A nanomotor with enhanced targeting behavior and its preparation method and application

Abstract

The present invention relates to a nanomotor with enhanced targeting behavior, a preparation method and an application thereof, wherein the nanomotor comprises a carrier, and two or more biological enzymes are modified on the surface of the carrier; the carrier is a micro-nano particle rich in active groups; the biological enzyme is an enzyme with a marker released by a physiological lesion as a substrate. In the nanomotor of the present invention, the specific catalysis of a plurality of biological enzyme-substrates is utilized to enhance the mobility of the nanomotor in a physiological environment; the difference in multiple microenvironments between the target and non-target areas and the chemotaxis synergy of a plurality of biological enzymes are utilized to enhance the targeting ability of the nanomotor to the target lesion site. The nanomotor of the present invention can overcome the off-target problem caused by single signal interference in a complex environment, improve the targeting rate, and has important significance for the application of micro-nano motors in the biomedical field.

Description

Technical Field

[0001] The present invention relates to the field of micro-nano motors, and in particular to a nano motor with enhanced targeting behavior and a preparation method and application thereof. Background Art

[0002] Bio-enzyme-driven micro-nanomotors can use chemical substrates in the physiological environment as fuel to propel themselves, and have stronger penetration and barrier-crossing capabilities than particles that rely solely on Brownian motion for passive diffusion; at the same time, their ability to chemotaxis to fuel concentration gradients is crucial for the development of intelligent biomedical micro-nanomotors.

[0003] However, most enzyme-driven micro-nanomotors reported so far are mostly concentrated in the micrometer scale. For example, CN108524941A discloses an enzyme-driven bottle-shaped nanomotor and its preparation method, which is several hundred nanometers in size, close to micrometers, and often requires high-concentration fuel to drive (usually at the mM or even M level). The large size and the need for high-concentration fuel result in low physiological adaptability. In recent years, although the chemotactic movement of motors under physiological concentration fuel gradients has been achieved by reducing the size, they all rely on a single fuel for driving. The extremely low substrate concentration in the physiological environment and the chemotactic behavior generated based on a single substrate concentration gradient make the targeting of enzyme-driven motors to lesions easily affected by the complex physiological environment. For example, tumor sites and inflammatory areas often have similar overexpressed markers (such as high concentrations of H2O2, DNA, and iNOS / ROS, etc. ( Nano Lett. 2021, 21, 8086 ; Adv. Mater. 2022, 34, 2206654; ACS Nano 2023, 17, 12573−12 ), it is difficult to distinguish tumors or inflammatory lesions by relying on the chemotaxis of a single characteristic substrate concentration gradient, which affects its targeting efficiency and limits its further application. Summary of the invention

[0004] In view of this, the present invention aims to provide a nanomotor with enhanced targeting behavior and a preparation method and application thereof, so as to solve the technical problems in the prior art that the nanomotor has low driving force at the physiological level, relies on a single signal gradient for target seeking, has low chemotaxis efficiency, and is easy to off-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 nanomotor with enhanced targeting behavior, including a carrier, and two or more biological enzymes modified on the surface of the carrier; the carrier is a micro-nanoparticle rich in active groups; the biological enzyme is an enzyme that uses a marker released by a physiological lesion as a substrate and has substrate concentration gradient chemotaxis.

[0006] In a second aspect, the present invention provides a method for preparing a nanomotor with enhanced targeting behavior, comprising the following steps: using a carrier, glutaraldehyde and two or more biological enzymes as raw materials, performing a glutaraldehyde chemical cross-linking reaction to prepare a nanomotor.

[0007] In a third aspect, the present invention provides an application of a nanomotor with enhanced targeting behavior in the preparation of anti-tumor drugs and / or cancer diagnostic and therapeutic drugs.

[0008] In a fourth aspect, the present invention provides a nanomotor with enhanced targeting behavior as a targeted active drug delivery carrier or a bioimaging agent.

