technical Field

[0001] The present application relates to the technical field of micro-nano devices, and in particular to a nanorobot capable of efficiently targeting and enriching inflammatory disease tissues and its application. Background Art

[0002] The in vivo delivery of tumor-targeted nanomedicines involves five steps: blood circulation, tumor tissue accumulation, penetration, cellular internalization, and intracellular drug release (CAPIR). These five steps together determine the efficacy of nanomedicines on tumor tissues. Existing nanomedicine delivery vehicles are unable to move autonomously, resulting in only 0.7% (median) of nanomedicines reaching the tumor area, 0.0014% entering tumor cells, and 2% of tumor cells coming into contact with nanomedicines. Therefore, compared with traditional treatment methods, the therapeutic effect of tumor-targeted nanomedicines has not been significantly improved.

[0003] Chemotactic nanorobots (also known as nanomotors) are artificial micro- and nanoscale devices that can convert other forms of energy into kinetic energy and sense chemical concentration gradients in the environment to achieve directional movement. Due to these properties, they are expected to revolutionize the field of active targeted drug delivery as drug delivery vehicles.

[0004] However, currently reported chemotactic nanorobots cannot be effectively driven within the blood circulation system, and their chemotactic sensitivity is weak, making them unable to effectively sense the low concentrations of chemical signals present in actual lesions. As a result, their targeting efficiency at the disease site is not significantly improved compared to inert carriers. These issues have greatly limited the application of nanorobots in the biomedical field. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present application provides a nanorobot that can efficiently target and enrich in inflammatory disease tissues and its application, aiming to solve the technical problems of effectively driving nanorobots as drug carriers in vivo and precisely targeting delivery.

[0006] In the first aspect, the embodiments of the present application provide a nanorobot that can efficiently target and enrich in inflammatory disease tissues, which includes nanoparticles and two or more enzymes connected to the nanoparticles, wherein at least one enzyme is used to drive the nanorobot in a physiological medium, and at least another enzyme is used to sense the site of inflammatory lesions and control the movement direction of the nanorobot.

[0007] In the technical solutions of the present application, the enzyme used to drive the nanorobot in a physiological medium (hereinafter referred to as the driving enzyme) is an enzyme that uses endogenous substances in the physiological medium as substrates, where endogenous substances include but are not limited to urea, glucose, uric acid, amino acids, lactose, etc. For example, in some embodiments of the present application, urea is used as the substrate, and the driving enzyme is urease (URE), which provides driving force for the nanorobot through a catalytic reaction.

[0008] In the technical solution of the present application, the enzyme used to sense the inflammatory lesion site and control the movement direction of the nanorobot (hereinafter referred to as chemotactic enzyme) is an enzyme that uses the biomarkers released from the inflammatory lesion site as substrates, wherein the biomarkers include but are not limited to hydrogen peroxide, glutathione, lactate, glucose, nucleic acid, adenosine triphosphate (ATP), H + Different types of inflammatory diseases release different biomarkers. For example, in some embodiments of the present application, where the inflammatory disease is a tumor, the chemoattractant may be catalase (CAT), which uses hydrogen peroxide released by tumor cells as a substrate. Based on the chemoattractant's specific sensing of the lesion site, the nanorobot's steering function is achieved.

[0009] In the technical solution of the present application, nanoparticles include but are not limited to simple substances, oxides or sulfide nanoparticles of metal elements such as gold, silver, palladium, platinum, manganese, iron, cerium, titanium, aluminum, zinc, etc., non-metallic inorganic nanoparticles such as silicon dioxide, organic nanoparticles such as dendrimers, liposomes, polydopamine. The nanoparticle size is preferably 10-1000nm, wherein 20-200nm is more excellent. As in some embodiments of the present application, the nanoparticle is nanogold. In addition, the nanoparticle can be hollow, solid or porous structure, and the shape of the nanoparticle can be spherical, bowl-shaped, rod-shaped or polygonal structure. As in some embodiments of the present application, the nanoparticle is a solid sphere.

[0010] In the technical solution of the present application, the function of the nanorobot can be enriched by connecting other functional substances to the nanoparticles. The other functional substances can specifically be therapeutic drugs for inflammatory disease tissues, and the therapeutic drugs include but are not limited to: chemotherapy drugs such as doxorubicin (DOX), cyclophosphamide, fluorouracil, gemcitabine, bleomycin, paclitaxel, dihydrochlorin E6 (Ce6), porphyrin mixtures (such as Mthpc), porphyrin derivatives (such as BPMppa), phthalocyanines, methylene blue, texaporphyrin and other photosensitizers, CTLA-4 inhibitors, bispecific antibodies, monoclonal antibodies, antibody-drug conjugates and other immunotherapy drugs, sodium iodide, yttrium, 177 lutetium oxide octreotide, radium chloride [223Ra], 131I-tositumomab and other radiotherapy drugs, glutathione peroxidase, superoxide dismutase, auranofin, curcumin, ligustrazine-curcumin hybrids and other antioxidants. The principle for using these therapeutic drugs is to ensure that they do not interfere with the synergistic action of the driving enzyme and chemotactic enzyme on the nanoparticles. For example, in some embodiments of the present application, antioxidants are also attached to the nanoparticles to scavenge reactive oxygen species at inflammatory sites. In other embodiments of the present application, photosensitizers are also attached to the nanoparticles, thereby creating a nanorobot capable of performing photodynamic therapy.

[0011] In the second aspect, the present application embodiment provides a drug delivery system, which includes a nanorobot provided by the present application. The nanoparticles in the nanorobot can load drugs. It is understandable that the drug can be connected to the surface of the nanoparticles by chemical bonds, or it can be present in the pores of porous nanoparticles, or it can be wrapped inside the nanoparticles, and the distribution mode is not limited. For example, it can be concentrated in a part of the nanoparticle area or dispersed over the entire surface of the nanoparticles. The specific situation can be determined by comprehensively considering factors such as the type of nanoparticles and the mechanism of action of the drug. The driving enzyme provides the main driving ability for the nanorobot, and the chemotactic enzyme provides the steering function of chemotaxis, so that the nanorobot can effectively target the lesion site in the physiological medium, thereby achieving the precise delivery of the drug.

[0012] Thirdly, embodiments of the present application provide a drug for the targeted treatment of inflammatory diseases, which uses the aforementioned nanorobot as a carrier and may also include pharmaceutically acceptable excipients. Experimental results show that after intravenous injection into mice, the nanorobots provided by this application can resist the random and irregular Brownian motion of nanoparticles and be effectively driven in physiological media (such as the blood circulation system or tumor microenvironment), significantly improving the efficiency of targeting the lesion site.

