Nanomotor capable of realizing efficient targeted enrichment in tissues with inflammatory diseases and application of nanomotor

Abstract

The invention discloses a nano-motor capable of realizing efficient targeted enrichment in inflammatory disease tissues and preparation and application thereof, the nano-motor is mainly formed by connecting two or more enzymes to nano-particles, at least one enzyme is used for driving the motor in a physiological medium, and at least one enzyme is used for driving the motor in the physiological medium. And at least another enzyme is used for sensing an inflammation focus part and controlling the movement direction of the motor. According to the invention, the synergistic effect of two enzymes is creatively utilized, so that the chemotactic sensitivity of the nano-motor and the targeting efficiency of the nano-motor on a focus part are remarkably improved; under better conditions, compared with a traditional single-enzyme chemotactic motor, the chemotactic ability of the nano motor is improved by more than 10 times, the targeting efficiency is remarkably improved by more than 20 times, and compared with an inert carrier, the targeting efficiency of the nano motor is remarkably improved by more than 200 times. The nano motor has ultrahigh plasticity, the preparation method is simple and efficient, and the nano motor has important significance in application of the micro-nano motor in the field of biological medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of micro-nano devices, and particularly relates to a nano-motor capable of efficiently targeting and enriching in inflammatory disease tissues, and a preparation method and application thereof. Background Art

[0002] A micro-nano motor is a micro-nano device at the nano-scale that can convert other forms of energy into kinetic energy and can move autonomously. Due to such characteristics, micro-nano motors are expected to revolutionize the field of active targeted drug delivery as drug delivery carriers.

[0003] However, the micro-nano motors reported currently cannot be effectively driven in the blood circulation system, and have weak chemotactic sensitivity, making it difficult to effectively respond to the chemical substance concentration gradient at the lesion site. At the same time, the targeting efficiency to the diseased site is not significantly improved compared with inert carriers. The above problems greatly limit the application of nano-motors in the biomedical field. Summary of the Invention

[0004] In view of this, the present invention aims to provide a chemotactic nano-motor that can be effectively driven in the blood circulation system and can efficiently target and enrich at the lesion site, so as to achieve precise and efficient drug delivery in vivo.

[0005] The technical solution of the present invention is specifically as follows:

[0006] The first aspect of the present invention provides a nano-motor capable of efficiently targeting and enriching in inflammatory disease tissues, which is mainly composed of two or more enzymes connected to nanoparticles, wherein at least one enzyme is used to drive the motor in a physiological medium, and at least another enzyme is used to sense the inflammatory lesion site and control the movement direction of the motor.

[0007] The inflammatory diseases described in the present invention include, but are not limited to, inflammatory diseases such as tumors, enteritis, pneumonia, arthritis, nephritis, etc. The enzymes described in the present invention can be biological enzymes and / or nano-enzymes, as long as they can drive the motor or control the movement direction of the motor through a catalytic reaction system in a physiological medium. The physiological media described in the present invention include, but are not limited to, blood.

[0008] In the nano-motor described in the present invention, the nanoparticles include, but are not limited to, nano-gold with different particle sizes, silicon dioxide (such as mesoporous silicon dioxide, etc.), liposomes, magnetic particles (such as magnetite, ferric oxide, etc.), polydopamine, etc. In some embodiments of the present invention, the nanoparticles are nano-gold.

[0009] In the nano-motor described in the present invention, the size of the nano-motor is 10-1000 nm, preferably 20-200 nm. For example, in some embodiments of the present invention, the particle size of the prepared nano-motor is between 90-100 nm.

[0010] In the nanomotor of the present invention, the enzyme used to drive the motor in a physiological medium can be one or more of urease, glucose oxidase, and other bioenzymes or nanoenzymes that use endogenous substances in the physiological medium as substrates. For example, in some embodiments of the present invention, the enzyme used to drive the motor in blood is urease, which catalyzes a reaction using urea in human blood as a substrate, thereby providing driving force for the nanomotor.

[0011] In the nanomotor of the present invention, the enzyme used to sense the inflammatory lesion site and control the movement direction of the motor is a bioenzyme or nanoenzyme that uses a biomarker released from the inflammatory lesion site as a substrate. For example, in some embodiments of the present invention, the inflammatory disease is a tumor, and the enzyme is catalase that uses hydrogen peroxide released from tumor cells as a substrate. Based on the specific sensing function of this enzyme for the lesion site, the present invention realizes the steering function of the nanomotor. Therefore, in practical applications, catalase can be replaced with other responsive components such as bioenzymes (e.g., glucose oxidase) and nanoenzymes (e.g., iron tetroxide, cerium dioxide) according to different targeted lesion sites.

