Nano-robot for targeted therapy of inflammatory diseases

Abstract

The invention discloses a nano-robot for targeted therapy of inflammatory diseases, the nano-robot comprises nano-particles, and at least two enzymes and anti-inflammatory molecules which are connected to the nano-particles, at least one enzyme is used for driving the nano-robot in a physiological medium, and the other enzyme is used for driving the nano-robot in a physiological medium. At least another enzyme is used for sensing an inflammatory part and controlling the movement direction of the nano-robot, and the anti-inflammatory molecules are used for removing active oxygen in the inflammatory part. Based on the synergistic effect of driving-orientation functions of different bio-enzymes, the enrichment efficiency of the nano-robot in an inflammatory part in a blood circulation system can be remarkably improved, meanwhile, active oxygen in the inflammatory part is removed by using the anti-inflammatory molecules alone or in cooperation with the enzymes, and the effect of remarkably improving and treating inflammatory diseases is achieved. The invention can provide a new way for targeted treatment of inflammatory diseases, and has the characteristics of high safety, excellent treatment effect and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanodevice preparation, and particularly relates to a nanorobot for targeted treatment of inflammatory diseases, and a preparation method and application thereof. BACKGROUND

[0002] Inflammation plays a key role in the immune response triggered by stimuli or injury, but excessive inflammatory response can cause tissue damage and organ dysfunction, and is associated with many inflammatory diseases. Inflammatory diseases bring health and economic burdens to communities worldwide, seriously affecting the quality of life of patients, and even leading to cancer risk. A large amount of evidence shows that excessive reactive oxygen species (ROS, including superoxide anion free radicals (•O2 – ) and hydrogen peroxide (H2O2)) are closely related to the occurrence and development of inflammation, so ROS scavenging is an effective strategy for treating inflammatory diseases. Nanodrug delivery systems show great potential in the treatment of inflammatory diseases, but the passive diffusion nanodrug delivery systems developed at present have very low targeting efficiency in the inflammatory lesion area, resulting in poor treatment effect and serious side effects.

[0003] Nanorobots with chemotaxis can sense the overexpressed chemicals in the inflammatory lesion area to achieve self-targeting, and are believed to bring revolutionary changes to the targeted treatment of inflammatory diseases. However, the existing chemotactic nanorobots have low chemotactic sensitivity, which makes them unable to effectively sense the low concentration of chemical signals in the lesion area; and compared with passive diffusion nanodrug delivery systems, their targeting efficiency in the inflammatory lesion area does not improve significantly, at most only increasing by 2-3 times. The above problems greatly limit the practical application of nanorobots in the field of biomedical active drug delivery. SUMMARY

[0004] Therefore, the present application aims to develop a nanorobot that can be used for targeted treatment of inflammatory diseases, which not only significantly improves the targeting efficiency in the inflammatory disease site, but also treats inflammatory diseases by effectively scavenging ROS.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: A nanorobot for targeted treatment of inflammatory diseases, comprising a nanoparticle and at least two enzymes and anti-inflammatory molecules connected to the nanoparticle; wherein at least one enzyme is used to drive the nanorobot in a physiological medium, at least another enzyme is used to sense the inflammatory site and control the movement direction of the nanorobot, and the nanorobot is driven in the physiological medium and enriched in the inflammatory site under the synergistic action of the above two enzymes; the anti-inflammatory molecules are used alone or in combination with any of the above enzymes to scavenge ROS in the inflammatory site.

[0006] In the nanorobot of the present application, the "enzyme" can be a biological enzyme and / or a nanoenzyme, as long as it can drive the nanorobot or control the movement direction of the nanorobot in a physiological medium through a catalytic reaction system; the "physiological medium" includes but is not limited to blood.

[0007] In the nanorobot of the present application, the nanoparticles include but are not limited to nanogold, silicon dioxide (such as mesoporous silica, etc.), liposomes, magnetic particles (such as ferroferric oxide, magnetite, etc.), polydopamine, etc. In some embodiments of the present application, the nanoparticles are nanogold.

[0008] In the nanorobot of the present application, the size of the nanorobot is preferably 10-1000 nm, and more preferably 20-200 nm.

