An enzyme-driven Janus nanomotor and its preparation method

Abstract

The present invention relates to the technical field of nanodevice preparation, and specifically to an enzyme-driven Janus nanomotor and a preparation method thereof, with noble metal nanoparticles as the basic skeleton of the motor, one side of which is connected to functional molecules, and the other side of which is connected to nanoparticles of biological enzymes. The enzyme-driven Janus nanomotor of the present invention is small in size, easy to enter and exit cell tissues, and has good biocompatibility, and thus has broad application prospects in the field of biological active drug delivery. Due to the special optical and electrical properties of noble metal nanoparticles, they have excellent performance in the fields of biosensing and disease treatment. The preparation method of the present invention has strong controllability, can meet the preparation of nanomotors driven by different sizes, different materials, and different enzymes, and has universality, which provides possibilities for subsequent biosensing and targeted drug delivery.

Description

Technical Field

[0001] The present invention relates to the technical field of nano device preparation, and in particular to an enzyme-driven Janus nano motor and a preparation method thereof. Background Art

[0002] Nanomotors are nanodevices that can convert other forms of energy into kinetic energy and move autonomously at the nanoscale. Due to their unique autonomous motion performance, they have important applications in drug loading, transportation and release, so nanomotors have great application prospects in the fields of biosensing and active drug delivery.

[0003] Among them, the preparation of motors is the most basic and important part in the research of micro / nanomotors. At present, a variety of technologies have been developed for the preparation of micro / nanomotors. For example, electrochemical deposition is used to prepare bimetallic rod-shaped, linear, and tubular motors, but this method is limited by the template and has a limited yield, and is only suitable for the preparation of micrometer motors; physical deposition is used to prepare a variety of Janus motors. The yield of motors prepared by this method is higher than that of electrochemical deposition, but it has certain difficulties in the preparation of nanomotors; in recent years, scientists have used chemical assembly methods to prepare a series of motors, which greatly enriched the types and functions of motors. Although nanomotors can be prepared, small-sized (<100nm) nanomotors cannot be prepared. Therefore, there are still huge challenges in the preparation of nano-sized motors.

[0004] Therefore, the present invention constructs an asymmetric structure at the nanoscale and combines nanoparticles with biological enzymes to prepare small-sized and size-controllable enzyme-driven Janus nanomotors, which will produce enhanced diffusion at a certain "fuel" concentration. Summary of the invention

[0005] One of the purposes of the present invention is to provide an enzyme-driven Janus nanomotor, which is small in size, nanoscale, easy to enter and exit cell tissues, has good biocompatibility, and has broad application prospects in the field of biological active drug delivery.

[0006] The second purpose of the present invention is to provide a method for preparing an enzyme-driven Janus nanomotor, which has strong controllability and can meet the preparation of nanomotors driven by different sizes, different materials, and different enzymes.

[0007] The third object of the present invention is to provide an application of an enzyme-driven Janus nanomotor.

[0008] The solution adopted by the present invention to achieve one of the purposes is: an enzyme-driven Janus nanomotor, with precious metal nanoparticles as the basic skeleton of the motor, one side connected to functional molecules, and the other side connected to biological enzymes.

[0009] Preferably, the noble metal is gold, platinum or palladium.

[0010] Preferably, the particle size of the noble metal nanoparticles is 10-100 nm.

[0011] Preferably, the functional molecule is polyethylene glycol or a nucleic acid molecule, and the functional molecule is bound to the noble metal nanoparticles via Au / Pt / Pd-S covalent bonds.

[0012] Preferably, the biological enzyme is any one of urease, glucose oxidase and catalase, and the biological enzyme is modified on the surface of the noble metal nanoparticles through a carboxyl group or an aldehyde group.