[0012] In some embodiments of the present invention, the enzyme used to drive the motor in a physiological medium is urease, and the enzyme used to sense tumor cells at the lesion site and control the movement direction of the motor is catalase. Urease provides the main driving ability for the nanomotor, and catalase provides the chemotactic steering function. Experimental results show that the movement performance of the nanomotor can be adjusted by changing the ratio of urease to catalase.

[0013] In the nanomotor of the present invention, the connection mode of the enzyme used to drive the motor in a physiological medium and the enzyme used to sense the inflammatory lesion site and control the movement direction of the motor on the nanoparticle can be any one of the following:

[0014] 1) The enzyme used to drive the motor in a physiological medium and the enzyme used to sense the inflammatory lesion site and control the movement direction of the motor are connected in layers on the same side of the nanoparticle;

[0015] 2) The enzyme used to drive the motor in a physiological medium and the enzyme used to sense the inflammatory lesion site and control the movement direction of the motor are connected in the same layer on the same side of the nanoparticle;

[0016] 3) The enzyme used to drive the motor in a physiological medium and the enzyme used to sense the inflammatory lesion site and control the movement direction of the motor are connected in layers or in the same layer on the entire surface of the nanoparticle.

[0017] In the above two cases of 1) and 2), the assembly positions of the two enzymes are consistent relative to the nanoparticles and are only connected to partial regions of the nanoparticles, forming an asymmetric structure. In the above case of 3), the two enzymes are directly connected to the entire surface of the nanoparticles, forming a relatively symmetric structure compared with 1) and 2).

[0018] Preferably, in the above two cases of 1) and 2), the other side of the nanomotor without enzymes has functional molecules for drug loading. The functional molecules include but are not limited to polyethylene glycol, and the drugs carried by the functional molecules include but are not limited to organic small molecules (such as DOX, etc.), biological macromolecules (antibodies, vaccines, antigens), etc.

[0019] The second aspect of the present invention provides a preparation method of the nanomotor in the above two cases of 1) and 2). Taking the nanomotor containing two enzymes and the nanoparticle being nanogold as an example, the preparation method includes the following steps:

[0020] S1. Prepare Janus nanogold particles;

[0021] S2. Biotinylate enzyme A and enzyme B to obtain biotinylated enzyme A and biotinylated enzyme B. Enzyme A is used to drive the motor in a physiological medium, and enzyme B is used to sense tumor cells at the lesion site and control the movement direction of the motor;

[0022] S3. Gradually couple biotinylated enzyme B and biotinylated enzyme A on the same side of the Janus nanogold particles by using streptavidin.

[0023] In the preparation method of the present invention, step S1 includes the following process:

[0024] S11. Prepare nanogold by the seed growth method;

[0025] S12. After mixing and preheating potassium persulfate, sodium 4-styrenesulfonate, water and ethanol, add a mixed solution of styrene and divinylbenzene (DVB), and then add the nanogold prepared in step S11 for reaction to obtain Janus nanogold particles.

[0026] In the preparation method of the present invention, it is convenient to obtain nanogold particles with different sizes by controlling the number of growth times when preparing nanogold by the seed growth method. In an embodiment of the present invention, nanogold particles of about 90 nm are selected to prepare Janus nanogold particles. In other embodiments, nanogold particles with other required particle sizes can also be selected for subsequent operations. The methods for preparing nanomotors using nanogold with different particle sizes are the same.

[0027] In some embodiments of the present invention, biotinylated enzyme A and biotinylated enzyme B are prepared on the same side of the nanomotor by a hierarchical assembly method. First, enzyme B with a steering function is assembled, and then enzyme A providing driving ability is assembled. At this time, step S3 of the preparation method includes the following process:

[0028] S31. Disperse Janus gold nanoparticles in water, add HO-PEG-SH (hydroxy-polyethylene glycol-thiol) solution and mix. Then add N,N-dimethylformamide (DMF) and SH-PEG-Biotin (thiol-polyethylene glycol-biotin), and mix with streptavidin and disperse in PB solution. Add biotinylated enzyme B and react.