[0009] In the nanorobot of the present application, the enzyme used to drive the nanorobot in a physiological medium (hereinafter referred to as a driving enzyme) includes urease, glucose oxidase and / or other enzymes that use endogenous substances in a physiological medium as substrates, and urease (URE) can be preferably used. Urease uses urea in the human physiological medium as a substrate for catalytic reaction, thereby effectively driving the nanorobot.

[0010] In the nanorobot of the present application, the enzyme used to sense the inflammatory site and control the movement direction of the nanorobot (hereinafter referred to as a chemotactic enzyme) is an enzyme that uses a biomarker (such as hydrogen peroxide) released by an inflammatory site as a substrate, and the enzyme includes but is not limited to catalase (CAT), glucose oxidase, and ferroferric oxide nanenzyme. In some embodiments of the present application, catalase is used as a chemotactic enzyme that responds to an inflammatory site, which uses hydrogen peroxide in blood as a substrate to achieve the enrichment of the nanorobot in the inflammatory site (such as the intestinal tract).

[0011] In the nanorobot of the present application, its diffusion coefficient under the urea concentration (~10 mM) in the human body is preferably not less than 1.0 μm 2 / s, thereby ensuring the effective targeting of the robot to the inflammatory site in the human blood circulation system.

[0012] In the nanorobot of the present application, the anti-inflammatory molecules include but are not limited to biological enzymes, nanoenzymes (such as cerium dioxide), anti-inflammatory small molecule drugs, and other substances that can be used to scavenge active oxygen in the inflammatory site, and the anti-inflammatory molecules can be one or more. In some embodiments of the present application, the anti-inflammatory molecules are superoxide dismutase (SOD), which can catalyze ROS to generate hydrogen peroxide, thereby achieving the treatment of inflammatory diseases by scavenging ROS in the inflammatory site.

[0013] Preferably, in the nanorobot of the present application, the anti-inflammatory molecule is superoxide dismutase, and the chemotactic enzyme is catalase; in this preferred case, the nanorobot is efficiently targeted and enriched at the inflammatory site, and the cascade reaction of superoxide dismutase and catalase effectively improves the ROS removal treatment effect.

[0014] More preferably, in the nanorobot of the present application, the anti-inflammatory molecule is superoxide dismutase, the chemotactic enzyme is catalase, and the driving enzyme is urease. In this preferred case, the nanorobot is driven and enriched at the inflammatory site under the synergistic action of urease and catalase, and efficiently removes ROS under the cascade reaction of superoxide dismutase and catalase. Among them, the use amount ratio of superoxide dismutase, catalase and urease in the nanorobot is 4:(1-4):1, which is the best.

[0015] In the nanorobot of the present application, the driving enzyme and the chemotactic enzyme can be connected to the nanoparticle in any of the following ways: 1) The driving enzyme and the chemotactic enzyme are connected on the same side of the nanoparticle in different layers; 2) The driving enzyme and the chemotactic enzyme are connected on the same side of the nanoparticle in the same layer; 3) The driving enzyme and the chemotactic enzyme are connected on the entire surface of the nanoparticle in different layers or in the same layer.

[0016] In the above cases 1) and 2), the assembly positions of the two enzymes relative to the nanoparticle are consistent and only connected to part of the area of the nanoparticle, forming an asymmetric structure. In the above case 3), the two enzymes are directly connected to the entire surface of the nanoparticle, forming a more symmetric structure relative to 1) and 2).

[0017] In the nanorobot of the present application, the connection mode and distribution mode of the anti-inflammatory molecule on the surface of the nanoparticle are not limited, such as being concentrated in part of the area of the nanoparticle, or being dispersed on the entire surface of the nanoparticle. When the anti-inflammatory molecule is a biological enzyme and / or a nanenzyme, it can be assembled on the same side of the nanoparticle in different layers or in the same layer with the driving enzyme and the chemotactic enzyme, or it can be assembled on the different side of the nanoparticle from the driving enzyme and the chemotactic enzyme, or it can be assembled on the entire surface of the nanoparticle in different layers or in the same layer with the driving enzyme and the chemotactic enzyme. Moreover, experimental data show that the assembly order of the three enzymes has no obvious effect on the motion performance, chemotactic performance and ROS removal performance of the nanorobot.

