Self-driven micro-nano motor with high chemotactic efficiency and preparation method and application thereof

Abstract

The invention relates to a self-driven micro-nano motor with high chemotactic efficiency and a preparation method and application thereof. The self-driven micro-nano motor comprises a carrier, and a responsive polymer chain and a biological enzyme which are connected to the surface of the carrier, the carrier is a micro-nano particle with rich active groups on the surface; a polymer in the responsive polymer chain is a high polymer material capable of responding to specific signal characteristics in a physiological environment; the biological enzyme has chemotaxis to a natural substrate in a physiological environment. In the self-driven micro-nano motor, the chemotaxis of the biological enzyme and the modulation of the responsive polymer chain on the surface property of the motor are combined, so that the autonomous target searching capability of the self-driven micro-nano motor is improved; according to the difference between a target spot and multiple microenvironments of a non-target area, gradient response to multiple signals in the target area can be realized, so that high chemotactic efficiency is achieved for a specific microenvironment, the problem of target missing caused by single signal interference in a complex environment is solved, and the targeting rate is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of micro-nano motors, and relates to a high-chemotactic efficiency self-driven micro-nano motor and a preparation method and application thereof. Background Art

[0002] Self-propelled micro-nanomotors are small in size and can use chemical energy in the surrounding environment to drive themselves. They have stronger penetration and barrier-crossing capabilities than particles that rely solely on Brownian motion for passive diffusion. They have significant advantages in application in closed or microscale environments and are expected to bring transformative technologies to biomedical fields including active drug transport, microsurgery, biosensors, and cell separation.

[0003] Unlike micro-nanomotors driven by external fields, self-driving micro-nanomotors do not require human intervention during their movement and are the easiest type of micro-nanomotors to achieve intelligence. However, the active targeting of self-driving micro-nanomotors currently developed mostly relies on their own chemotaxis to respond to the intrinsic signal gradients in the environment, such as the intrinsic glucose and hydrogen peroxide gradients in the physiological environment (Adv. Healthcare Mater. 2020, 9, 1901710; Small 2020, 16,2002732; Angew. Chem. Int. Ed. 2022, 61, e202116013). Although the targeting ability of self-driving micro-nanomotors has been improved to a certain extent, this single signal gradient response is particularly susceptible to complex environmental constraints. For example, high concentrations of hydrogen peroxide in the physiological environment exist simultaneously in inflammation and tumor areas. Only the concentration gradient of hydrogen peroxide in normal tissue and diseased tissue is used as the response signal (Mater. Chem. Front. 2021, 5, 4474-4501; Carbohyd. Polym. 2022, 285, 119252), which can easily cause the motor to miss the tumor lesion and affect its task completion. Therefore, it is crucial to design and construct self-driven micro-nanomotors to improve their chemotaxis efficiency in complex physiological environments and achieve high targeting rate and high utilization rate of micro-nanomotors to specific target areas, which is crucial for the development of a new generation of active drug delivery technology. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned technical deficiencies, provide a self-driven micro-nano motor with high chemotaxis efficiency and its preparation method and application, and solve the technical problems in the prior art that the target seeking of the self-driven micro-nano motor depends on a single signal gradient, has low chemotaxis efficiency, and is easy to miss the target.

[0005] In order to achieve the above technical purpose, the technical solution provided by the present invention is: In the first aspect, the present invention provides a self-propelled micro-nano motor with high chemotaxis efficiency, which includes a carrier, and a responsive polymer chain and a biological enzyme connected to the surface of the carrier; the carrier is a micro-nano particle with rich active groups on the surface; the polymer in the responsive polymer chain is a high molecular material that can respond to specific signal characteristics in the physiological environment; the biological enzyme has chemotaxis to natural substrates in the physiological environment.

[0006] In a second aspect, the present invention provides a method for preparing a self-propelled micro-nano motor with high chemotaxis efficiency, comprising the following steps: S1, dissolving a carrier and a reactant in a first solvent, adding an initiator dropwise to react, and separating an intermediate product after the reaction is completed; S2, mixing the intermediate product, a responsive monomer molecule, and a second solvent, adding a ligand and a metal halide, and using an atom transfer radical polymerization method to obtain a carrier with a surface modified with a responsive polymer chain; S3, using a glutaraldehyde chemical crosslinking method to connect a biological enzyme to the carrier with a surface modified with a responsive polymer chain to obtain a self-propelled micro-nano motor.