[0013] Fourthly, embodiments of the present application provide a medical device comprising the nanorobot provided herein. The nanorobot, through the synergistic action of multiple enzymes, is capable of autonomous movement within a living organism and efficiently targeting inflammatory lesions. Therefore, the nanorobot can be used to construct a miniature medical device and perform interventional therapy on a living organism.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) Compared with existing chemotactic nanorobots, this application innovatively utilizes the synergistic effect of multiple enzymes to significantly improve the chemotactic sensitivity of the nanorobot and the targeting efficiency to inflammatory disease tissues, so that the nanorobot is effectively enriched in inflammatory disease tissues in physiological media; for example, compared with traditional single-enzyme chemotactic nanorobots (such as catalase chemotactic nanorobots), its chemotactic ability is improved by more than 10 times, and its targeting efficiency is improved by more than 20 times; compared with inert carriers, its targeting efficiency is improved by more than 200 times.

[0016] (2) The nanorobot of the present application has a wide range of applications and ultra-high plasticity; based on the nanorobot structure provided by the present application, nanoparticles, functional substances, driving enzymes and / or chemotactic enzymes can be replaced as needed without affecting their functions, thereby achieving efficient targeting and multifunctional treatment approaches for different diseases and different lesion sites.

[0017] (3) The preparation method of the nanorobot of the present application is simple and efficient, which is conducive to large-scale production.

[0018] (4) The nanorobots of the present application have good biosafety. At the same time, given the excellent targeting efficiency of the nanorobots, they can significantly reduce the amount of drugs used, thereby significantly reducing toxic side effects and significantly improving the therapeutic effect of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] To more clearly illustrate the technical solution of this application, the following is a brief introduction to the drawings used in this application. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0020] FIG1 is a schematic diagram of the structure and driving mechanism of the nanorobot provided in this application;

[0021] FIG2 is a schematic diagram of the structure and driving mechanism of the nanorobot in Example 1 of the present application;

[0022] FIG3 is a transmission electron micrograph and elemental analysis diagram of the nanorobot prepared in Example 2 of the present application;

[0023] FIG4 is a diagram showing the motion trajectory of the nanorobot in urea solutions of different concentrations, a diagram showing the change in mean square displacement and diffusion coefficient with urea concentration, and a diagram showing the change in diffusion coefficient with different ratios of urease to catalase in Example 2 of the present application;

[0024] FIG5 is a graph showing the chemotactic ability test results of the nanorobot in Example 4 of the present application;

[0025] FIG6 is a graph showing the detection results of the targeting efficiency of the nanorobot in vivo in Example 4 of the present application;

[0026] FIG7 is a graph showing the chemotaxis ability test results of the nanorobot in Example 6 of the present application;

[0027] FIG8 is a schematic diagram of the structure and driving mechanism of the nanorobot in Example 7 of the present application;

[0028] FIG9 is a transmission electron micrograph and elemental analysis diagram of the nanorobot prepared in Example 7 of the present application;

[0029] FIG10 is a graph showing the chemotactic ability test results of the nanorobot in Example 7 of the present application;

[0030] FIG11 is a schematic diagram of the structure and mechanism of action of the nanorobot in Example 9 of the present application;

[0031] Figure 12 is a transmission electron microscope image (A) and an elemental analysis image (B) of the nanorobot in Example 10 of the present application.

[0032] FIG13 is a graph showing the results of testing the motility of different nanorobots in a urea solution of physiological concentration in Example 11 of the present application;

[0033] FIG14 is a graph showing the detection results of the targeting efficiency of the nanorobot to colon tissue in mice in Example 12 of the present application;

[0034] FIG15 is a diagram showing the therapeutic effect of the nanorobot on colitis in mice in Example 12 of the present application;

[0035] FIG16 is a diagram showing the safety evaluation results of the nanorobot in vivo in Example 12 of the present application;

[0036] FIG17 is a schematic diagram of the structure and driving mechanism of the nanorobot in Example 13 of the present application;

[0037] FIG18 is a diagram showing the morphology and structure of the nanorobot prepared in Example 14 of the present application;

[0038] FIG19 is a comparison of the motility of different nanorobots in Example 15 of the present application in a urea solution with physiological concentrations;

[0039] FIG20 is a graph showing the detection results of the nanorobot's targeting efficiency against breast cancer in Example 16 of the present application;

[0040] FIG21 is a graph showing the results of detecting the tumor-suppressing effect of the nanorobot on tumor-bearing mice in Example 16 of the present application;

[0041] FIG22 is a graph showing the biosafety test results of the nanorobot in Example 16 of the present application when treating tumor-bearing mice;

[0042] FIG23 is a graph showing the chemotactic ability test results of the nanorobot in Example 17 of the present application;

[0043] Figure 24 is a graph showing the chemotaxis ability test results of the nanorobot in Example 18 of the present application. DETAILED DESCRIPTION

[0044] The following embodiments of the technical solution of the present application will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application and are therefore only examples and are not intended to limit the scope of protection of the present application.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned figure descriptions are intended to cover non-exclusive inclusions.

[0046] In the specification and claims of this application, the terms "one" and "another" are only used to distinguish different objects and cannot be understood as indicating or implying relative importance or implicitly indicating a specific order or primary and secondary relationship of the indicated technical features; the term "multiple" refers to more than two (including two); the term "room temperature" means that the temperature can be within the range of 4-40°C; the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: the existence of A alone, the existence of A and B at the same time, and the existence of B alone.

[0047] In order to solve the technical problems that existing nanorobots cannot be effectively driven in physiological media and have weak chemotactic sensitivity, and at the same time have low targeting efficiency to disease sites, the present application provides a nanorobot that can efficiently target and enrich in inflammatory disease tissues. As shown in Figure 1, the nanorobot has a multi-enzyme structure, specifically including nanoparticles and multiple enzymes connected to the nanoparticles, wherein at least one enzyme is a driving enzyme and is used to drive the nanorobot in a physiological medium, and at least another enzyme is a chemotactic enzyme and is used to sense tumor cells in the lesion site and control the movement direction of the nanorobot. Through the synergistic action of multiple enzymes, the chemotactic sensitivity and targeting efficiency of the nanorobot in the physiological medium are significantly improved.

[0048] In the technical solutions of the present application, inflammatory diseases include but are not limited to tumors, enteritis, pneumonia, arthritis, nephritis, etc. For example, in some embodiments of the present application, the inflammatory disease is a tumor; while in other embodiments of the present application, the inflammatory disease is specifically enteritis.

[0049] In the technical solutions of the present application, physiological media include, but are not limited to, blood, tumor tissue microenvironment, tissue fluid, etc. For example, in some embodiments of the present application, the physiological medium is blood. For example, in other embodiments of the present application, the physiological medium is the tumor tissue microenvironment.

[0050] In the technical solution of the present application, the enzyme in the nanorobot can be a biological enzyme and / or a nanozyme, as long as it can drive the nanorobot or control the movement direction of the nanorobot through a catalytic reaction system in a physiological medium.