[0018] The second aspect of the present application provides a method for preparing a nanorobot for targeted treatment of inflammatory diseases. Taking a nanoparticle of nanogold as an example, and the nanorobot containing a driving enzyme, a chemotactic enzyme and an enzyme for removing active oxygen, the method specifically comprises the following steps: S1, preparing a double-sided nanogold particle; S2, biotinylate the three enzymes, and then use streptavidin to simultaneously or gradually couple the three enzymes on the same side of the Janus nanogold particles.

[0019] It can be understood that the nanorobot prepared by the above method has the driving enzyme, the chemotactic enzyme and the enzyme for removing active oxygen assembled on the same side of the nanoparticle in the same layer or in different layers, and forms an asymmetric structure.

[0020] Preferably, in the above method, the step S1 comprises the following process: S11, prepare nanogold by a seed growth method; S12, mix potassium persulfate, 4-styrene sulfonic acid sodium salt, water and ethanol, preheat, then add a mixed solution of styrene and divinylbenzene, and then add nanogold to react, so as to obtain Janus nanogold particles.

[0021] Preferably, the step S2 is specifically: S21, biotinylate the driving enzyme, the chemotactic enzyme and the enzyme for removing active oxygen respectively; S22, disperse the Janus nanogold particles in water, add a Biotin-PEG-SH solution, then add N,N-dimethylformamide (DMF) and mPEG-SH, and then mix and disperse the streptavidin in a PB solution; S22, take the above PB solution, add the first biotinylated enzyme to react, take the reaction solution, mix and disperse the streptavidin (SA) in a PB solution, then add the second biotinylated enzyme to react, take the reaction solution, mix and disperse the SA in a PB solution, and finally add the third biotinylated enzyme to react; Or, take the above PB solution, directly add a mixed solution of the three biotinylated enzymes to react; Or, take the above PB solution, first add one or two biotinylated enzymes to react, take the reaction solution, mix and disperse the SA in a PB solution, and then add the remaining biotinylated enzymes to react.

[0022] The third aspect of the present application provides another method for preparing a nanorobot for targeted treatment of inflammatory diseases, taking the nanoparticle as nanogold, and the nanorobot containing one driving enzyme, one chemotactic enzyme and one enzyme for removing active oxygen as an example, which specifically comprises the following steps: S1, prepare nanogold by a seed growth method; S2, biotinylate the three enzymes; S3, disperse the nano gold particles in water, add Biotin-PEG-SH, mix with streptavidin and disperse in PB solution, and then couple three kinds of biotinylated enzymes (the coupling condition is the same as above); It can be understood that, in the nano robot prepared by the above method, the driving enzyme, the chemotactic enzyme and the enzyme for removing active oxygen are assembled on the whole surface of the nano particle in the same layer or different layers, forming a relatively symmetrical structure. However, experimental data show that the nano robot with an asymmetric structure has a higher targeting efficiency for inflammatory sites.

[0023] The fourth aspect of the present application provides the use of the nano robot for targeted treatment of inflammatory diseases in the preparation of a drug for treating inflammatory diseases.

[0024] The fifth aspect of the present application provides a drug for targeted treatment of inflammatory diseases, one of the effective components of which is the nano robot provided by the present application. It can be understood that the drug can also include a pharmaceutically acceptable dressing.

[0025] For the drug for targeted treatment of inflammatory diseases provided by the present application, the preferred administration mode is intravenous injection. Experimental data of the present application show that, after the SOD / CAT / URE driving nano robot is intravenously injected into a mouse, through the synergistic effect of the double enzymes, the nano particle can resist random Brownian motion, achieve the effect of enhanced chemotaxis, significantly improve the targeting efficiency for inflammatory sites, and, in combination with anti-inflammatory molecules, effectively relieve inflammatory damage in the organism and significantly improve the treatment effect of inflammatory diseases.

[0026] Compared with the prior art, the present application has the following beneficial effects: (1) Compared with the existing nano robot, the present application innovatively utilizes the synergistic effect of double enzymes to construct a nano robot with significantly improved chemotactic sensitivity and targeting efficiency for inflammatory sites, and further connects anti-inflammatory molecules for removing active oxygen at the inflammatory site to obtain a nano robot for targeted treatment of inflammatory diseases.

[0027] (2) The type of the carrier (i.e. the nano particle), the chemotactic enzyme and the driving enzyme in the nano robot can be changed according to the actual needs of the type of inflammatory disease, the administration mode, etc. without affecting the overall function, so it has ultra-high plasticity and a wide range of applications.