[0007] In a third aspect, the present invention provides an application of a self-propelled micro-nano motor with high chemotactic efficiency as a self-actuated actuator for targeted drug delivery.

[0008] Compared with the prior art, the beneficial effects of the present invention include: In the self-driving micro-nano motor of the present invention, the chemotaxis of biological enzymes and the modulation of the motor surface properties by responsive polymer chains are combined to enhance the autonomous targeting ability of the self-driving micro-nano motor; it has modulability and can respond to multiple signal gradients in the target area according to the differences in multiple microenvironments of the target point and the non-target area, thereby having high chemotaxis efficiency for a specific microenvironment, and overcoming the off-target problem caused by single signal interference in a complex environment, thereby improving the targeting rate; the micro-nano motor with high chemotaxis efficiency proposed by the present invention can be used for tumor-targeted drug delivery. The preparation method of the present invention is simple and has strong controllability. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is the infrared spectrum of the polydopamine nanoparticles in Example 1; Figure 2 It is a schematic diagram of the simulated environmental concentration gradient; Figure 3 is the chemotactic motion trajectory of the self-propelled nanomotor in Example 1 under different species concentration gradients; Figure 4 is the movement rate of the self-propelled nanomotor in Example 1 in solutions of different pH values; Figure 5 is the movement speed of the self-propelled nanomotor in Example 1 in solutions with different H2O2 concentrations; Figure 6is the contact angle of water droplets of different pH on the surface of the self-propelled nanomotor in Example 1; Figure 7 is the change in particle size of the self-propelled nanomotor in Example 1 in solutions with different pH values; Figure 8 is the movement rate of the self-propelled nanomotors with different chain lengths in Example 1 and Example 2 at pH=5; Fig. 9 is the chemotactic fluorescence intensity of the self-propelled nanomotor in Example 1 at different concentration gradients; Fig.10 This is a fluorescence image of the chemotaxis of different types of cells by the self-propelled nanomotor in Example 1. DETAILED DESCRIPTION

[0010] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0011] In view of the shortcomings of existing self-driving micro-nano motors that rely on a single signal gradient for target seeking, have low chemotaxis efficiency and are easily off-target, the present invention provides a self-driving micro-nano motor with high chemotaxis efficiency and a preparation method thereof. The self-driving micro-nano motor can respond to multiple signal gradients in a complex environment, has the characteristics of high chemotaxis efficiency and can be modulated on demand, thereby improving the targeting rate of the self-driving micro-nano motor to the target area.

[0012] In the first aspect, the present invention provides a self-propelled micro-nano motor with high chemotaxis efficiency, which includes a carrier, and a responsive polymer chain and a biological enzyme connected to the surface of the carrier; the carrier is a micro-nano particle with rich active groups on the surface; the polymer in the responsive polymer chain is a high molecular material that can respond to specific signal characteristics in the physiological environment; the biological enzyme has chemotaxis to natural substrates in the physiological environment.

[0013] The main mechanism of action of the present invention is: (1) The present invention uses micro-nanoparticles with rich amino and hydroxyl groups on the surface as carriers, which have more surface active sites and are conducive to the grafting of polymer chains and the loading of biological enzymes; (2) Using natural substrates in the physiological environment as "fuel" to drive the movement of biological enzymes, the surface is grafted with polymer chains that respond to specific physiological signals in the target environment. Through the chemotaxis of biological enzymes to high concentrations of "fuel" and the change in the hydrophilicity and hydrophobicity of responsive polymer chains, the surface properties of the motor are modulated to achieve chemotaxis-enhanced movement of self-propelled micro-nano motors to the target area, thereby improving the autonomous targeting ability of self-propelled micro-nano motors.

[0014] (3) The self-propelled micro-nanomotor obtained by the present invention is modulatable. Different biological enzymes or responsive polymers can be selected according to the differences in the multiple microenvironments of the target site and the non-target area to construct a self-propelled micro-nanomotor with high chemotactic efficiency for a specific microenvironment, thereby enhancing targeted drug delivery.

[0015] Preferably, the size of the carrier (micro-nanoparticles with rich active groups on the surface) is 50 nm to 5 µm.