[0013] The solution adopted by the present invention to achieve the second purpose is: a method for preparing the enzyme-driven Janus nanomotor, comprising the following steps:

[0014] (1) Preparing noble metal-polystyrene eccentric particles using noble metal nanoparticles and styrene monomers, wherein the polystyrene only covers a portion of the surface of the noble metal nanoparticles, and the portion of the noble metal nanoparticles not covered by the polystyrene is an exposed surface;

[0015] (2) using Au / Pt / Pd-S covalent bonds to modify the functional molecules on the exposed surface of the noble metal-polystyrene eccentric particles to obtain functional molecule-noble metal-polystyrene particles;

[0016] (3) removing polystyrene from the functional molecule-noble metal-polystyrene particles obtained in step (2) to obtain functional molecule-noble metal eccentric particles, wherein the portion of the noble metal nanoparticles not connected to the functional molecule is an exposed surface;

[0017] (4) Modifying the exposed surface of the noble metal nanoparticles with aldehyde groups or carboxyl groups, and then modifying the exposed surface with biological enzyme nanoparticles through condensation reaction or amide reaction to obtain the enzyme-driven Janus nanomotor.

[0018] Preferably, in step (1), the noble metal nanoparticles are prepared by a redox seed growth method.

[0019] Preferably, in the step (1), the specific preparation method of the noble metal-polystyrene eccentric particles is: under an inert atmosphere, a mixed solution of an initiator, a stabilizer, ethanol and water is heated to 65-75°C, and then a mixed solution of styrene monomer and DVB with a volume ratio of 200:1 is added, and after the system is stabilized, an excess of noble metal nanoparticles is added, stirred until completely combined, and centrifuged to obtain the noble metal-polystyrene eccentric particles.

[0020] Preferably, in the step (3), the functional molecule-noble metal-polystyrene particles are added to N,N-dimethylformamide or tetrahydrofuran, mixed evenly, left to stand and then washed to obtain the functional molecule-noble metal eccentric particles.

[0021] The solution adopted by the present invention to achieve the third purpose is: an application of the enzyme-driven Janus nanomotor, wherein the enzyme-driven Janus nanomotor is used as a drug carrier.

[0022] The present invention has the following advantages and beneficial effects:

[0023] 1. The enzyme-driven Janus nanomotor of the present invention is small in size, easy to enter and exit cell tissues, and has good biocompatibility, and thus has broad application prospects in the field of biological active drug delivery.

[0024] 2. The enzyme-driven Janus nanomotor of the present invention has excellent performance in the fields of biosensing and treatment due to the special optical and electrical properties of the precious metal nanoparticles.

[0025] 3. The preparation method of the present invention has strong controllability and can meet the preparation of nanomotors driven by different sizes, different materials, and different enzymes.

[0026] 4. The preparation method of the present invention is suitable for the preparation of small-sized nanomotors and has universal applicability, providing possibilities for subsequent biosensing and targeted drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the preparation process of the enzyme-driven nanomotor in Example 1 of the present invention;

[0028] Figure 2 Transmission electron microscopy and UV-visible spectra of gold nanoparticles of different sizes in Example 1 of the present invention;

[0029] Figure 3 The scanning electron microscope and transmission electron microscope of the Au-PS eccentric structures of different sizes prepared in Example 1 of the present invention;

[0030] Figure 4 Transmission electron microscopy images of urease-driven Janus nanogold motors of different sizes prepared in Example 1 of the present invention, wherein A is 10 nm, B is 50 nm, and C is 90 nm;

[0031] Figure 5 The diagram is a motion trajectory diagram of a 10 nm urease-driven nano-gold motor in urea solutions of different concentrations, and a diagram showing changes in mean square displacement and diffusion coefficient with urea concentration in Example 2 of the present invention;

[0032] Figure 6The diagram is a motion trajectory diagram of a 50 nm urease-driven nano-gold motor in urea solutions of different concentrations, and a diagram showing changes in mean square displacement and diffusion coefficient with urea concentration in Example 3 of the present invention;

[0033] Figure 7 The diagram is a motion trajectory diagram of the 90 nm urease-driven nano-gold motor in urea solutions of different concentrations, and a diagram showing changes in mean square displacement and diffusion coefficient with urea concentration in Example 4 of the present invention;

[0034] Figure 8 The motion trajectory diagram of the 50 nm glucose oxidase-driven nano-gold motor in urea solutions of different concentrations, and the change diagram of the mean square displacement and diffusion coefficient with the urea concentration in Example 5 of the present invention;

[0035] Fig. 9 The diagram is a motion trajectory diagram of a 50 nm catalase-driven nano-gold motor in urea solutions of different concentrations, and a diagram showing changes in mean square displacement and diffusion coefficient with urea concentration in Example 6 of the present invention;