[0051] In the technical solutions of the present application, nanoparticles include, but are not limited to, gold nanoparticles, nanosilica, liposomes, magnetic nanoparticles, polydopamine nanoparticles, and the like. In specific embodiments, nanoparticles of different types and sizes can be selected according to actual needs, wherein the size of the nanoparticles is preferably 10-1000 nm, and more preferably 20-200 nm. For example, in some embodiments of the present application, the nanoparticles are gold nanoparticles of 90-100 nm.

[0052] In the technical solution of the present application, the driving enzyme uses endogenous substances in the physiological medium as substrates, which provide driving force for the nanorobot through catalytic reactions. Endogenous substances in the physiological medium include but are not limited to urea, glucose, etc. For example, in some embodiments of the present application, the driving enzyme is urease (URE), which uses urea in the blood as a substrate to catalyze reactions and drive the nanorobot to move in the blood; preferably, the diffusion coefficient of the nanorobot at the human body urea concentration (~10mM) is not less than 1.0μm 2When the concentration of GOx is less than 100 μg / s, it can effectively ensure its effective targeting to the inflammatory site in the human blood circulation system. For another example, when glucose is used as the endogenous substance, the driving enzyme can be glucose oxidase (GOx).

[0053] In the technical solution of this application, chemotactic enzymes use biomarkers released from inflammatory lesions as substrates. Based on the specificity of chemotactic enzymes for biomarkers, the nanorobot's steering function is realized, thereby accurately targeting the inflammatory lesions. Different inflammatory diseases release different biomarkers. For example, in some embodiments of this application, catalase is selected as the chemotactic enzyme for hydrogen peroxide released by tumor cells.

[0054] In the technical solution of the present application, the connection mode of the driving enzyme and the chemotactic enzyme on the nanoparticles can be any of the following:

[0055] 1) The kinase and chemoattractant are layered and attached to the same side of the nanoparticle;

[0056] 2) the kinase and chemoattractant are attached to the same side of the nanoparticle in the same layer;

[0057] 3) The driving enzyme and the chemotactic enzyme are connected in layers or in the same layer on the entire surface of the nanoparticle.

[0058] In scenarios 1) and 2), the assembly positions of the kinase and chemoattractant are consistent relative to the nanoparticle, and they are only attached to a portion of the nanoparticle, forming an asymmetric structure. In scenario 3), the two enzymes are directly attached to the entire surface of the nanoparticle, forming a relatively symmetrical structure relative to scenarios 1) and 2). (It should be noted that since the chemical reactions of the two enzymes are not completely consistent, the symmetry described in this application is not completely symmetrical, but rather a relatively symmetrical state compared to the asymmetric structures of scenarios 1) and 2).

[0059] The experimental data of this application show that when the driving enzyme and chemoattractant are layered or connected to the same side of the nanoparticle or the entire surface, the chemotactic ability of the nanorobot can be significantly improved. The only difference is the different ratio requirements of the driving enzyme and chemoattractant. For example, when the driving enzyme and chemoattractant are connected on the same side in layers, and the driving enzyme and chemoattractant are urease and catalase respectively, the driving ability is strongest when the ratio of urease to catalase is 1:1. However, when connected in the same layer, the amount of urease needs to be increased to achieve similar technical effects. Preferably, nanorobots with asymmetric structures are more effective.

[0060] In the technical solution of the present application, the kinase and the chemotactic enzyme can be linked to the nanoparticles through the action of streptavidin and biotin. For example, in some embodiments of the present invention, the nanoparticles are gold nanoparticles, and the method for linking the kinase and the chemotactic enzyme to the gold nanoparticles is as follows: preparing asymmetric Au-PS eccentric particles, mixing the Au-PS eccentric particles with HO-PEG-SH (hydroxy-polyethylene glycol-thiol), then adding N,N-dimethylformamide (DMF) and SH-PEG-Biotin (thiol-polyethylene glycol-biotin), then adding streptavidin, and finally adding biotinylase to react; wherein the molar ratio of the Au-PS eccentric particles, HO-PEG-SH, SH-PEG-Biotin and streptavidin is 1:1-50000:1-50000:1-50000, and preferably 1:2-10000:2-10000:2-1000.

[0061] In the technical solution of the present application, the nanorobot also includes therapeutic drugs to achieve targeted treatment of inflammatory disease tissues; the therapeutic drugs may be chemotherapy drugs, radiotherapy drugs, immune drugs, antioxidant drugs, photosensitizers, etc.

[0062] For example, in some embodiments of the present invention, the therapeutic drug is an antioxidant (i.e., an anti-inflammatory molecule), and the anti-inflammatory molecule is used alone or in conjunction with a driving enzyme / chemoattractant enzyme to clear ROS at the inflammatory site. A large body of evidence indicates that excessive reactive oxygen species (ROS, including superoxide anion radicals (·O2–) and hydrogen peroxide (H2O2)) are closely related to the occurrence and development of inflammation. Therefore, further introducing functional substances capable of clearing ROS into the structure of nanorobots is an effective strategy for treating inflammatory diseases.

[0063] The anti-inflammatory molecules in this application are selected from biological enzymes, nanozymes (such as ceria), anti-inflammatory small molecule drugs, and other substances that can be used to remove reactive oxygen species at inflammatory sites. For example, in some embodiments of this application, the anti-inflammatory molecule is superoxide dismutase (SOD), which can catalyze ROS to produce hydrogen peroxide, thereby treating inflammatory diseases by removing ROS at inflammatory sites; at the same time, if the chemotactic enzyme is catalase, the cascade reaction of superoxide dismutase and catalase helps further enhance the ROS removal effect.

[0064] As in other embodiments of the present application, the therapeutic drug is a photosensitizer, which is connected to the nanoparticles and induces cancer cell death under light conditions. As a small molecule, the photosensitizer has no effect on the enzyme drive and enzyme chemotaxis of the nanorobot of the present application, and after the photosensitizer is simultaneously connected to the nanoparticles, the resulting nanorobot can achieve photodynamic therapy at inflammatory sites (such as tumors). In the specific implementation process, the type and quantity of the photosensitizer can be replaced according to actual needs. For example, in some embodiments of the present application, the photosensitizer is Ce6.

[0065] It is understood that the therapeutic drug attached to the nanoparticle can be a single drug or multiple drugs, and the manner in which the therapeutic drug is attached and distributed on the nanoparticle is not limited. For example, the therapeutic drug can be concentrated in a portion of the nanoparticle or dispersed over the entire surface of the nanoparticle. For example, when the therapeutic drug is an enzyme (such as superoxide dismutase), it can be layered or assembled in the same layer with the kinase or chemotactase on the same side or different sides of the nanoparticle, or it can be layered or assembled in the same layer with the kinase or chemotactase on the entire surface of the nanoparticle.