[0029] S32. Take the reaction solution obtained in step S31, mix it with streptavidin (SA) and disperse in PB solution. Add biotinylated enzyme A and react, and then wash to obtain the nanomotor.

[0030] In some other embodiments of the present invention, biotinylated enzyme A and biotinylated enzyme B are simultaneously prepared on the same side of the nanomotor by a mixed coupling method. At this time, step S3 of the preparation method includes the following process:

[0031] Disperse Janus gold nanoparticles in water, add HO-PEG-SH (hydroxy-polyethylene glycol-thiol) solution and mix. Then add N,N-dimethylformamide (DMF) and SH-PEG-Biotin (thiol-polyethylene glycol-biotin), and mix with streptavidin and disperse in PB solution. Add a mixture of biotinylated enzyme B and biotinylated enzyme A and react.

[0032] The example data of the present invention show that when enzyme A and enzyme B are connected to the same side of the nanoparticle in a hierarchical or same-layer manner, the chemotactic ability of the nanomotor can be significantly improved. The difference is only in the ratio requirements for enzyme A and enzyme B. For example, when connected in a hierarchical manner, the driving ability is the strongest when the ratio of urease to catalase is 1:1, while when connected in the same layer, the amount of urease needs to be increased to achieve a similar technical effect.

[0033] The third aspect of the present invention provides a preparation method of the nanomotor in the case of 3). Taking the nanomotor containing two enzymes and the nanoparticle being gold nanoparticles as an example, the preparation method includes the following steps:

[0034] S1. Prepare gold nanoparticles by the seed growth method.

[0035] S2. Disperse the gold nanoparticles in water, add SH-PEG-Biotin, and mix with streptavidin and disperse in PB solution. Couple biotinylated enzyme B and biotinylated enzyme A separately or simultaneously, and then wash to obtain the nanomotor.

[0036] The fourth aspect of the present invention provides the application of the nano - motor described in the present invention as a drug delivery carrier in the preparation of drugs or medical devices. The experimental results of the present invention show that after the dual - enzyme nano - motor prepared by the present invention is intravenously injected into mice, it can resist the random and irregular Brownian motion of nanoparticles, be effectively driven in the blood circulation system, and the targeting efficiency to the lesion site is much higher than that of inert carriers and single - enzyme - driven nano - motors.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) Compared with existing chemotactic motors, the present invention innovatively utilizes the synergistic effect of dual enzymes, significantly improving the chemotactic sensitivity of the nano - motor and the targeting efficiency to inflammatory disease tissues; for example, compared with traditional single - enzyme chemotactic motors (such as catalase - chemotactic nano - motors), its chemotactic ability is increased by more than 10 times, and its targeting efficiency is significantly increased by more than 20 times; compared with inert carriers, its targeting efficiency is significantly increased by more than 200 times.

[0039] (2) In the nano - motor of the present invention, catalase can sense the concentration gradient of tumor cells, and the addition of urease improves the motility. The combination of the two can not only drive effectively but also chemotax, and at the same time, the motility is significantly improved, enabling the nano - motor to be effectively driven by the urea concentration in the human blood circulation system.

[0040] (3) Based on the nano - motor structure provided by the present invention, the types of carriers, functional molecules, and / or chemotactic bio - enzymes can be replaced as needed without affecting its function, thereby realizing targeted drug delivery to different diseases and different lesion sites (such as tumors, inflammations, etc.). That is, the nano - motor of the present invention has a wide range of applications and extremely high plasticity.

[0041] (4) The preparation method of the nano - motor of the present invention is simple and efficient. Due to the significantly improved targeting efficiency of the nano - motor, the resulting toxic and side effects are significantly reduced, and the drug treatment effect is also significantly improved. Description of the Drawings

[0042] Figure 1 It is a schematic diagram of the structure and driving mechanism of the chemotactic nano - motor prepared in Example 1 of the present invention;

[0043] Figure 2 It is a transmission electron microscope image and elemental analysis diagram of the chemotactic nano - motor prepared in Example 1 of the present invention;

[0044] Figure 3 It is a movement trajectory diagram of the nano - motor in different concentration urea solutions in Example 2 of the present invention, a graph of the mean square displacement and diffusion coefficient changing with urea concentration, and a graph of the diffusion coefficient change with different ratios of urease to catalase;

[0045] Figure 4 This is a comparison chart of the chemotactic effects of the nanomotor in Example 3 of the present invention, other single-enzyme-driven motors, and inert carriers in a 3D chip;