[0016] Preferably, the carrier includes polydopamine with rich amino and hydroxyl groups on the surface. The loading of biological enzymes depends on the active sites on the surface of the carrier, so the present invention uses organic matter as a carrier, which is more conducive to the loading of biological enzymes.

[0017] Preferably, the biological enzyme includes any one or more of catalase, glucose oxidase, urease, superoxide dismutase, and lactate oxidase. The present invention can use natural substrates in a physiological environment as "fuel" to drive the movement of the corresponding biological enzyme, and the "fuel" is a natural substrate that can be catalyzed by the biological enzyme, such as hydrogen peroxide, glucose, urea, superoxide anion, lactic acid, glutathione, etc. at physiological concentrations. Correspondingly, the biological enzyme can also be selected from a variety of enzymes, such as glucose oxidase using glucose as fuel, catalase using hydrogen peroxide as fuel, urease using urea as fuel, superoxide dismutase using superoxide anion as fuel, etc.

[0018] Preferably, the content of biological enzyme loaded on the surface of the self-propelled micro-nanomotor is 200-900 mg / g, including but not limited to 200 mg / g, 220 mg / g, 260 mg / g, 280 mg / g, 300 mg / g, 350 mg / g, 400 mg / g, 450 mg / g, 500 mg / g, 550 mg / g, 600 mg / g, 650 mg / g, 680 mg / g, 700 mg / g, 720 mg / g, 750 mg / g, 800 mg / g, 850 mg / g, 900 mg / g and the like.

[0019] Preferably, the polymer in the responsive polymer chain is a high molecular material that can respond to any one or more specific signal characteristics of pH, temperature, ions, natural substrates (such as hydrogen peroxide, glucose, urea, superoxide anion, lactic acid, glutathione, etc.) in the physiological environment.

[0020] Further preferably, the polymer includes polydimethylaminoethyl methacrylate (PDMAEMA), which can respond to the pH in a physiological environment and perform hydrophilic-hydrophobic conversion within a pH range (pH = 5 to 8).

[0021] Preferably, the length of the responsive polymer chain is 10 to 100 nm, including but not limited to 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, etc.

[0022] In a second aspect, the present invention provides a method for preparing a self-propelled micro-nano motor with high chemotactic efficiency, comprising the following steps: S1, dissolving the carrier A and the reactants in a first solvent, adding an initiator dropwise to react, and separating to obtain an intermediate product B with surface-connected initiation sites after the reaction is completed; S2, mixing the intermediate product B, the responsive monomer molecule and the second solvent, adding the ligand and the metal halide, and using the atom transfer radical polymerization method to obtain the carrier C with the surface modified with the responsive polymer chain; S3, using glutaraldehyde chemical cross-linking method, connect the biological enzyme to the carrier C to obtain a self-driven micro-nano motor.

[0023] In the present invention, a reaction system is first constructed by mixing reactants with a carrier, and an initiator is added to form an initiation site on the carrier surface. Then, a ligand and a metal halide are used as a catalytic system to carry out controllable polymerization at the initiation site on the carrier surface to complete the modification of the responsive polymer chain on the surface. Then, a glutaraldehyde chemical cross-linking method is used to connect one side of the aldehyde groups at both ends of glutaraldehyde to the carrier of the surface-modified responsive polymer chain, and the other side is connected to a biological enzyme to obtain a self-driven micro-nano motor.

[0024] The polymer chain can provide a certain steric hindrance to the biological enzyme, which is beneficial to the loading of the enzyme. At the same time, the biological enzyme is easily affected by the environment and thus inactivated. The organic reagents required for atom transfer radical polymerization will affect the enzyme activity. Therefore, the present invention first grafts the responsive polymer chain and then loads the biological enzyme, effectively ensuring the activity of the biological enzyme.

[0025] Preferably, in step S1, the reactants include triethylamine and 2-dimethylaminopyridine, and the carrier A includes polydopamine nanoparticles; the ratio of carrier A, triethylamine and 2-dimethylaminopyridine is 8 g: (55-65) mL: (45-55) mL, including but not limited to 8 g: 55 mL: 45 mL, 8 g: 55 mL: 50 mL, 8 g: 55 mL: 55 mL, 8 g: 60 mL: 45 mL, 8 g: 65 mL: 45 mL, 8 g: 60 mL: 50 mL, 8 g: 60 mL: 55 mL, 8 g: 65 mL: 55 mL, etc.