[0066] Given that the nanorobots provided in this application have excellent chemotactic sensitivity and targeting efficiency, they can be used to prepare drugs, drug delivery systems, medical devices and other products for the targeted treatment of different inflammatory diseases, and are widely used in the field of biomedicine technology.

[0067] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used without manufacturer's indication are all commercially available conventional products.

[0068] Example 1

[0069] This example provides a nanorobot (denoted as CAT / URE-driven nanorobot), whose structure and driving mechanism are shown in Figure 2. This nanorobot uses gold nanoparticles, urease as the driving enzyme, and catalase as the chemoattractant enzyme. The urease and catalase are layered and assembled on one side of the gold nanoparticle using polyethylene glycol, biotin, and streptavidin, forming an asymmetric structure.

[0070] Example 2

[0071] For the nanorobot in Example 1, this example provides a preparation method comprising the following steps:

[0072] (1) Preparation of gold nanoparticles.

[0073] Synthesis of 10 nm gold nanoparticles: Take 1 mM chloroauric acid solution, stir and heat in an oil bath until boiling, then heat for another 10 minutes after boiling. Quickly add 38.8 mM sodium citrate aqueous solution, continue heating for 10 minutes, and then stop heating.

[0074] Initial growth of gold nanoparticles: Add a certain amount of 10 nm gold nanoparticle seed solution, a certain amount of 60 mM sodium citrate solution, and 142.5 mL of deionized water into a three-necked flask, heat and stir at 90 °C, and after the system stabilizes, add a certain amount of 25 mM chloroauric acid solution twice. After 30 minutes, the initial growth of gold nanoparticles is completed.

[0075] Subsequent growth of gold nanoparticles: Take out about 1 / 3 of the sample solution and add an equal amount of deionized water to the original three-necked flask. After the temperature stabilizes, add a certain amount of 60mM sodium citrate solution. After stabilization, add a certain amount of 25mM chloroauric acid solution twice and react for 30 minutes.

[0076] By controlling the number of growth times, gold nanoparticles of different sizes can be obtained. In this embodiment, gold nanoparticles with a particle size of 90 nm are specifically selected.

[0077] (2) Preparation of asymmetric Au-PS eccentric particles.

[0078] 1 mL of 50 mg / mL potassium persulfate solution, 1 mL of 6 mg / mL 4-styrenesulfonic acid sodium salt solution, 2.5 mL of water, and 16.5 mL of ethanol were added to a three-necked flask and preheated at 70°C for 10 minutes. Then, 200 μL of a mixed solution (styrene and divinylbenzene = 200:1, v / v) was added. After 2 minutes, 3 mL of nanogold was added. The reaction was continued for 4 hours and collected by centrifugation to obtain asymmetric Au-PS eccentric particles.

[0079] (3) Preparation of biotinylated urease and biotinylated catalase.

[0080] Mix the catalase solution with the biotinylation reagent, shake it evenly at room temperature, wash it five times with deionized water, dissolve it in PB solution, and store it at 4°C for later use. Mix the urease solution with the biotinylation reagent, shake it evenly at room temperature, wash it five times with deionized water, dissolve it in PB solution, and store it at 4°C for later use.

[0081] (4) Preparation of nanorobots.

[0082] The Au-PS eccentric particles obtained in step (2) were dispersed in 6 mL of ultrapure water. 2 mL of Au-PS solution was added to 0.1 mL of HO-PEG-SH (10 mg / mL) solution and mixed and shaken. 1 mL of N,N-dimethylformamide was added to the sample, dispersed in 500 μL of ultrapure water, and 100 μL of SH-PEG-Biotin (10 mg / mL) solution was added. After shaking, the mixture was dispersed in PB solution (10 mM, pH = 7.4). 100 μL of the above solution was added to 100 μL of streptavidin solution (0.02 mg / mL), mixed, and dispersed in PB solution (10 mM, pH = 7.4). 100 μL of the above solution was added to 100 μL of biotinylated catalase solution and shaken for reaction. Subsequently, SA and an equal amount of biotinylated urease were coupled. After the reaction was complete, the nanorobot was washed to obtain the nanorobot. The ratio of biotinylated catalase to biotinylated urease was 1:1.

[0083] Figure 3 shows transmission electron microscopy (TEM) images and elemental analysis of the nanorobot prepared in this example. Figure 3A shows a brightfield image of the chemotactic nanorobot under TEM mode, while Figure 3B shows the distribution of gold, nitrogen, and iron under darkfield and elemental analysis. The images show that catalase and urease are evenly distributed on one side of the gold nanorobot, forming an asymmetric structure.

[0084] The nanorobot prepared in this example was placed in 0, 1, 5, 10, 15, 20, 25, and 50 mM urea solutions to test its mobility in urea solutions of different concentrations. Its motion trajectory is shown in Figure 4A, and its mean square displacement (MSD = |r(t)-r(0)| 2 , where r is the position at a certain moment) is shown in Figure 4B, and the change of its diffusion coefficient (diffusion coefficient = MSD / 4t) is shown in Figure 4C. It can be seen from the figure that: with the increase of urea concentration, the moving distance of the nanorobot is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and it can be effectively driven at the human body urea concentration (~10mM).

[0085] Example 3

[0086] Referring to Example 2, this example obtained nanorobots with different ratios of urease to catalase (urease: catalase = 1:1-20) by changing the dosage of biotinylated catalase and biotinylated urease, and detected the effect of the relative dosage of urease and catalase on the mobility of the nanorobots.

[0087] In the CAT / URE-driven nanorobots, urease provides the primary driving force, while hydrogen peroxide only provides the chemotactic steering function. The results of the nanorobot's motility performance when the urease:catalase ratios were 1:1, 1:4, and 1:8, respectively, are shown in Figure 4D. A 1:1 urease:catalase ratio showed the strongest driving force, while gradually reducing the urease ratio resulted in a significant decrease in driving force.

[0088] Example 4

[0089] Taking tumors, an inflammatory disease, as an example, this example demonstrates the targeting efficiency of the nanorobots described in Example 1 on tumor tissues in both in vitro and in vivo environments, as follows:

[0090] (1) Preparation of control product.

[0091] In this example, an inert carrier, urease-driven nanorobots, and catalase-driven nanorobots are used as controls, and the preparation methods of the inert carrier, urease-driven nanorobots, and catalase-driven nanorobots refer to Example 2, with the only difference being that: the urease and catalase in the inert carrier are both inactivated by heat, the catalase in the urease-driven nanorobot is inactivated by heat, and the urease in the catalase-driven nanorobot is inactivated by heat.

[0092] (2) In vitro testing.