[0046] Figure 5 This is a comparison chart of the targeting efficiencies of the nanomotor in Example 4 of the present invention, other single-enzyme-driven motors, and inert carriers in in-vivo experiments;

[0047] Figure 6 This is a comparison chart of the chemotactic effects of the nanomotor in a 3D chip under different ratios of urease to catalase in Example 6 of the present invention;

[0048] Figure 7 This is a schematic diagram of the structure and driving mechanism of the chemotactic nanomotor prepared in Example 7 of the present invention;

[0049] Figure 8 This is a transmission electron microscope image and elemental analysis chart of the chemotactic nanomotor prepared in Example 7 of the present invention;

[0050] Figure 9 This is the chemotactic effect chart of the nanomotor in a 3D chip in Example 8 of the present invention. Detailed implementation manners

[0051] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific implementation manners described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms "including" and "having" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusion.

[0053] The terms "one kind", "another kind", "enzyme A", and "enzyme B" in the specification and claims of the present invention are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly specifying a specific order or primary-secondary relationship of the indicated technical features.

[0054] Existing reported micro-nano motors (such as chemotactic motors driven by GSH, pH, hydrogen peroxide, etc.) cannot be effectively driven in the blood circulation system, have weak chemotactic sensitivity, and there is no obvious improvement in the targeting efficiency to the diseased site compared with inert carriers.

[0055] To solve the technical problems that limit the practical application of micro-nano motors, the present invention provides a nano-motor with a multi-enzyme structure. This nano-motor can be effectively driven in the blood circulation system and can be efficiently targeted and enriched at the inflammatory lesion site. Its structure is specifically as follows: It is mainly composed of two or more enzymes connected to nanoparticles. At least one enzyme is used to drive the motor in the physiological medium, and at least one other enzyme is used to sense tumor cells at the lesion site and control the movement direction of the motor. Through the synergistic effect of the two enzymes, the chemotactic sensitivity and targeting efficiency of the nano-motor are significantly improved.

[0056] In the present invention, the enzyme used to drive the motor in the physiological medium is preferably urease but not limited to urease, as long as this enzyme can effectively drive the nano-motor in vivo. The type of enzyme used to sense the inflammatory lesion site and control the movement direction of the motor in the present invention needs to be specifically selected according to the lesion site or disease type.

[0057] For the convenience of description below, the nano-motor in some embodiments of the present invention is taken as an example for illustration.

[0058] Please refer to Figure 1 , Figure 1 which is a schematic diagram of the structure and driving mechanism of the chemotactic nano-motor prepared in Example 1 of the present invention. This nano-motor uses nano-gold as the main body of the motor. On one side of the nano-gold, urease and catalase are assembled using polyethylene glycol, biotin, and streptavidin. The polyethylene glycol on the other side of the nano-gold is used for drug loading; the catalase in the nano-motor is used to sense the signal chemotaxis, and the urease uses body fluid to drive the motor, thereby achieving the efficient targeted enrichment of the nano-motor at the lesion site.

[0059] Please refer to Figure 7 , Figure 7 which is a schematic diagram of the structure and driving mechanism of the chemotactic nano-motor prepared in Example 7 of the present invention. This nano-motor uses nano-gold as the main body of the motor. On the entire surface of the nano-gold, urease and catalase are assembled using polyethylene glycol, biotin, and streptavidin; the catalase in the nano-motor is used to sense the signal chemotaxis, and the urease uses body fluid to drive the motor, thereby achieving the efficient targeted enrichment of the nano-motor at the lesion site.

[0060] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention and cannot be construed as a limitation to the present invention.

[0061] For those not specifying specific technologies or conditions in the following embodiments, they are all carried out according to the technologies or conditions described in the literature in this field or according to the product instructions; for the reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0062] Example 1

[0063] In this embodiment, the nanomotor was prepared through the following steps:

[0064] (1) Preparation of gold nanoparticles.

[0065] Synthesis of 10 nm gold nanoparticles: Take 1 mM chloroauric acid solution, stir and heat it in an oil bath until boiling, then heat for another 10 min after boiling. Quickly add 38.8 mM sodium citrate aqueous solution, continue heating and reacting for 10 min, and then stop heating.