[0026] Preferably, in step S1, the initiator is 2-bromoisobutyryl bromide; the ratio between carrier A and initiator is 8 g: (45-55) mL, including but not limited to 8 g: 45 mL, 8 g: 48 mL, 8 g: 50 mL, 8 g: 52 mL, 8 g: 55 mL, etc.

[0027] Preferably, in step S1, the ratio between carrier A and the first solvent is 8 g: (4000-6000) mL, including but not limited to 8 g: 4000 mL, 8 g: 4500 mL, 8 g: 5000 mL, 8 g: 5500 mL, 8 g: 6000 mL, etc.; the first solvent includes tetrahydrofuran.

[0028] Preferably, in step S1, the reaction conditions are: temperature of 10-30°C, including but not limited to 10°C, 15°C, 20°C, 25°C, 30°C, etc., and time of 18-30 h, including but not limited to 18h, 20h, 22h, 24h, 26h, 28h, 30h, etc.

[0029] Preferably, in step S2, the ratio between the intermediate product B and the responsive monomer molecule is 2 g: (2.5-6) ​​mL, including but not limited to 2 g: 2.5 mL, 2 g: 3 mL, 2 g: 4 mL, 2 g: 5 mL, 2 g: 6 mL, etc.; the mass ratio of the intermediate product B and the ligand is 2: (20-24), including but not limited to 2:20, 2:21, 2:22, 2:23, 2:24, etc.

[0030] Further preferably, the responsive monomer molecule includes dimethylaminoethyl methacrylate (DMAEMA); the ligand includes 2,2-bipyridine, the metal halide includes cuprous bromide, and the mass ratio of 2,2-bipyridine to cuprous bromide is 2:1.

[0031] Preferably, in the atom transfer radical polymerization method of step S2, the polymerization temperature is 10-30°C, including but not limited to 10°C, 15°C, 20°C, 25°C, 30°C, etc., and the polymerization time is 1-3h, including but not limited to 1h, 1.5h, 2h, 2.5h, 3h, etc. The present invention controls the chain length of the responsive polymer chain by the polymerization time, wherein the chain lengths mentioned are all average chain lengths.

[0032] Preferably, in step S2, the ratio between the intermediate product B and the second solvent is 2 g: (4000-6000) mL, including but not limited to 2 g: 4000 mL, 2 g: 4500 mL, 2 g: 5000 mL, 2 g: 5500 mL, 2 g: 6000 mL, etc.; the second solvent includes a mixed solution of methanol and water, and the volume ratio is (2.5-3.5): 2, for example, selected from 2.5:2, 2.8:2, 3:2, 3.2:2, 3.5:2, etc.

[0033] Preferably, in the glutaraldehyde chemical cross-linking method of step S3, the carrier C and glutaraldehyde are first subjected to a aldehyde reaction in a third solvent, and an aldehyde intermediate is obtained by washing; the aldehyde intermediate is then subjected to an enzyme fixation reaction with a biological enzyme in a third solvent to obtain a self-driven micro-nano motor; wherein the ratio between the carrier C and glutaraldehyde is 1 mg: (0.05-0.15) mL, including but not limited to 1 mg: 0.05 mL, 1 mg: 0.1 mL, 1 mg: 0.15 mL, etc.; the mass ratio of the carrier C and the biological enzyme is 1: (0.4-4), including but not limited to 1: 0.4, 1: 0.8, 1: 1, 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, etc.

[0034] Further preferably, the ratio between carrier C and the third solvent is 1 mg: (0.5-1.5) mL, including but not limited to 1 mg: 0.5 mL, 1 mg: 0.8 mL, 1 mg: 1 mL, 1 mg: 1.2 mL, 1 mg: 1.5 mL, etc.; the third solvent uses PBS buffer with a concentration of 0.01 M (pH 7.4).

[0035] Further preferably, the aldehyde reaction time is 1.5 to 2.5 h, including but not limited to 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, etc.; the enzyme immobilization reaction time is 14 to 18 h, including but not limited to 14 h, 15 h, 16 h, 17 h, 18 h, etc.; the reaction temperature is 10 to 30 ° C, including but not limited to 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, etc.