[0036] Fig.10 The motion trajectory diagram of the 50 nm urease-driven Janus nanogold motor (the functional molecule on one side becomes a nucleic acid chain) in urea solutions of different concentrations, and the variation diagram of the mean square displacement and diffusion coefficient with urea concentration in Example 7 of the present invention;

[0037] Fig.11 The diagram shows the motion trajectory of the 50 nm urease-driven nanoplatinum motor in urea solutions of different concentrations, and the variation of the mean square displacement and diffusion coefficient with the urea concentration in Example 8 of the present invention. DETAILED DESCRIPTION

[0038] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0039] Example 1

[0040] The preparation process of the enzyme-driven Janus nanomotor in this embodiment is as follows Figure 1 As shown, it mainly includes three parts: (1) preparation of gold nanoparticles of different sizes, (2) preparation of asymmetric Au-PS (gold-polystyrene) eccentric particles, and (3) preparation of urease-driven Janus gold nanomotor of different sizes.

[0041] (1) Preparation of gold nanoparticles of different sizes: Synthesis of 10 nm gold nanoparticles (in other embodiments, gold nanoparticles of other sizes can also be synthesized as needed): Take 1 mM chloroauric acid solution, stir and heat to boiling in an oil bath, heat for another 10 min after boiling, quickly add 38.8 mM sodium citrate aqueous solution, continue heating for 10 min, and then stop heating;

[0042] Initial 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 into a three-necked flask. Heat and stir at 90 °C. After the system stabilizes, add a certain amount of 25 mM chloroauric acid solution in two portions. After 30 minutes, the initial growth of gold nanoparticles is completed.

[0043] 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, and after the temperature stabilizes, add a certain amount of 60mM sodium citrate solution. After stabilization, add a certain amount of 25mM chloroauric acid solution twice and react for 30 minutes. By controlling the number of growth times, gold nanoparticles of different sizes can be obtained.

[0044] (2) Preparation of asymmetric Au-PS eccentric particles. First, assemble the nitrogen device and condenser, and adjust the nitrogen volume. Prepare 50 mg / mL potassium persulfate solution and 6 mg / mL 4-styrenesulfonate sodium salt solution. Then, add 2.5 mL of water and 16.5 mL of ethanol to the three-necked flask, connect the nitrogen device, and then add 1 mL of potassium persulfate solution and 1 mL of 4-styrenesulfonate sodium salt solution. Stir and stabilize for 20 minutes. Turn on the heating device and heat to 70°C. After heating to 70, add 200 μL of the mixed solution (V styrene :V DVB =200:1), and after the system stabilized. After shaking the nanogold, add 3mL of nanogold (~1.4×10 11 After 4 hours of reaction, the asymmetric Au-PS eccentric particles were obtained by centrifugation.

[0045] (3) Preparation of enzyme-driven Janus nano-gold motors of different sizes. The obtained Au-PS particles were dispersed in 6 mL of ultrapure water. 2 mL of Au-PS solution was taken, and 0.1 mL of HO-PEG-SH (hydroxy-polyethylene glycol-thiol) solution (10 mg / mL) was added and mixed evenly. The mixture was shaken at room temperature overnight and then washed with ultrapure water. 1 mL of DMF was added to the sample, mixed evenly, allowed to stand for 15 min, washed with ultrapure water, and finally dispersed in 2 mL of ultrapure water. 10 μL of N-acetylcysteine ​​(10 mM) was added and mixed evenly. The mixture was shaken at room temperature overnight. Take 500 μL of the above solution, wash it 3 times with PB buffer, and then disperse it in 120 μL PB buffer. Quickly add 20 μL N-hydroxysuccinimide (concentration 0.08M) and 10 μL (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (carbodiimide, concentration 0.08M) to the above test tube, mix well, and shake and react at room temperature for 30 minutes. Prepare 1 mg / 50 μL urease solution, glucose oxidase solution and catalase solution respectively, take 50 μL of each enzyme solution and add it to the above test tube, mix well, and shake and react at room temperature overnight. After the reaction is completed, wash it 3 times in ultrapure water to obtain urease-driven nanomotors, glucose oxidase-driven nanomotors and catalase-driven nanomotors.