[0066] Primary growth of gold nanoparticles: Take 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 and add them to a three-necked flask. Heat and stir at 90 °C. After the system is stable, add a certain amount of 25 mM chloroauric acid solution in two portions. After 30 min, the primary growth of gold nanoparticles is completed.

[0067] Subsequent growth of gold nanoparticles: Take out about 1 / 3 of the sample solution, add an equal amount of deionized water to the original three-necked flask. After the temperature is stable, add a certain amount of 60 mM sodium citrate solution. After stabilization, add a certain amount of 25 mM chloroauric acid solution in two portions and react for 30 min.

[0068] 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 were selected for subsequent experimental operations.

[0069] (2) Preparation of asymmetric Au-PS eccentric particles.

[0070] Add 1 mL of 50 mg / mL potassium persulfate solution, 1 mL of 6 mg / mL sodium 4-styrenesulfonate solution, 2.5 mL of water, and 16.5 mL of ethanol to a three-necked flask. After preheating at 70 °C for 10 minutes, then add 200 μL of the mixed solution (V styrene :V DVB = 200:1). After 2 minutes, add 3 mL of gold nanoparticles and continue reacting for 4 hours. Centrifuge and collect to obtain asymmetric Au-PS eccentric particles.

[0071] (3) Preparation of biotinylated urease and biotinylated catalase.

[0072] Mix the catalase solution with the biotinylation reagent and shake well at room temperature. Wash it with deionized water 5 times and dissolve it in PB solution for use at 4 °C. Mix the urease solution with the biotinylation reagent and shake well at room temperature. Wash it with deionized water 5 times and dissolve it in PB solution for use at 4 °C.

[0073] (4) Preparation of chemotactic nanomotors.

[0074] Disperse the Au-PS eccentric particles obtained in step (2) in 6 mL of ultrapure water. Take 2 mL of the Au-PS solution, add 0.1 mL of HO-PEG-SH solution (10 mg / mL), and mix and oscillate; add 1 mL of N,N-dimethylformamide to the sample, disperse it in 500 μL of ultrapure water, add 100 μL of SH-PEG-Biotin solution (10 mg / mL), and after oscillation, disperse it in PB solution (10 mM, pH = 7.4). Take 100 μL of the above solution and add 100 μL of streptavidin solution (0.02 mg / mL), mix and disperse it in PB solution (10 mM, pH = 7.4); take 100 μL of the above solution and add 100 μL of biotinylated catalase solution and oscillate to react. Subsequently, continue to couple SA and an equal amount of biotinylated urease. After the reaction is completed, wash to obtain the nanomotors.

[0075] Figure 2 Figure 4 shows the transmission electron microscope image and elemental analysis image of the nanomotors prepared in this example. Figure 2 A is the bright-field image of the chemotactic nanomotors in the STEM mode of the transmission electron microscope. Figure 2 B is the distribution of gold, nitrogen, and iron elements under dark field and elemental analysis. According to the image, catalase and urease are evenly distributed on one side of the motor, forming an asymmetric structure.

[0076] Example 2

[0077] In this example, the motility of the nanomotors obtained in Example 1 in urea solutions with different concentrations was detected. The experimental process and detection results are as follows:

[0078] Place the nanomotors prepared in Example 1 in urea solutions of 0, 1, 5, 10, 15, 20, 25, and 50 mM respectively. Their movement trajectories are as shown in Figure 3 A, and their mean square displacement changes are as shown in Figure 3 B, and their diffusion coefficient changes are as shown in Figure 3 C. It can be seen from the figure that as the urea concentration increases, the moving distance of the motor gradually increases, and the slope of the mean square displacement and the diffusion coefficient also gradually increase, and can be effectively driven at the human urea concentration (~10 mM).

[0079] Referring to Example 1, in this example, by changing the amounts of biotinylated catalase and biotinylated urease, nanomotors with different ratios of urease to catalase were obtained, and the influence of the relative amounts of urease and catalase on the motility of the nanomotors was detected. In the nanomotors of the present invention, urease provides the main driving ability, while hydrogen peroxide only provides the chemotactic steering function. The results of testing the motility performance with different ratios of urease to catalase are as shown in Figure 3As shown in D: when the ratio of urease to catalase is 1:1, the driving ability is the strongest. Gradually reducing the ratio of urease will cause a significant decline in the driving ability.