[0093] The nanorobot was placed in a 3D chip, and the absorbance changes in areas 3, 4, and 5 were observed to determine the chemotactic ability of the nanorobot. The design of the 3D chip is shown in Figure 5A. The left reservoir area is filled with a gel matrix containing MCF-7 cells (tumor cells), while the right reservoir is filled with a gel matrix containing MCF-10A cells (normal cells). The channels are filled with mouse plasma.

[0094] Figure 5B shows a comparison of the chemotactic abilities of different nanorobots. The test results show that the nanorobot provided in the present application (prepared in Example 2) is selective and can spontaneously move toward the cancer cell area rather than toward normal cells. The chemotactic ability of this nanorobot is significantly enhanced, which is more than 10 times stronger than that of traditional single-enzyme chemotactic nanorobots.

[0095] (3) In vivo testing.

[0096] The nanorobots were injected intravenously into tumor-bearing mice, and the mice were killed 24 hours later. The gold content in the tumor tissue was detected by ICP-MS, and the targeting efficiency of the nanorobots on the tumor was calculated.

[0097] The targeting efficiency of different nanorobots is shown in Figure 6. The targeting efficiency of the nanorobot provided in the present application (prepared in Example 2) is as high as 69.09% ID / g (targeting efficiency (% ID / g) = (gold content in tumor tissue measured by ICP-MS ÷ gold content of injected nanorobot × 100%) ÷ weight of tumor tissue), which is 209 times that of an inert carrier, 246 times that of a urease-driven nanorobot with only motor function, and 23 times that of a catalase-driven nanorobot.

[0098] Example 5

[0099] This example provides a nanorobot, which differs from the nanorobot structure shown in Example 1 only in that urease and catalase are assembled in the same layer on one side of the nanogold through polyethylene glycol, biotin and streptavidin to obtain a single-layer dual-enzyme nanorobot.

[0100] Example 6

[0101] For the nanorobot in Example 5, this example provides a preparation method comprising the following steps:

[0102] (1) Preparation of gold nanoparticles is the same as step (1) in Example 2.

[0103] (2) The preparation of asymmetric Au-PS eccentric particles is consistent with step (2) of Example 2.

[0104] (3) The preparation of biotinylated urease and biotinylated catalase is the same as step (3) in Example 2.

[0105] (4) The preparation of the chemotactic nanorobot is different from step (4) of Example 2 only in that after the nanoparticles are coupled with streptavidin, biotinylated urease and biotinylated catalase are simultaneously mixed and coupled.

[0106] Based on the above method, nanorobots with different ratios of urease to catalase (urease: catalase = 1:0.1-20) were obtained by changing the amounts of biotinylated catalase and biotinylated urease in step (4).

[0107] Using the 3D chip shown in Figure 5A, single-layer dual-enzyme nanorobots prepared in this example were tested in vitro at different ratios. The transport performance of nanorobots with urease:catalase ratios of 1:1, 2:1, 3:1, and 4:1, respectively, is shown in Figure 7. The test results show that by adjusting the urease:catalase ratio to the appropriate ratio, the single-layer dual-enzyme nanorobots, like the double-layer dual-enzyme nanorobots, can not only selectively and spontaneously move toward cancer cells, but also significantly enhance their chemotactic ability.

[0108] Example 7

[0109] This example provides a nanorobot, whose structure and driving mechanism are shown in Figure 8. The nanorobot uses gold nanoparticles, urease as the driving enzyme, and catalase as the chemoattractant enzyme. The urease and catalase are layered and assembled on the entire surface of the gold nanoparticles using polyethylene glycol, biotin, and streptavidin, forming a relatively symmetrical structure.

[0110] Example 8

[0111] For the nanorobot in Example 7, this example provides a preparation method comprising the following steps:

[0112] (1) Preparation of gold nanoparticles is the same as step (1) in Example 2.

[0113] (2) The preparation of biotinylated urease and biotinylated catalase was the same as step (3) in Example 2.

[0114] (3) Preparation of chemotactic nanorobots.

[0115] The gold nanoparticles obtained in step (1) were dispersed in 6 mL of ultrapure water. 2 mL of the gold nanoparticle solution was added to 100 μL of SH-PEG-Biotin (10 mg / mL) solution, and the mixture was dispersed in a PB solution (10 mM, pH = 7.4) after oscillation. 100 μL of the above solution was added to 100 μL of streptavidin (0.02 mg / mL) solution, mixed, and dispersed in a PB solution (10 mM, pH = 7.4). 100 μL of the above solution was added to 100 μL of biotinylated catalase solution and oscillated for reaction. Subsequently, SA and biotinylated urease were coupled. After the reaction was complete, the nanorobot was washed to obtain the nanorobot. The ratio of biotinylated catalase to biotinylated urease was 1:1.

[0116] Figure 9 shows transmission electron microscopy (TEM) images and elemental analysis of the nanorobot prepared in this example. Figure 9A shows a brightfield image of the chemotactic nanorobot under TEM mode, while Figure 9B shows the distribution of gold, nitrogen, and iron under darkfield and elemental analysis. The images show that catalase and urease are distributed on both sides of the nanorobot, forming a relatively symmetrical structure.

[0117] The symmetrical dual-enzyme-driven nanorobot prepared in this example was tested in vitro using the 3D chip shown in Figure 5A. As shown in Figure 10, the chemotactic ability of the nanorobot was significantly enhanced, more than five times that of a conventional single-enzyme nanorobot.

[0118] Example 9

[0119] This example provides a nanorobot (denoted as SOD / CAT / URE-driven nanorobot), which is based on the nanorobot structure shown in Example 1 and is simultaneously connected to an anti-inflammatory molecule. Its structure and driving mechanism are shown in Figure 11: nanogold is used as nanoparticles, urease is used as the driving enzyme, catalase is used as the chemotactic enzyme, and superoxide dismutase is used as the anti-inflammatory molecule. Urease, catalase and superoxide dismutase are layered and assembled on one side of the nanogold through polyethylene glycol, biotin and streptavidin.

[0120] In the nanorobot structure of this example, the catalytic reaction of urease and urea is used to effectively drive the nanorobot in the biological environment (such as blood), and catalase is used to sense the hydrogen peroxide concentration gradient at the inflammatory site (i.e., the chemotactic source) to produce a chemotactic effect. The synergistic effect of urease and catalase enables the nanorobot to be efficiently and accurately enriched at the inflammatory site, and then the reactive oxygen is efficiently removed through the cascade reaction of superoxide dismutase and catalase.

[0121] Example 10

[0122] For the nanorobot in Example 9, this example provides a preparation method comprising the following steps:

[0123] (1) Preparation of gold nanoparticles: Refer to the method of step (1) of Example 2 to obtain gold nanoparticles with a particle size of 90 nm.

[0124] (2) The preparation of asymmetric Au-PS eccentric particles is consistent with step (2) of Example 2.