[0028] (3) The nano robot prepared by urease and catalase can be administered by intravenous injection, which can be effectively driven by the urea concentration in the human blood circulation system, and through the catalytic reaction of catalase and hydrogen peroxide, the nano robot produces chemotaxis and moves towards the inflammatory site.

[0029] (4) The present application couples superoxide dismutase as an anti-inflammatory molecule on the basis of the urease and catalase constructed nanorobot, and then uses the cascade reaction between superoxide dismutase and catalase to efficiently remove the active oxygen in the inflammatory site, thereby significantly improving the treatment effect.

[0030] (5) The nanorobot of the present application has excellent treatment effect on the inflammatory site, and the mouse in vivo experiment shows that the nanorobot constructed by urease, catalase and superoxide dismutase can effectively relieve the clinical symptoms of colitis, and the treatment effect is significantly better than other nanorobots. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The structure and mechanism of the nanorobot for targeted treatment of inflammatory diseases in Example 1.

[0032] Figure 2 The transmission electron microscope image (A) and element analysis diagram (B) of the SOD / CAT / URE driven nanorobot prepared in Example 2.

[0033] Figure 3 The movement ability comparison of the nanorobot with different proportions of catalase and urease in the physiological concentration urea solution in Example 3.

[0034] Figure 4 The targeted efficiency results of the SOD / CAT / URE driven nanorobot on the inflammatory intestinal site in Example 4.

[0035] Figure 5 The treatment effect diagram of the SOD / CAT / URE driven nanorobot on the inflammatory intestinal disease in Example 5.

[0036] Figure 6 The safety evaluation results of the SOD / CAT / URE driven nanorobot in the organism in Example 5. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be described clearly and completely in combination with the drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the specification and the claims of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The use of the terms "including," "comprising," or "having" and variations thereof herein is intended to be equivalent to the term "consisting of to describe one embodiment of the application. The terms "a," "another," "an," "other," and "at least one" used in the specification and in the claims, are used "by way of example," rather than "by way of limitation," to illustrate information. The term "the" as used in the specification and in the claims, is used in the sense of "including" and "involving."

[0039] Excessive ROS is closely related to the occurrence and development of inflammatory diseases (such as inflammatory bowel disease, arthritis and pneumonia, etc.), so removing ROS is an effective strategy for treating inflammatory diseases. Using nanorobots as drug carriers to deliver anti-inflammatory molecules to inflammatory sites is a feasible approach, however, the targeting efficiency of existing nanorobots to the diseased site in the body is limited by many factors, so that its targeting efficiency has not been significantly improved relative to passive diffusion of nanomedicine carriers, which is not conducive to practical application. Therefore, the present application develops a nanorobot for targeted treatment of inflammatory diseases, the structure of which is specifically as follows: it comprises a nanoparticle and at least two enzymes and anti-inflammatory molecules connected to the nanoparticle; 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 site and control the movement direction of the nanorobot, and the nanorobot is driven in the physiological medium and enriched in the inflammatory site under the synergistic action of the above two enzymes, and the anti-inflammatory molecules are used to remove active oxygen in the inflammatory site.

[0040] The present application significantly improves the chemotactic sensitivity and targeting efficiency of the nanorobot to the inflammatory site in the physiological medium through the synergistic action of the two enzymes, so as to efficiently and accurately deliver the anti-inflammatory molecules to the inflammatory site, and the anti-inflammatory molecules achieve the effect of treating inflammatory diseases by removing ROS.

[0041] Some specific examples are listed below, it should be noted that the examples described below are exemplary and are used to explain the present application, and cannot be understood as limiting the present application. If the specific technology or condition is not specified in the following examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction; if the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by market purchase.

[0042] Example 1 This example provides a nanorobot for targeted treatment of inflammatory diseases, the structure and mechanism of action of which are as shown in Figure 1 The nanorobot uses gold nanoparticles as the main body, and urease, catalase and superoxide dismutase are assembled on one side of the gold nanoparticles by using polyethylene glycol, biotin and streptavidin.