[0080] Example 3

[0081] In this example, an inert carrier, a urease-driven motor, and a catalase-driven motor were used as controls to detect the chemotactic ability of the nanomotors of the present invention towards tumor tissues in mouse plasma. The preparation methods of the inert carrier, the urease-driven motor, and the catalase-driven motor refer to Example 1, with the only difference being that: both urease and catalase in the inert carrier were heat-inactivated, catalase in the urease-driven motor was heat-inactivated, and urease in the catalase-driven motor was heat-inactivated.

[0082] The experimental process and test results of this example are as follows:

[0083] The nanomotors prepared in Example 1 were placed in a 3D chip, and the changes in the absorbance of the motors were observed in regions 3, 4, and 5 respectively to determine the chemotactic ability of the motors. The design of the 3D chip is as Figure 4 shown in A. The left reservoir area was filled with a gel matrix containing MCF-7 cells (tumor cells), while the right reservoir was filled with a gel matrix containing MCF-10A cells (normal cells), and the channel was filled with mouse plasma. Figure 4 B shows the comparison of the chemotactic abilities of different control groups. The chemotactic nanomotors prepared in Example 1 have selectivity and can spontaneously move towards the cancer cell area but not towards normal cells. The chemotactic ability of this motor is significantly enhanced, more than 10 times stronger than that of traditional single-enzyme chemotactic nanomotors.

[0084] Example 4

[0085] In this example, an inert carrier, a urease-driven motor, and a catalase-driven motor were used as controls to detect the targeting efficiency of the nanomotors of the present invention through in-vivo experiments on tumor-bearing mice. The inert carrier, the urease-driven motor, and the catalase-driven motor were the same as in Example 3. The experimental process and test results are as follows:

[0086] The nanomotors prepared in Example 1 were injected into tumor-bearing mice via intravenous injection. After 24 hours, the mice were sacrificed, and the targeting efficiency of the motors towards the tumor was tested by ICP-MS. The gold content in the tumor tissue was detected by ICP-MS. The targeting efficiencies of different control groups are as Figure 5 shown. The targeting efficiency of the chemotactic nanomotors prepared in Example 1 is as high as 69.09% ID / g, which is 209 times that of the inert carrier, 246 times that of the urease-driven motor with only the movement function, and 23 times that of the traditional catalase-driven motor.

[0087] Example 5

[0088] Different from Example 1, the nanomotors in this example were prepared through the following steps:

[0089] (1) Preparation of gold nanoparticles, which was the same as step (1) in Example 1.

[0090] (2) Preparation of asymmetric Au-PS eccentric particles, which was the same as step (2) in Example 1.

[0091] (3) Preparation of biotinylated urease and biotinylated catalase, which was the same as step (3) in Example 1.

[0092] (4) Preparation of chemotactic nanomotors. The difference from step (4) in Example 1 was only that after the nanoparticles were conjugated with streptavidin, biotinylated urease and biotinylated catalase were mixed simultaneously.

[0093] Compared with Example 1, in this example, urease and catalase were simultaneously linked to the nanomotors, and a monolayer dual-enzyme motor was obtained.

[0094] Example 6

[0095] Referring to Example 3, in this example, the chemotactic ability of the monolayer dual-enzyme motor prepared in Example 5 towards tumor tissues in mouse plasma was detected. The specific detection process and results were as follows:

[0096] The prepared nanomotors were placed in a 3D chip, and the changes in the absorbance of the motors were observed in regions 3, 4, and 5 respectively to determine the chemotactic ability of the motors. The design of the 3D chip was as Figure 4 shown in A. The left reservoir area was filled with a gel matrix containing MCF-7 cells (tumor cells), while the right reservoir was filled with a gel matrix containing MCF-10A cells (normal cells), and the channel was filled with mouse plasma. Figure 6 showed the comparison of the chemotactic abilities of different control groups. It can be seen from Figure 6 that by changing the ratio of urease to catalase to an appropriate value, the monolayer dual-enzyme motor was similar to the bilayer dual-enzyme motor. It could not only selectively move spontaneously towards the cancer cell region, but also significantly enhance its chemotactic ability.

[0097] Example 7

[0098] Different from Example 1, the nanomotors in this example were prepared through the following steps:

[0099] (1) Preparation of gold nanoparticles, which was the same as step (1) in Example 1.

[0100] (2) Preparation of biotinylated urease and biotinylated catalase, which was the same as step (3) in Example 1.

[0101] (3) Preparation of chemotactic nanomotors.