[0036] In a third aspect, the present invention provides an application of a self-propelled micro-nano motor with high chemotactic efficiency as a self-actuated actuator for targeted drug delivery.

[0037] In some embodiments, the preparation method of the present invention adopts atom transfer radical polymerization to graft responsive polymer chains to modify the surface of the carrier; and then adopts glutaraldehyde chemical cross-linking method to load biological enzymes, and the content of biological enzymes is controlled by the feed ratio of enzymes. The high chemotactic efficiency self-driven micro-nano motor obtained by the present invention is used as an active delivery system to improve the targeting rate of the target target and can be applied to highly targeted tumors.

[0038] In some preferred embodiments, the present invention uses biological enzymes and responsive polymer chains to construct micro-nano motors with multiple signal gradient responsiveness to improve their chemotaxis efficiency to the target area. Micro-nano motors modified by biological enzymes have chemotaxis to high-concentration substrates, such as catalase has chemotaxis to hydrogen peroxide, and other biological enzymes are similar; and the surface-grafted poly (dimethylaminoethyl methacrylate) polymer chain makes the self-driven micro-nano motor appear hydrophobic under neutral conditions (pH = 7.0) and hydrophilic under slightly acidic conditions (pH = 6.5). The generated surface tension gradient enhances the chemotaxis movement of the motor to the target area (such as high hydrogen peroxide concentration, low pH area). Therefore, the present invention realizes the enhanced chemotaxis movement of the self-driven micro-nano motor to the target area through the chemotaxis of the biological enzyme to the high-concentration "fuel" and the change of the hydrophilicity and hydrophobicity of the responsive polymer chain, thereby improving the autonomous target-seeking ability. The present invention uses the difference in multiple microenvironments between the target point and the non-target area to construct a self-driven micro-nano motor with high chemotaxis efficiency, which is expected to be applied to targeted drug delivery and improve the drug targeting rate.

[0039] The present invention is further described in detail below through specific examples. Among them, the raw materials not specifically noted are all commercially available raw materials; the polydopamine carrier is a mesoporous bowl-shaped polydopamine nanoparticle prepared by a dual template method, specifically referring to the literature Angew. Chemie Int. Ed. 2018, 57, 6176–6180. Example 1 A method for preparing a self-driven micro-nano motor with high chemotactic efficiency comprises the following steps: S1, 8 mg of carrier polydopamine nanoparticles A, 0.06 mL of triethylamine, and 0.05 mL of 2-lutidine were dissolved in 5 mL of tetrahydrofuran, and 0.05 mL of 2-bromoisobutyryl bromide was added dropwise, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the intermediate product B was separated; S2, 2 mg of intermediate product B, 0.004 mL of responsive monomer molecule DMAEMA and 5 mL of mixed solution (methanol and water in a volume ratio of 3:2), 22 mg of 2,2-bipyridine and 11 mg of copper bromide were added, and the reaction was carried out at room temperature by atom transfer radical polymerization (ATRP polymerization). The ATRP polymerization time was controlled to be 2 h to obtain the carrier C with surface modified responsive polymer chain; the particle size distribution was tested by DLS, and the TEM transmission electron microscope was used for auxiliary testing, and the chain length of the responsive polymer chain was measured to be about 35 nm; S3, 1 mg of carrier C with surface modified responsive polymer chains was washed twice with 10 mM PBS buffer, ultrasonically dispersed in 0.9 mL 10 mM PBS buffer, ultrasonically treated for 10 min, and then 0.1 mL glutaraldehyde was added, and the particles were shaken at 25 ° C for 2 h. Then, the treated particles were washed three times with PBS buffer, dispersed in 1 mL 10 mM PBS buffer, and 3 mg catalase (CAT) was added, and the particles were shaken at 25 ° C for 16 h. After the reaction, the particles were washed three times with water to complete the loading of biological enzymes on the carrier C with surface modified responsive polymer chains, and a self-driven micro-nano motor was obtained. The protein detection kit was used for testing, and the content of biological enzymes on the load was measured to be about 435 mg / g; a self-driven micro-nano motor was obtained.

[0040] Example 2 The only difference from Example 1 is that the ATRP polymerization time is adjusted to 10 min, 30 min, 60 min, and 90 min, respectively, so that the chain lengths of the grafted responsive polymer chains are 10 nm, 25 nm, 35 nm, and 50 nm, respectively. The other steps and conditions are the same as in Example 1.