[0043] In the nanorobot of the present example, the catalytic reaction of urease with urea in the body fluid (such as blood) is utilized to effectively drive the nanorobot in the biological body environment, the chemotaxis of the nanorobot is generated by utilizing the hydrogen peroxide concentration gradient of the inflammatory site (i.e. chemotactic source) sensed by the hydrogen peroxide enzyme, and the synergistic effect of urease and hydrogen peroxide enzyme enables the nanorobot to be efficiently and accurately enriched at the inflammatory site through blood circulation; after being enriched at the inflammatory site, the active oxygen is efficiently removed through the cascade reaction of superoxide dismutase and hydrogen peroxide enzyme.

[0044] Example 2 The present example provides a method for preparing the SOD / CAT / URE driven nanorobot of the structure shown in Example 1, which specifically comprises the following steps: (1) Preparation of nanogold of different sizes.

[0045] Synthesis of 13 nm nanogold: 100 mL of deionized water and a certain amount of 100 mM chloroauric acid aqueous solution were added to a 250 mL three-necked round-bottom flask, and after stirring and heating in a 150 °C oil bath for 15 minutes, a certain amount of 38.8 mM sodium citrate aqueous solution was added and heated for 10 minutes.

[0046] Primary growth of nanogold: 142.5 mL of deionized water, a certain amount of 60 mM sodium citrate aqueous solution and a certain amount of 13 nm nanogold were sequentially added to a three-necked round-bottom flask, and after stirring and preheating in a 90 °C oil bath for 10 minutes, a certain amount of 25 mM chloroauric acid aqueous solution was added in two portions and heated for 30 minutes.

[0047] Subsequent growth of nanogold: 55 mL of the above solution, 53 mL of deionized water and a certain amount of 60 mM sodium citrate were sequentially added to a three-necked round-bottom flask, and after stirring and heating to 90 °C, a certain amount of 25 mM chloroauric acid aqueous solution was added in two portions and reacted for 30 minutes. By controlling the number of growth times, nanogold particles of different sizes can be obtained.

[0048] In the present example, nanogold with a particle size of 90 nm is selected for subsequent experimental operations, and in other examples, nanogold with other desired particle sizes can also be selected for subsequent operations.

[0049] (2) Preparation of asymmetric Au-PS eccentric particles.

[0050] 16.5 mL of ethanol, 2.5 mL of deionized water, 1 mL of 50 mg / mL potassium persulfate aqueous solution and 1 mL of 6 mg / mL sodium p-styrenesulfonate aqueous solution were sequentially added to a 25 mL three-necked flask, and after stirring and preheating in a 70 °C oil bath for 10 minutes, a mixture of styrene and divinylbenzene (200:1, v / v) was added to the flask and reacted, 3 mL of nanogold was added after 2 minutes, and the reaction was continued for 4 hours. Asymmetric Au-PS eccentric particles were obtained by centrifugation.

[0051] (3) Preparation of biotinylated superoxide dismutase, catalase and urease.

[0052] The superoxide dismutase solution was mixed with the biotinylating reagent and shaken at room temperature for 2 hours. It was washed with deionized water 5 times and re-dispersed in a PB solution and stored at 4°C for standby. The biotinylation of catalase and urease was the same as that of superoxide dismutase, which will not be repeated here.

[0053] (4) Preparation of SOD / CAT / URE nanorobot.

[0054] The obtained Au-PS particles were dispersed in 5 mL ultrapure water, 1 mL Au-PS solution was taken, 0.1 mL 10 mg / mL Biotin-PEG-SH solution was added and shaken; 1 mL N,N-dimethylformamide was added to the sample, which was dispersed in 500 μL deionized water, 0.1 mL 10 mg / mL mPEG-SH solution was added, and shaken and then dispersed in a PB solution (10 mM, pH=7.4). 100 μL of the above solution was taken and added to 100 μL streptavidin solution (0.02 mg / mL), which was shaken and then dispersed in a PB solution (10 mM, pH=7.4). 100 μL of the above solution was taken and added to 100 μL biotinylated superoxide dismutase solution and shaken. Biotinylated catalase and urease were assembled on the surface of the gold nanoparticles by repeating this step. After the reaction was completed, the SOD / CAT / URE driven nanorobot was obtained by washing. In the nanorobot of this embodiment, the amount ratio of SOD, CAT and URE was 4:1:1.