[0102] Disperse the gold nanoparticles obtained in step (1) in 6 mL of ultrapure water. Take 2 mL of the gold nanoparticle solution, add 100 μL of SH-PEG-Biotin solution (10 mg / mL), and after oscillation, disperse it in PB solution (10 mM, pH = 7.4). Take 100 μL of the above solution and add 100 μL of streptavidin solution (0.02 mg / mL), and after mixing, disperse it in PB solution (10 mM, pH = 7.4); take 100 μL of the above solution and add 100 μL of biotinylated catalase solution and oscillate for reaction. Subsequently, continue to couple SA and biotinylated urease. After the reaction is completed, wash to obtain the nanomotors.

[0103] Figure 7 For the schematic diagram of the structure and driving mechanism of the obtained symmetric chemotactic nanomotors, Figure 8 For the transmission electron microscope image and elemental analysis image of the nanomotors prepared in this example, Figure 8 A is the bright-field image of the chemotactic nanomotors in the STEM mode of the transmission electron microscope. Figure 8 B is the distribution of gold, nitrogen, and iron elements under dark field and elemental analysis. According to the image, catalase and urease are distributed on both sides of the motor, forming a symmetric structure (it should be noted that since the chemical reactions of the two enzymes are not completely the same, the symmetry described in this example is not completely symmetric, but a relatively symmetric state compared to the asymmetric structure of Example 1).

[0104] Example 8

[0105] Referring to Example 3, in this example, the chemotactic ability of the nanomotors prepared in Example 7 towards tumor tissues in mouse plasma was detected. The experimental process and detection results of this example are as follows:

[0106] Place the nanomotors prepared in Example 7 in a 3D chip, and observe the change in the absorbance of the motors in regions 3, 4, and 5 respectively to determine the chemotactic ability of the motors. The design of the 3D chip is as Figure 4 shown in A. 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), and the channel is filled with mouse plasma. Figure 8 Shows the chemotactic ability of this motor, and the chemotactic ability of this motor is significantly enhanced, more than 5 times stronger than that of traditional single-enzyme chemotactic nanomotors.

[0107] All the raw materials listed in the present invention, as well as the upper and lower limits, interval values of the raw materials of the present invention, and the upper and lower limits, interval values of the process parameters can achieve the present invention, and the examples are not listed one by one here.

[0108] In summary, the nanomotor provided by the present invention has ultra-high chemotactic sensitivity. Under optimal conditions, compared with traditional chemotactic motors, the sensitivity of the nanomotor of the present invention is increased by 10 times. Moreover, it has ultra-high targeting efficiency for tumor regions in vivo. Compared with inert carriers, the targeting efficiency of the nanomotor of the present invention is increased by more than 200 times. Compared with traditional chemotactic motors, the targeting efficiency of the nanomotor of the present invention is increased by more than 20 times. It can be seen that the present invention has important significance for the application of micro-nanomotors in the fields of biomedicine and micro-nano manipulation.

[0109] It should be noted that the above embodiments are only a part of the embodiments of the present invention rather than all of them, and are only used to illustrate the technical solutions of the present invention rather than to limit them; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

Claims

1. A nanomotor capable of highly targeted enrichment in inflammatory disease tissues, characterized in that, It is mainly composed of two or more enzymes linked to nanoparticles, where at least one enzyme is used to drive the motor in a physiological medium, and at least one other enzyme is used to sense the inflammatory lesion site and control the direction of motor movement.

2. The nanomotor capable of highly targeted enrichment in inflammatory disease tissues according to claim 1, wherein The size of the nanomotor is 10 - 1000 nm.

3. The nanomotor capable of highly targeted enrichment in inflammatory disease tissues according to claim 1, characterized in that, The enzymes used to drive the motor in a physiological medium include urease, glucose oxidase, and / or other enzymes that use endogenous substances in the physiological medium as substrates; the enzymes used to sense the inflammatory lesion site and control the direction of motor movement are enzymes that use biomarkers released from the inflammatory lesion site as substrates, and they include catalase.

4. The nanomotor capable of highly efficient targeted enrichment in inflammatory disease tissues according to claim 1, wherein Functional molecules for drug loading are linked to the nanomotor, and the functional molecules include polyethylene glycol.

5. Use of the nanomotor capable of highly targeted enrichment in inflammatory disease tissues as described in any one of claims 1 - 4 as a drug delivery carrier in the preparation of drugs or medical devices.

Images