[0046] The transmission electron microscope and ultraviolet absorption images of the gold nanoparticles of different sizes are shown in Figure 2. Figure 2 A-2D, the average Au particle size is 10nm, 50nm, and 90nm respectively; the morphology of the Au-PS asymmetric structure under scanning electron microscopy and transmission electron microscopy is shown in Figure 3 As shown in A and 3B, it has an asymmetric structure, and the PS particle size is 260-280nm, with an average value of 270nm;

[0047] The transmission electron microscopy of the different sizes of urease-driven Janus nanogold motors is shown in Figure 4 As can be seen from the figure, urease-driven Janus nanogold motors of different sizes have good dispersion in the solution.

[0048] Example 2

[0049] The 10 nm urease-driven Janus nanogold motor obtained in Example 1 was placed in 0, 2, 4, 6, 8, 10, 20, 50 mM urea solution. Its motion trajectory is shown in Figure 5 As shown in A, its mean square displacement changes as follows Figure 5 B, its diffusion coefficient changes as Figure 5 As shown in C, it can be seen from the figure that: with the increase of urea concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the urea concentration is 10mM.

[0050] Example 3

[0051] The 50 nm urease-driven Janus nanogold motor obtained in Example 1 was placed in 0, 2, 4, 6, 8, 10, 20, and 50 mM urea solutions. Figure 6 As shown in A, its mean square displacement changes as follows Figure 6 B, its diffusion coefficient changes as Figure 6 As shown in C, it can be seen from the figure that: with the increase of urea concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the urea concentration is 10mM.

[0052] Example 4

[0053] The 90 nm urease-driven Janus nanogold motor obtained in Example 1 was placed in 0, 2, 4, 6, 8, 10, 20, 50 mM urea solutions. Its motion trajectory is shown in Figure 7 As shown in A, its mean square displacement changes as follows Figure 7 B, its diffusion coefficient changes as Figure 7 As shown in C, it can be seen from the figure that: with the increase of urea concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the urea concentration is 10mM.

[0054] Example 5

[0055] The 50 nm glucose oxidase-driven Janus nanogold motor obtained in Example 1 was placed in 0, 20, 40, 60, 100, 150, 250, 500 mM glucose solutions. Its motion trajectory is shown in Figure 8 As shown in A, its mean square displacement changes as follows Figure 8 B, its diffusion coefficient changes as Figure 8 As shown in C, it can be seen from the figure that: with the increase of glucose concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the glucose concentration is 250mM.

[0056] Example 6

[0057] The 50 nm catalase-driven Janus nanogold motor obtained in Example 1 was placed in 0, 0.05, 0.1, 0.15, 0.2, 0.5, 0.75, and 1 M hydrogen peroxide solutions. Fig. 9 As shown in A, its mean square displacement changes as follows Fig. 9 B, its diffusion coefficient changes as Fig. 9 As shown in C, it can be seen from the figure that: with the increase of hydrogen peroxide concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the hydrogen peroxide concentration is 0.2M.

[0058] Example 7

[0059] The polyethylene glycol on one side of the 50 urease-driven Janus nanogold motor obtained in Example 1 was replaced with a nucleic acid chain and placed in 0, 2, 4, 6, 8, 10, 20, 50 mM urea solution. Fig.10 As shown in A, its mean square displacement changes as follows Fig.10 B, its diffusion coefficient changes as Fig.10 As shown in C, it can be seen from the figure that: with the increase of urea concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the urea concentration is 10mM.

[0060] Example 8

[0061] Preparation of nano-platinum: First, add 1% sodium citrate and 0.05% citric acid to a boiling chloroplatinic acid hexahydrate (0.2%) solution, and add 5.5 mL of freshly prepared sodium borohydride (0.08%) solution containing 1% sodium citrate and 0.05% citric acid for 30 seconds. After 10 minutes, the product was cooled to room temperature to obtain a seed solution; then, a certain amount of seed solution was added. Part of the chloroplatinic acid solution, 1% sodium citrate and 1.25% L-ascorbic acid were added to obtain nano-platinum with a particle size of 50 nm under heating conditions.