[0125] (3) Preparation of biotinylated superoxide dismutase, urease, and catalase, refer to step (3) of Example 2.

[0126] (4) Preparation of SOD / CAT / URE driven nanorobots.

[0127] Au-PS eccentric particles were dispersed in 5 mL of ultrapure water. 1 mL of the Au-PS solution was added to 0.1 mL of a 10 mg / mL SH-PEG-Biotin solution and the mixture was shaken. 1 mL of N,N-dimethylformamide was added to the sample, which was then dispersed in 500 μL of deionized water. 0.1 mL of a 10 mg / mL mPEG-SH solution was then added and shaken, and the mixture was then dispersed in a 10 mM PB solution (pH = 7.4). 100 μL of this solution was added to 100 μL of a 0.02 mg / mL streptavidin solution, mixed, and then dispersed in a 10 mM PB solution (pH = 7.4). 100 μL of this solution was then added to 100 μL of a biotinylated superoxide dismutase solution and shaken. This step was repeated to assemble biotinylated catalase and urease onto the gold nanoparticle surface. After the reaction, the SOD / CAT / URE-driven nanorobot was obtained by washing. The ratio of biotinylated SOD, CAT and URE is 4:1:1.

[0128] Figure 12 shows a transmission electron micrograph (TEM) and elemental analysis of the SOD / CAT / URE-driven nanorobot prepared in this example (scale: 100 nm). Figure 12A shows a brightfield image of the SOD / CAT / URE-driven nanorobot under TEM mode, while Figure 12B shows the distribution of gold, sulfur, nitrogen, iron, and zinc under darkfield and elemental analysis. The images indicate that superoxide dismutase, catalase, and urease are asymmetrically distributed on one side of the nanorobot, forming an asymmetric structure.

[0129] Example 11

[0130] Referring to Example 10, this example prepared SOD / CAT / URE nanorobots with different ratios of catalase and urease (catalase: urease = 1-20:1) by changing the dosage ratio of biotinylated catalase and biotinylated urease in step (4).

[0131] The motility of the SOD / CAT / URE-driven nanorobots in physiological urea solutions was tested at different catalase / urease ratios. Specifically, the nanorobots were placed in a 10mM urea solution and their diffusion coefficients were measured to examine the effect of the relative amounts of catalase and urease on their motility. The results for catalase / urease ratios of 2:1, 4:1, and 1:1, respectively, are shown in Figure 13. A 1:1 ratio of catalase and urease exhibited the strongest driving force, while gradually decreasing the urease ratio resulted in a decrease in driving force.

[0132] Example 12

[0133] Taking the inflammatory disease of colitis as an example, this example demonstrates the targeting efficiency and therapeutic effect of the nanorobot shown in Example 9 in the organism.

[0134] (1) Preparation of control product.

[0135] In this example, inert particles, URE-driven nanorobots, and SOD / CAT-driven nanorobots were used as controls, and the preparation method of the control substances was referred to Example 10, with the only difference being that the urease, catalase, and superoxide dismutase in the inert particles were all inactivated by high temperature, the catalase and superoxide dismutase in the URE-driven nanorobots were all inactivated by high temperature, and the urease in the SOD / CAT-driven nanorobots was inactivated by high temperature.

[0136] (2) Construct a mouse model of ulcerative colitis.

[0137] This case was established by induction with dextran sodium sulfate (2.5% w / v) for 7 days.

[0138] (3) Detection of targeting efficiency.

[0139] The SOD / CAT / URE-driven nanorobots prepared in Example 10 and a control product were dissolved in PBS buffer to prepare different nanorobot injections. The nanorobots were then injected intravenously into a colitis mouse model at a dose of 200 μL at 1.5 mg / kg. The mice were killed 3, 6, 12, and 24 hours later. The gold content in the colonic tissue was measured by ICP-MS, and the targeting efficiency of the nanorobots against colitis was calculated.

[0140] The test results are shown in Figure 14, where Figure 14A shows the targeting efficiency of the nanorobots at different times, and Figure 14B shows the targeting efficiency of different control groups at 6 hours. As shown in Figure 14, the SOD / CAT / URE-driven nanorobots reached saturation targeting efficiency for colitis 6 hours after intravenous injection, with a targeting efficiency of 5.01% ID / g, 28 times that of inert particles and 25 times that of SOD / CAT-driven nanorobots.

[0141] (4) Detection of treatment effects.

[0142] The SOD / CAT / URE-driven nanorobots prepared in Example 10 and a control product were dissolved in PBS buffer to prepare nanorobot injections. The injections were then injected intravenously into colitis mice at a dose of 200 μL at a concentration of 1.5 mg / kg. The mice were injected every other day for four times. The colon, major organs (heart, liver, spleen, lungs, and kidneys), and blood were collected, and colon length was measured and photographed.

[0143] Figure 15 shows the therapeutic effect of SOD / CAT / URE-driven nanorobots in mice with colitis. Figure 15A shows an image of the colon, Figure 15B shows the colon length, and Figure 15C shows a hematoxylin-eosin-stained pathological section of the colon tissue (scale: 200 μm). As shown in Figure 15 , after treatment with SOD / CAT / URE-driven nanorobots, the colon length of the mice returned to normal (6.6±0.3 cm), and the typical inflammatory symptoms of colon shortening and colon damage were significantly improved, effectively alleviating the clinical symptoms of colitis in the mice.

[0144] The blood and major organs of healthy mice and mice treated with SOD / CAT / URE-driven nanorobots were tested for biosafety assessment. The test results are shown in Figure 16. Figure 16A shows the blood biochemical index detection of healthy mice and mice after nanorobot treatment, and Figure 16B shows the hematoxylin-eosin stained histological sections of the major organs of healthy mice and mice after nanorobot treatment. As can be seen from Figure 16, there is no significant difference in the levels of blood biochemical indicators of mice treated with nanorobots and healthy mice, and no obvious systemic toxicity was found in the hematoxylin-eosin stained sections of the heart, liver, spleen, lungs and kidneys.

[0145] Example 13

[0146] This example provides a nanorobot that can be used for photodynamic therapy (denoted as Ce6-CAT / URE driven nanorobot). Based on the nanorobot structure shown in Example 1, it is also connected to a photosensitizer. Its structure and driving mechanism are shown in Figure 17: nanogold is used as nanoparticles, urease is used as the driving enzyme, catalase is used as the chemotactic enzyme, and Ce6 is used as the photosensitizer. Urease and catalase are layered and assembled on one side of the nanogold through polyethylene glycol, biotin and streptavidin, and Ce6 is connected to streptavidin.

[0147] In the nanorobot structure of this example, catalase is used to sense signal chemotaxis, and urease uses body fluids to drive the nanorobot, thereby achieving efficient targeted enrichment of the nanorobot in inflammatory disease tissues, and then treating them through Ce6-mediated photodynamic therapy.