[0041] Performance Testing 1. The polydopamine nanoparticles used in step S1 of Example 1 were subjected to infrared testing. The results are as follows: Figure 1 As shown; Figure 1 The results show that the surface of the obtained polydopamine is rich in amino groups and hydroxyl groups.

[0042] 2. The chemotactic movement of particles was characterized using an inverted optical microscope (Leica DMI 3000 M), e.g. Figure 2 The environmental concentration gradient was simulated as shown. The self-driving nanomotor obtained in Example 1 was placed in an inlet a environment with a pH of 7.4 and a H2O2 concentration of 50 µM, and an agar block fully swollen in pH 6.5 and 90 µM H2O2 was placed in an outlet b environment to construct a pH and H2O2 double concentration gradient (△pH+△c(H2O2)) to observe the chemotactic enhanced movement of the nanomotor. At the same time, the no concentration gradient (Control), pH concentration gradient (△pH), and H2O2 concentration gradient (△c(H2O2)) were used as the comparison outlet c to observe the changes in the chemotactic behavior of the nanomotor.

[0043] in, Figure 3 This is a diagram of the chemotactic motion trajectory of the self-propelled nanomotor of Example 1 under different types of concentration gradients. In the motion trajectory within 15 seconds, the chemotactic path of the nanomotor in the dual gradient environment (△pH+△c(H2O2)) is longer, indicating that the nanomotor undergoes chemotactic enhanced motion in the dual concentration gradient environment.

[0044] 3. Investigate the effect of environmental pH on the movement rate of nanomotors: The self-propelled nanomotor of Example 1 was dispersed in 10 mM H2O2 buffers of different pH values ​​at a concentration of 0.2 mg / mL. The movement rate of the self-propelled nanomotor was related to the pH value of the environment, such as Figure 4 As shown in the figure, the movement rate of the self-driving nanomotor under different pH conditions is also greatly affected. At pH = 7, the movement rate is the lowest. As the pH decreases, the speed of the self-driving nanomotor increases significantly. The lower the pH value, the higher the movement rate, indicating that the responsiveness of the polymer chain affects the movement behavior of the self-driving nanomotor.

[0045] 4. Investigate the effect of fuel concentration on the movement rate of self-propelled nanomotors: The self-propelled nanomotor of Example 1 was dispersed in 0, 0.05, 0.09, 0.5, 1, 10, and 50 mM H2O2 solutions at a concentration of 0.2 mg / mL. Figure 5 is the movement rate of the self-driving nanomotor under different H2O2 concentrations. The higher the H2O2 concentration, the faster the nanomotor moves. This shows that the driving force of the self-driving nanomotor comes from the H2O2 concentration in the environment, and its movement behavior changes with the change of the environmental H2O2 concentration.

[0046] 5. Investigate the effect of environmental pH on the hydrophilicity and hydrophobicity of nanomotors: The self-driving nanomotor in Example 1 was spread on the surface of a glass plate at a concentration of 1 mg / mL, and the contact angles of water droplets at pH = 5, 6, 6.5, 7, 7.4, and 8 on the surface of the self-driving nanomotor were tested. Figure 6 As shown in the figure, the contact angles of droplets of different pH values ​​on the surface of the self-propelled nanomotor are different, indicating that the surface of the nanomotor exhibits different hydrophilic and hydrophobic properties under different pH conditions. Under alkaline conditions, it exhibits a hydrophobic state, and under acidic and neutral conditions, it exhibits a hydrophilic state, and as the pH increases, the contact angle increases.

[0047] 6. Investigate the effect of environmental pH on the particle size of nanomotors: The self-propelled nanomotor in Example 1 was placed in a buffer solution at pH = 5, 6, 7, or 8. Figure 7 As shown in the figure, the particle size increases significantly with the decrease of pH. In an acidic environment, the responsive polymer chain is protonated and the chain is in an extended state. The hydrated particle size is the smallest under neutral pH conditions. Under alkaline conditions, the responsive polymer chain is deprotonated and the chain is in a contracted state. The particles are easy to aggregate, which increases the hydrated particle size. Therefore, when the environmental pH changes, the polymer chain on the surface of the self-propelled nanomotor stretches and contracts, which affects the particle size.