[0055] Figure 2 The transmission electron microscope (TEM) image and element analysis image (scale: 100 nm) of the SOD / CAT / URE driven nanorobot prepared in this embodiment are as follows, Figure 2 A is the bright field image of the SOD / CAT / URE driven nanorobot under the STEM mode of the transmission electron microscope, Figure 2 B is the distribution of gold elements, sulfur elements, nitrogen elements, iron elements and zinc elements under the dark field and element analysis. According to the image, superoxide dismutase, catalase and urease are asymmetrically distributed on one side of the nanorobot, forming an asymmetric structure.

[0056] Example 3 With reference to Example 2, different amount ratios of biotinylated catalase and biotinylated urease were used in step (4) to prepare SOD / CAT / URE driven nanorobots with different ratios of catalase and urease.

[0057] The movement ability of the nanorobots obtained in Example 2 and this example in a physiological concentration urea solution was detected, and the experimental process and detection results are as follows: The nanorobots were placed in a 10 mM urea solution, and the diffusion coefficient (MSD (mean square displacement) = |r(t) - r(0) 2 , wherein r is the position at a certain time, and the diffusion coefficient is MSD / 4t) was tested to detect the influence of the relative amount of catalase and urease on the movement ability of the nanorobots.

[0058] In the nanorobots of the present application, urease provides the main driving ability, and hydrogen peroxide provides the chemotactic turning function. The test movement performance of the nanorobots with different proportions of catalase and urease is shown in Figure 3 , and the driving ability is the strongest when the proportion of catalase and urease is 1:1. Gradually reducing the proportion of urease will lead to a decrease in driving ability, and it is shown that the nanorobots of the present application can be effectively driven in the urea concentration of the human body (~10 mM).

[0059] Example 4 This example takes the constructed ulcerative colitis mouse model as an example to detect the targeting efficiency of the nanorobots of the present application to the inflammatory sites in vivo.

[0060] The method for establishing the ulcerative colitis mouse model is as follows: sodium dextran sulfate (2.5% w / v) is used to induce for 7 days.

[0061] The SOD / CAT / URE driven nanorobots prepared in Example 2 and other nanorobots were respectively dissolved in a PBS physiological buffer solution to prepare different nanorobot injection solutions. Then the nanorobots were injected into the colitis mice in vivo through intravenous injection at an injection amount of 200 μL, 1.5 mg / kg, and the mice were killed after 3, 6, 12 and 24 hours, respectively. The gold content in the colon tissue was detected by ICP-MS, and then the targeting efficiency of the nanorobots to the colitis was calculated.

[0062] The detection results are shown in Figure 4 , wherein Figure 4 A is the targeting efficiency of the nanorobots at different times, Figure 4 B is the targeting efficiency of different control groups at 6 hours. The targeting uptake efficiency (%ID / g) = (the gold content in the colon detected by ICP-MS ÷ the gold content of the injected nanorobots × 100%) ÷ the weight of the colon. It can be seen from Figure 4 that the targeting efficiency of the SOD / CAT / URE driven nanorobots prepared in Example 2 to the colitis reaches saturation after intravenous injection for 6 hours, and the targeting efficiency is 5.01%ID / g, which is 28 times that of inert particles and 25 times that of SOD / CAT driven nanorobots.

[0063] In this embodiment, the preparation methods of inert particles, URE-driven nanorobots, and SOD / CAT-driven nanorobots are the same as in Embodiment 2, with the only difference being that: the URE, CAT, and SOD coupled in the inert particles are all deactivated by high temperature; the CAT and SOD coupled in the URE-driven nanorobots are all deactivated by high temperature; and the URE coupled in the SOD / CAT-driven nanorobots is deactivated by high temperature.

[0064] Example 5 Taking the nanorobot prepared in Example 2 as an example, this example tested the therapeutic effect of the nanorobot on inflammatory bowel disease.

[0065] The SOD / CAT / URE driven nanorobots and other nanorobots (same as in Example 3) prepared in Example 2 were dissolved in PBS physiological buffer solution to prepare nanorobot injection solutions. The injection solutions were then administered intravenously to colitis mice at a dose of 200 μL (1.5 mg / kg) every other day for four days. The mice were then euthanized, and the colon, major organs (heart, liver, spleen, lungs, and kidneys), and blood were collected. The colon length was measured and photographed.