[0148] Example 14

[0149] For the photodynamic therapy nanorobot in Example 13, this example provides a preparation method comprising the following steps:

[0150] (1) Preparation of gold nanoparticles: Referring to the method of step (1) of Example 2, gold nanoparticles with a particle size of 90 nm were obtained.

[0151] (2) The preparation of asymmetric Au-PS eccentric particles is consistent with step (2) of Example 2.

[0152] (3) The preparation of biotinylated urease and catalase is the same as step (3) in Example 2.

[0153] (4) Preparation of photodynamic therapy nanorobots.

[0154] Au-PS eccentric particles were dispersed in 6 mL of ultrapure water. 2 mL of Au-PS solution was added to 0.1 mL of HO-PEG-SH solution (10 mg / mL) and the mixture was shaken. 1 mL of N,N-dimethylformamide was added to the sample, which was then dispersed in 500 μL of ultrapure water. 100 μL of SH-PEG-Biotin solution (10 mg / mL) was added and shaken, and the mixture was then dispersed in PB solution (10 mM, pH = 7.4). 100 μL of this solution was added to 100 μL of streptavidin solution (0.02 mg / mL), mixed, and then dispersed in PB solution (10 mM, pH = 7.4). 100 μL of this solution was then added to 100 μL of biotinylated catalase solution and shaken. Subsequently, SA and biotinylated urease were coupled to produce a dual-enzyme-driven nanorobot. The ratio of biotinylated urease to biotinylated catalase was 1:1.

[0155] 0.2 μg of Ce6 was dissolved in 120 μL of dimethyl sulfoxide (DMSO), and 20 μL of 10 mM EDC and 10 μL of 10 mM NHS were added. The mixture was shaken for half an hour, and then the dual-enzyme-driven nanorobot was added. The mixture was shaken overnight. After the reaction was completed, the nanorobot was washed to obtain the photodynamic therapy nanorobot.

[0156] Figure 18 shows the morphological structure of the photodynamic therapy nanorobot prepared in this example. Figures 18A and 18B are transmission electron microscopy (TEM) and elemental analysis images of the robot (scale: 100 nm), respectively. The images show that catalase and urease are asymmetrically distributed on one side of the nanorobot, forming an asymmetric structure. Figure 18C shows the UV absorption spectrum of the photodynamic therapy nanorobot before and after modification with Ce6. The image shows that the coupled nanorobot has a clear Ce6 characteristic peak at 400 nm, indicating successful Ce6 coupling.

[0157] Example 15

[0158] Referring to Example 14, this example prepared photodynamic therapy nanorobots with different ratios of urease and catalase (urease: catalase = 1:1-20) by changing the dosage ratio of biotinylated catalase and biotinylated urease in step (4).

[0159] The diffusion coefficients of the photodynamic therapy nanorobots in 10 mM urea solution were detected under different enzyme ratios. The results when the urease:catalase ratios were 1:1, 1:4 and 1:8, respectively, are shown in Figure 19. The driving ability is strongest when the urease:catalase ratio is 1:1. Gradually reducing the urease ratio will lead to a significant decrease in the driving ability. At the same time, it shows that the nanorobot of the present invention can be effectively driven at the human body urea concentration (~10 mM).

[0160] Example 16

[0161] Taking breast cancer, an inflammatory disease, as an example, this example demonstrates the targeting efficiency and therapeutic effect of the nanorobot shown in Example 13 in the organism.

[0162] (1) Preparation of control product.

[0163] In this example, the Ce-6 inert carrier, Ce6-URE-driven nanorobot and Ce6-CAT-driven nanorobot were used as controls, and the preparation method of the control samples referred to Example 14, with the only difference being that the urease and catalase in the Ce-6 inert carrier were both inactivated by heat, the catalase in the Ce6-URE-driven nanorobot was inactivated by heat, and the urease in the Ce6-CAT-driven nanorobot was inactivated by heat.

[0164] (2) Detection of targeting efficiency.

[0165] The photodynamic therapy nanorobots prepared in Example 14 were resuspended in a PBS solution and injected intravenously into 4T1 breast cancer-bearing mice (the dosage of the nanorobots was 10 μg / mouse, of which the amount of Ce6 was approximately 0.0312 μg by UV quantification). The mice were killed after 6 h, 12 h, and 24 h, respectively. The gold content of the tumor tissue was detected by ICP-MS and the uptake efficiency was calculated. Figure 20A is an image of the change in tumor uptake efficiency over time. As can be seen from Figure 20A, as time increases, the uptake of the photodynamic therapy nanorobot by the tumor gradually increases, reaching a peak of 26.37% ID at 24 h.

[0166] The photodynamic therapy nanorobots prepared in Example 14 and their control substances were resuspended in PBS solution and injected intravenously into 4T1 breast cancer-bearing mice (the dosage was 10 μg / mouse). The mice were killed after 24 hours, and the targeting efficiency of the nanorobots on the tumors was tested by ICP-MS. Figure 20B shows the targeting efficiency results of the photodynamic therapy nanorobots and their control groups. As can be seen from Figure 20B, the targeting efficiency of the Ce6-CAT / URE-driven nanorobots is as high as 63.61% ID / g, which is 302 times that of the Ce-6 inert carrier, 219 times that of the Ce6-URE-driven nanorobots with only motor function, and 22 times that of the Ce6-CAT-driven nanorobots.

[0167] The vertical axis in Figure 20A represents the absolute uptake efficiency, calculated as follows: ICP-MS gold content measured after grinding and digestion of tumor tissue / ICP-MS gold content of the injected nanorobot solution. The vertical axis in Figure 20B represents the relative uptake efficiency, calculated as follows: absolute uptake efficiency / tumor weight.

[0168] (3) Detection of treatment effects.

[0169] The photodynamic therapy nanorobots prepared in Example 14 and their control substances were resuspended in PBS solution and then injected intravenously into 4T1 breast cancer tumor-bearing mice (10 μg / mouse). After 24 hours, the photodynamic therapy nanorobots were illuminated with a 660 nm laser at 0.18 W / cm 2 Irradiation was performed for 10 minutes, repeated every three days for three times, and mice were sacrificed 48 hours later. Tumor size and body weight were recorded daily, and H&E staining of organs such as the heart, liver, spleen, lung, and kidney was performed.

[0170] The tumor inhibition effect is shown in Figure 21. Figure 21A shows the changes in mouse tumors over time, Figure 21B shows images of different tissues, and Figure 21C shows tumor weight. As shown in Figure 21, while the Ce6-CAT / URE nanorobot has a Ce6 loading two orders of magnitude lower than other reported nanorobots, its tumor inhibition effect remains highly significant, reaching a tumor inhibition efficiency of 92.7%.