[0048] 7. Investigate the effect of polymer chain length on the movement rate in nanomotors: The self-propelled nanomotors in Example 1 and Example 2 (with surface modified chain lengths of 10 nm, 25 nm, 35 nm, and 50 nm, respectively) were placed in a buffer solution at pH = 5 and moved autonomously. The rate of change of movement speed at pH = 5 is as follows: Figure 8 As shown, the longer the chain length, the greater the rate of change of its speed, indicating that the change in the movement speed of the nanomotor in a pH gradient environment is related to the length of the polymer chain modified on the surface of the nanomotor.

[0049] 8. Investigate the effect of double concentration gradient on the chemotaxis of nanomotors: The self-driving nanomotor in Example 1 was modified with fluorescein isothiocyanate (FITC) fluorescence (the modification was completed after the nanomotor was added with excess FITC and oscillated), and placed in different types of concentration gradient environments (control group Control, pH gradient group △pH, H2O2 gradient group △H2O2, double gradient group △pH+△H2O2, the specific concentration gradient is the same as the above 2), and the fluorescence intensity under different concentration gradients was statistically analyzed from 0 to 60 minutes, as shown in FIG. Fig. 9 As shown in the figure, the fluorescence intensity of the H2O2 gradient group is greater than that of the pH gradient group and the control group, indicating that both the biological enzyme and the responsive polymer chain are selective for the target area, and the biological enzyme plays a major driving role; and the fluorescence intensity under the double concentration gradient (△pH+△H2O2) is significantly higher than that under the single concentration gradient, indicating that the biological enzyme and the responsive polymer chain play a synergistic role in improving the selectivity of the nanomotor for the target area. In the double concentration gradient environment, the aggregation of nanomotors increases, showing chemotaxis-enhanced behavior, thus overcoming the off-target caused by single signal interference in a complex environment and improving the targeting rate.

[0050] 9. Investigate whether nanomotors have chemotactic enhancement behavior on tumor cells: L929 normal cells and A549 tumor cells with good growth status were cultured at 5×10 6 / mL for lysis, and extract cell lysate. A microfluidic channel was used to construct a dual concentration gradient of the physiological environment with the lysate of A549 tumor cells, and the cell lysate of L929 normal cells was used as the control group. The self-driving nanomotor in Example 1 was FITC fluorescently modified (operation 8 as above), and placed in different concentration gradient environments generated by L929 cells and A549 cells. The L929 cell lysate represented a neutral microenvironment simulating the normal tissue microenvironment, and the A549 cell lysate represented a low pH and high H2O2 environment simulating the tumor microenvironment. After 60 minutes, the fluorescence changes around the two groups of cell environments were compared, where the cell size was about 10μm, the nanomotor particle size was about 300nm, and the nanomotor could not be observed under an inverted fluorescence microscope, but the fluorescence intensity could be used to judge whether the nanomotor had chemotaxis enhancement behavior (more particles moved and gathered at the cells, resulting in an increase in fluorescence intensity).

[0051] like Fig.10 As shown, the fluorescence intensity in the tumor cell A549 environment is significantly higher, indicating that the nanomotor has a chemotactic enhancement behavior towards the dual concentration gradient generated by the tumor cells.

[0052] In summary, the present invention provides a self-driven micro-nano motor with high chemotaxis efficiency and a preparation method thereof. The self-driven micro-nano motor is driven by a biological enzyme, and the micro-nano particles with responsive polymers grafted on the surface can be used to improve the targeting rate to the target environment; the preparation method includes: grafting the responsive polymer chain on the surface of the nanoparticles rich in hydroxyl or amino groups through atomic free radical transfer, and cross-linking the loaded biological enzyme with glutaraldehyde. The present invention utilizes the modulation of the motor surface properties by the responsive polymer chain and the chemotaxis of the biological enzyme to the high concentration substrate to achieve the chemotaxis-enhanced movement of the nanomotor to the target area and improve the chemotaxis efficiency. Compared with the prior art, the present invention has the following advantages: First, it can achieve multi-signal gradient response in the target area and improve the chemotaxis efficiency; second, the chemotaxis can be modulated; third, it can overcome the off-target caused by the interference of a single signal in a complex environment and improve the targeting rate.