[0066] Figure 5 The therapeutic effect of SOD / CAT / URE-driven nanorobots in mice with colitis. Figure 5 A is a picture of the colon and Figure 5 B represents the length of the colon. Figure 5 C is a hematoxylin-eosin stained pathological section of colon tissue (scale bar is 200 μm). According to... Figure 5 As can be seen, after treatment with the nanorobots of this invention, the colon length of mice returned to normal (6.6±0.3cm), and the typical inflammatory symptoms of colon shortening and colon damage were significantly improved, effectively alleviating the clinical symptoms of colitis mice.

[0067] Simultaneously, blood and major organs were collected from healthy mice and mice treated with SOD / CAT / URE-driven nanorobots for biosafety assessment.

[0068] Test results as follows Figure 6 As shown, Figure 6 A represents the blood biochemical parameters of healthy mice and mice after the completion of nanorobot treatment. Figure 6 B shows hematoxylin-eosin stained histological sections of major organs from healthy mice and mice after nanorobot treatment. Figure 6 It was found that the blood biochemical indicators of mice treated with nanorobots were not significantly different from those of healthy mice, and no obvious systemic toxicity was observed in the hematoxylin-eosin stained sections of the heart, liver, spleen, lungs and kidneys.

[0069] The raw materials listed in the present application, and the upper and lower limits, interval values of each raw material of the present application, and the upper and lower limits, interval values of process parameters can all realize the present application, and examples are not listed one by one.

[0070] In summary, the nanorobot provided by the present application based on the synergistic effect of double enzymes can be efficiently and accurately enriched in the inflammatory site in the blood circulation system, and the targeting efficiency is significantly higher than that of inert particles and single enzyme driven nanorobots; at the same time, the cascade reaction of catalase and superoxide dismutase is used to efficiently remove active oxygen, so as to significantly improve the effect of treating inflammatory diseases (such as colitis).

[0071] It should be noted that the above examples are only a part of the embodiments of the present application but not all the embodiments, and are used to illustrate the technical solutions of the present application but not to limit; based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

Claims

1. A nanorobot for targeted therapy of inflammatory diseases, characterized in that, The invention includes nanoparticles and at least two enzymes and an anti-inflammatory molecule 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 site of inflammation and control the direction of movement of the nanorobot, and the anti-inflammatory molecule is used alone or in synergy with any of the enzymes to scavenge reactive oxygen species at the site of inflammation.

2. The nanorobot according to claim 1, characterized in that, The enzymes used to drive nanorobots in physiological media are urease, glucose oxidase, and / or other enzymes that use endogenous substances in physiological media as substrates.

3. The nanorobot according to claim 1, characterized in that, The enzyme used to sense inflammatory sites and control the direction of movement of nanorobots is an enzyme that uses biomarkers released from inflammatory sites as substrates, including hydrogen peroxide.

4. The nanorobot according to claim 1 or 3, characterized in that, The anti-inflammatory molecule is any one or more of the following: biological enzymes, nanozymes, and anti-inflammatory small molecule drugs.

5. The nanorobot according to claim 4, characterized in that, The anti-inflammatory molecule is superoxide dismutase, and the enzyme used to sense inflammatory sites and control the movement direction of nanorobots is catalase.

6. The nanorobot according to claim 5, characterized in that, The enzyme used to drive nanorobots in physiological media is urease.

7. The nanorobot according to claim 1, characterized in that, The nanoparticles are any one of gold nanoparticles, silicon dioxide, liposomes, magnetic particles, and polydopamine.

8. The nanorobot according to claim 1, characterized in that, The motion diffusion coefficient of the nanorobot is ≥1.0 μm 2 / s.

9. The use of the nanorobots for targeted therapy of inflammatory diseases as described in any one of claims 1-8 in the preparation of medicaments for treating inflammatory diseases.

10. The application according to claim 9, characterized in that, The drug can be administered via any one of the following methods: intravenous injection, intraperitoneal injection, intramuscular injection, or oral administration.

11. The application according to claim 9, characterized in that, The inflammatory diseases mentioned include inflammatory bowel disease.

12. A drug for targeted treatment of inflammatory diseases, characterized in that, It contains the nanorobots described in any one of claims 1-8.

13. The medicament according to claim 12, characterized in that, It contains pharmaceutically acceptable excipients.

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