[0171] The biosafety profile is shown in Figure 22 . Figure 22A shows the changes in mouse weight over time, Figure 22B shows images of mouse tissue sections, and Figure 22C shows blood biochemical parameters. These results demonstrate that the Ce6-CAT / URE-driven nanorobot has good biosafety.

[0172] Example 17

[0173] This example provides a nanorobot capable of photodynamic therapy (denoted as a Ce6-GOx / URE-driven nanorobot). The nanorobot differs from the nanorobot described in Example 13 only in that glucose oxidase replaces catalase. The Ce6-GOx / URE-driven nanorobot exhibits enhanced chemotaxis in response to a source of glucose concentration.

[0174] A Ce6-GOx / URE driven nanorobot was prepared with reference to Example 14, and the following tests were performed on it:

[0175] Referring to Example 15, the diffusion coefficient of the Ce6-GOx / URE driven nanorobot in 10 mM urea solution was measured to be 2.63 μm 2 / s.

[0176] The Ce6-GOx / URE-driven nanorobot was placed in a 3D chip, and the changes in absorbance were observed in areas 3, 4, and 5, respectively, to determine the robot's chemotactic ability. The design of the 3D chip is shown in Figure 23A. The left reservoir area is filled with a gel matrix containing 10mM glucose, while the right reservoir is filled with a gel matrix containing deionized water, and the channel is filled with a urea (10mM) aqueous solution. Figure 23B shows the chemotactic ability of the Ce6-GOx / URE-driven nanorobot and its control (the preparation of the control refers to Example 16). It can be seen that the Ce6-GOx / URE-driven nanorobot is selective and can spontaneously move toward the overexpressed glucose area rather than in the other direction. The chemotactic ability of the robot is significantly enhanced, more than 10 times stronger than that of the traditional single-enzyme chemotactic nanorobot.

[0177] Example 18

[0178] This example provides a nanorobot (denoted as a Ce6-GGT / URE-driven nanorobot) that can be used for photodynamic therapy. Compared to the nanorobot described in Example 13, the only difference is that catalase is replaced with glutamyl transpeptidase (GGT). The Ce6-GGT / URE-driven nanorobot is a nanorobot that produces enhanced chemotaxis in response to a source of glutathione concentration.

[0179] A Ce6-GGT / URE driven nanorobot was prepared with reference to Example 14, and the following tests were performed on it:

[0180] Referring to Example 15, the diffusion coefficient of the Ce6-GGT / URE driven nanorobot in 10 mM urea solution was measured to be 2.59 μm 2 / s.

[0181] Referring to the test method in Example 17, the chemotactic ability of the Ce6-GGT / URE-driven nanorobot was tested using a 3D chip, and the results are shown in Figure 24. The test results show that the Ce6-GGT / URE-driven nanorobot is selective and can spontaneously move toward the glutathione-overexpressing area but not in the other direction. In addition, the chemotactic ability of the robot is significantly enhanced, which is more than 10 times stronger than that of the traditional single-enzyme chemotactic nanorobot.

[0182] All raw materials listed in this application, as well as the upper and lower limits and interval values ​​of each raw material in this application, and the upper and lower limits and interval values ​​of the process parameters in this application can realize this application, and the embodiments are not listed one by one here.

[0183] In summary, the nanorobots constructed using multiple enzymes in this application have ultra-high chemotactic sensitivity and ultra-high targeting efficiency in organisms. They can overcome the complex environment in various physiological media (such as the blood circulatory system and tumor microenvironment) and efficiently enrich in the target inflammatory site. At the same time, the nanorobots can further carry different functional substances, such as photosensitizers, antioxidants, chemotherapy drugs, etc., so that targeted treatment of various diseases can be achieved through multiple pathways, and have very good clinical application potential.

[0184] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A nanorobot capable of highly efficient targeted enrichment in inflammatory disease tissues, characterized in that, It includes nanoparticles and at least two enzymes attached to the nanoparticles, wherein at least one enzyme is used to drive the nanorobot in a physiological medium, and at least another enzyme is used to sense the inflammatory lesion site and control the movement direction of the nanorobot.

2. The nanorobot according to claim 1, wherein, The enzyme used to drive the nanorobot in a physiological medium is an enzyme that uses an endogenous substance in the physiological medium as a substrate, and the enzyme used to sense the inflammatory lesion site and control the movement direction of the nanorobot is an enzyme that uses a biomarker released from the inflammatory lesion site as a substrate.

3. The nanorobot according to claim 2, wherein The endogenous substances include glucose, lactose, lipids, amino acids, urea, uric acid, and water, and the biomarkers include hydrogen peroxide, glutathione, lactic acid, glucose, nucleic acid, adenosine triphosphate, H + .

4. The nanorobot according to claim 1, characterized in that, The nanorobot further includes a therapeutic drug, and the therapeutic drug includes doxorubicin, cyclophosphamide, fluorouracil, gemcitabine, bleomycin, paclitaxel, chlorin e6, porphyrin mixture photosensitizer, porphyrin derivative photosensitizer, phthalocyanine photosensitizer, methylene blue, texaphyrin photosensitizer, CTLA-4 inhibitor, bispecific antibody immunotherapeutic, monoclonal antibody immunotherapeutic, antibody-drug conjugate, sodium iodide, yttrium, 177Lu-oxytocin, radium chloride [223Ra], 131I-tositumomab, glutathione peroxidase, superoxide dismutase, auranofin, curcumin, ligustrazine-curcumin hybrid.

5. The nanorobot according to claim 4, characterized in that The connection mode of the therapeutic drug to the nanoparticles includes: connecting to the surface of the nanoparticles, connecting to the pores of the porous nanoparticles, and being encapsulated inside the nanoparticles; the distribution mode of the therapeutic drug in the nanoparticles includes: concentrating in some regions of the nanoparticles and dispersing on the entire surface of the nanoparticles.

6. The nanorobot according to claim 1, characterized in that, The nanoparticles include elemental, oxide or sulfide nanoparticles of metal elements such as gold, silver, palladium, platinum, manganese, iron, cerium, titanium, aluminum, and zinc, inorganic non-metallic nanoparticles, and organic nanoparticles; the nanoparticles are of hollow, solid or porous structure, and their shape is spherical, bowl-shaped, rod-shaped or multi-sided structure.

7. The application of the nanorobot according to any one of claims 1-6 in the preparation of a drug for treating inflammatory diseases, and the nanorobot improves the targeting efficiency and therapeutic effect of the drug.

8. The application according to claim 7, wherein The inflammatory diseases include tumors and enteritis.

9. A drug for targeted treatment of inflammatory diseases, characterized in that, The drug uses the nanorobot according to any one of claims 1-3 as a delivery carrier.

10. A drug delivery system, characterized in that, It contains the nanorobot according to any one of claims 1-3.

11. A medical device, characterized in that, It contains the nanorobot according to any one of claims 1-3.

Images