[0053] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A self-driven micro-nano motor with high chemotactic efficiency, characterized in that: The self-driving micro-nano motor comprises a carrier, and a responsive polymer chain and a biological enzyme connected to the surface of the carrier; The carrier is a micro-nano particle with rich active groups on the surface; The polymer in the responsive polymer chain is a high molecular material that can respond to specific signal characteristics in a physiological environment; The biological enzyme has chemotaxis to natural substrates in a physiological environment.

2. The high chemotactic efficiency self-driven micro-nano motor according to claim 1, characterized in that: The size of the carrier is 50 nm to 5 µm; The carrier comprises polydopamine with rich amino groups and hydroxyl groups on its surface.

3. The high chemotactic efficiency self-driven micro-nano motor according to claim 1, characterized in that: The biological enzyme includes any one or more of catalase, glucose oxidase, urease, superoxide dismutase, and lactate oxidase; The natural substrate includes any one or more of hydrogen peroxide, glucose, urea, superoxide anion, lactic acid, and glutathione.

4. The high chemotactic efficiency self-driven micro-nano motor according to claim 1, characterized in that: The specific signal characteristics include any one or more of pH, temperature, ions, and natural substrates; The chain length of the responsive polymer chain is 10 to 100 nm.

5. The high chemotactic efficiency self-driven micro-nano motor according to claim 4, characterized in that: The polymer in the responsive polymer chain includes polydimethylaminoethyl methacrylate.

6. The method for preparing a self-propelled micro-nano motor with high chemotactic efficiency according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, dissolving the carrier and the reactant in a first solvent, adding an initiator dropwise to react, and separating to obtain an intermediate product after the reaction is completed; S2, mixing the intermediate product, the responsive monomer molecule and the second solvent, adding the ligand and the metal halide, and using the atom transfer radical polymerization method to obtain a carrier with a surface-modified responsive polymer chain; S3, using glutaraldehyde chemical cross-linking method, biological enzymes were connected to the carrier with surface modified responsive polymer chains to obtain self-driven micro-nanomotors.

7. The method for preparing a self-propelled micro-nano motor with high chemotactic efficiency according to claim 6, characterized in that: In step S1, the reactants include triethylamine and 2-dimethylaminopyridine, and the carrier includes polydopamine nanoparticles; The ratio between the carrier, triethylamine and 2-dimethylaminopyridine is 8 g: (55-65) mL: (45-55) mL; The initiator is 2-bromoisobutyryl bromide; the ratio between the carrier and the initiator is 8 g: (45-55) mL; The first solvent includes tetrahydrofuran.

8. The method for preparing a self-propelled micro-nano motor with high chemotactic efficiency according to claim 6, characterized in that: In step S2, the ratio between the intermediate product and the responsive monomer molecule is 2 g:(2.5-6) ​​mL; the mass ratio between the intermediate product and the ligand is 2:(20-24); The responsive monomer molecule includes dimethylaminoethyl methacrylate; the ligand includes 2,2-bipyridine, the metal halide includes cuprous bromide, and the mass ratio of 2,2-bipyridine to cuprous bromide is 2:1; In the atom transfer radical polymerization method, the polymerization temperature is 10 to 30°C and the polymerization time is 1 to 3 hours; The second solvent includes a mixed solution of methanol and water.

9. The method for preparing a self-propelled micro-nano motor with high chemotactic efficiency according to claim 6, characterized in that: In the glutaraldehyde chemical crosslinking method of step S3, the carrier with the surface-modified responsive polymer chain and glutaraldehyde are first subjected to an aldehyde reaction in a third solvent, and then washed to obtain an aldehyde intermediate; the aldehyde intermediate is then subjected to an enzyme fixation reaction with a biological enzyme in a third solvent to obtain a self-driven micro-nano motor; Wherein, the ratio between the carrier of the surface-modified responsive polymer chain and the glutaraldehyde is 1 mg: (0.05-0.15) mL; the mass ratio between the carrier of the surface-modified responsive polymer chain and the biological enzyme is 1: (0.4-4); The time of the aldehyde reaction is 1.5 to 2.5 h; the time of the enzyme immobilization reaction is 14 to 18 h; the reaction temperature is 10 to 30°C; The third solvent includes PBS buffer.

10. Use of the high chemotactic efficiency self-propelled micro-nanomotor as claimed in any one of claims 1 to 5 as a self-propelled actuator for targeted drug delivery.

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