[0062] According to the method of Example 1, a 50 nm urease-driven Janus nano-platinum motor was obtained, and the obtained 50 nm urease-driven Janus nano-platinum motor was placed in 0, 2, 4, 6, 8, 10, 20, 50 mM urea solution. Its motion trajectory is shown in Fig.11 As shown in A, its mean square displacement changes as follows Fig.11 B, its diffusion coefficient changes as Fig.11 As shown in C, it can be seen from the figure that: with the increase of urea concentration, the moving distance of the motor is gradually increasing, the mean square displacement slope and the diffusion coefficient are also gradually increasing, and they reach stability when the urea concentration is 10mM.

[0063] The raw materials listed in the present invention, as well as the upper and lower limits and interval values ​​of the raw materials of the present invention, and the upper and lower limits and interval values ​​of the process parameters (such as temperature, time, etc.) can all realize the present invention, and the embodiments are not listed one by one here.

[0064] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. An enzyme-driven Janus nanomotor, characterized in that: The motor is based on precious metal nanoparticles, one side of which is connected to a functional molecule and the other side is connected to a biological enzyme. The functional molecule is polyethylene glycol or a nucleic acid molecule.

2. The enzyme-driven Janus nanomotor according to claim 1, characterized in that: The noble metal is gold, platinum or palladium.

3. The enzyme-driven Janus nanomotor according to claim 1, characterized in that: The particle size of the noble metal nanoparticles is 10-100 nm.

4. The enzyme-driven Janus nanomotor according to claim 1, characterized in that: The functional molecules are combined with the noble metal nanoparticles through at least one of Au-S covalent bonds, Pt-S covalent bonds, and Pd-S covalent bonds.

5. The enzyme-driven Janus nanomotor according to claim 1, characterized in that: The biological enzyme is any one of urease, glucose oxidase and catalase, and the biological enzyme is modified on the surface of the noble metal nanoparticles through a carboxyl group or an aldehyde group.

6. A method for preparing an enzyme-driven Janus nanomotor according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Preparing noble metal-polystyrene eccentric particles using noble metal nanoparticles and styrene monomers, wherein the polystyrene only covers a portion of the surface of the noble metal nanoparticles, and the portion of the noble metal nanoparticles not covered by the polystyrene is an exposed surface; (2) modifying the exposed surface of the noble metal-polystyrene eccentric particles with functional molecules by using at least one of Au-S covalent bonds, Pt-S covalent bonds, and Pd-S covalent bonds to obtain functional molecule-noble metal-polystyrene particles; (3) removing polystyrene from the functional molecule-noble metal-polystyrene particles obtained in step (2) to obtain functional molecule-noble metal eccentric particles, wherein the portion of the noble metal nanoparticles not connected to the functional molecule is an exposed surface; (4) Modifying the exposed surface of the noble metal nanoparticles with aldehyde groups or carboxyl groups, and then modifying the exposed surface with biological enzyme nanoparticles through condensation reaction or amide reaction to obtain the enzyme-driven Janus nanomotor.

7. The method for preparing the enzyme-driven Janus nanomotor according to claim 6, characterized in that: In the step (1), the noble metal nanoparticles are prepared by a redox seed growth method.

8. The method for preparing the enzyme-driven Janus nanomotor according to claim 6, characterized in that: In the step (1), the specific preparation method of the noble metal-polystyrene eccentric particles is: under an inert atmosphere, a mixed solution of an initiator, a stabilizer, ethanol and water is heated to 65-75° C., and then a mixed solution of styrene monomer and DVB with a volume ratio of 200:1 is added. After the system is stabilized, an excess of noble metal nanoparticles is added, stirred until the mixture is completely combined, and centrifuged to obtain the noble metal-polystyrene eccentric particles.

9. The method for preparing the enzyme-driven Janus nanomotor according to claim 6, characterized in that: In the step (3), the functional molecule-precious metal-polystyrene particles are added to N,N-dimethylformamide or tetrahydrofuran, mixed evenly, left to stand and then washed to obtain the functional molecule-precious metal eccentric particles.

10. An application of the enzyme-driven Janus nanomotor according to any one of claims 1 to 5 or the enzyme-driven Janus nanomotor prepared by the preparation method according to any one of claims 6 to 9, characterized in that: The enzyme-driven Janus nanomotor is used as a drug carrier.

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