[0009] Compared with the prior art, the beneficial effects of the present invention include: (1) Compared with existing chemotaxis motors, the nanomotor of the present invention innovatively utilizes the specific catalysis of multiple biological enzymes and substrates to enhance the driving force of the nanomotor, achieve effective driving in physiological concentration fuels, and overcome the problem of insufficient driving force caused by low concentration of a single fuel substrate; (2) The nanomotor of the present invention innovatively utilizes dual enzyme synergistic chemotaxis, using urea, which is overexpressed in liver cancer tissue, and hydrogen peroxide, which is overexpressed in all tumor microenvironments, as fuel, significantly improving the nanomotor's targeting efficiency to liver cancer cells and overcoming the off-target problem that may be caused by a single signal response in a complex environment; (3) The nanomotor carrier and the surface-modified biological enzyme in the present invention can be replaced according to the application scenario, thereby achieving efficient targeting of different tumors and different diseases, and expanding the application to the fields of precise drug delivery, imaging, etc.; (4) The preparation method of the present invention is simple, and the driving force and driving efficiency of the obtained nanomotor are modulatable and highly controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The characterization of the amino functional carrier and the resulting nanomotor used in Example 1 of the present invention, including (A) infrared spectrum, (B) zeta potential; Figure 2 This is a characterization of the nanomotor obtained in Example 1 of the present invention, wherein (A) is a TEM image, and (B) is an EDS image, and the scale is 50 nm; Figure 3 It is a characterization of the movement behavior of the nanomotor obtained in Example 1 of the present invention in urea solutions of different concentrations; wherein (A) MSD curve, (B) diffusion coefficient; Figure 4 It is a characterization of the movement behavior of the nanomotor obtained in Example 1 of the present invention in hydrogen peroxide solutions of different concentrations; wherein (A) MSD curve; (B) diffusion coefficient; Figure 5 It is the characterization of the movement behavior of the nanomotors with different ratios of catalase to urease in the dual-fuel system at the physiological level of the present invention; Figure 6 is a schematic diagram of the chemotaxis assay of the present invention; Figure 7 The chemotaxis of the nanomotor under different gradients, including (A) trajectory within 10 s, (B) center of mass displacement, and (C) directionality; I: control group (control), II: hydrogen peroxide gradient, III: urea gradient, and IV: dual-fuel gradient; the black solid line in IV represents the sum of the values ​​of the single gradient experimental group; Figure 8 is a schematic diagram of the microfluidic device of the present invention; Fig. 9 It is the selective targeted enrichment of the nanomotor of the present invention; Fig.10 It is a comparison of the targeted fluorescence effects of the nanomotor of the present invention on normal cells, liver cancer cells and breast cancer cells; Fig.11 The figure shows the normalized fluorescence intensity comparison of the nanomotor of the present invention on normal cells, liver cancer cells and breast cancer cells. DETAILED DESCRIPTION

[0011] 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.

[0012] In view of the shortcomings of existing nanomotors that rely on a single fuel to drive, have low driving force in a physiological environment, and rely on a single signal gradient for target seeking, have low chemotaxis efficiency, and are easy to miss the target, the present invention provides a nanomotor with improved driving force and chemotaxis efficiency. The motor is composed of a carrier and two or more biological enzymes modified on its surface, and can utilize multi-enzyme synergy to achieve effective driving in a physiological environment and efficient targeting of liver cancer cells or breast cancer cells. The preparation and operation of the nanomotors of the present invention are simple, and the universality is high. It can be modulated as needed to improve the targeting efficiency of different target areas.

[0013] In the first aspect, the present invention provides a nanomotor with enhanced targeting behavior, including a carrier, and two or more biological enzymes modified on the surface of the carrier; the carrier is a nanoparticle rich in active groups; the biological enzyme is an enzyme that uses markers released by physiological lesions as substrates and has substrate concentration gradient chemotaxis.

[0014] Preferably, the particle size of the carrier is 10 nm to 1000 nm.

[0015] Preferably, the carrier includes a non-metallic oxide or polymer having an asymmetric structure or intrinsic asymmetry.

[0016] Further preferably, the carrier comprises Au / SiO2 nanoparticles functionalized with an asymmetric structure and amino groups, and the loading of the enzyme depends on the bridging effect of glutaraldehyde, the aldehyde group at one end reacts with the amino group on the surface of the carrier, and the aldehyde group at the other end reacts with the amino acid residue of the enzyme.

[0017] More preferably, the preparation steps of amino-functionalized Au / SiO2 nanoparticles include: dispersing Au / SiO2 nanoparticles in anhydrous ethanol to obtain an Au / SiO2 dispersion with a concentration of 0.4-0.6 mg / mL, adding aminopropyltriethoxysilane (APTES) under ultrasonic conditions, mixing evenly, condensing and refluxing at 100-120°C for 12-24 h under stirring conditions, and obtaining amino-functionalized Au / SiO2 nanoparticles by centrifugation and washing.

[0018] More preferably, the Au / SiO2 nanoparticles are Janus-structured Au / SiO2 nanoparticles prepared by partially depositing silicon dioxide on the surface of gold nanoparticles.

[0019] Preferably, the biological enzyme is an enzyme with a physiological marker released by a physiological lesion as a substrate, wherein at least one biological enzyme can respond to a physiological marker specific to the lesion site. The type of biological enzyme in the present invention can be specifically selected according to the type of tumor or the type of disease. The "fuel" of the nanomotor in the present invention may include but is not limited to characteristic physiological markers released by lesions such as hydrogen peroxide, urea, superoxide anion, lactic acid, glutathione, etc. Correspondingly, the biological enzyme includes a mixture of at least two of any combination and ratio of biological enzymes such as catalase, urease, superoxide dismutase, lactate oxidase, glutathione, etc.

[0020] The present invention uses two or more biological enzymes that use markers released by physiological lesions (such as breast cancer or liver cancer) as substrates to construct a nanomotor that is driven under a physiological environment and has enhanced targeting behavior. Micro-nanomotors modified with biological enzymes have chemotaxis to high-concentration substrates, for example, nanomotors modified with catalase have chemotaxis to high-concentration hydrogen peroxide, and the same is true for others. Therefore, the present invention utilizes the synergy of the chemotaxis of multiple biological enzymes to high-concentration "fuels" to achieve chemotactic enhancement of nanomotors to target areas and improve their targeting efficiency to tumor cells. The present invention can select different combinations of biological enzymes according to the differences in the lesion environment, and is expected to be applied to fields such as drug delivery and bioimaging to improve the targeting rate of nanomedicines.

[0021] Preferably, the content of biological enzyme loaded on the surface of the nanomotor is 50-250 mg / g, including but not limited to 50 mg / g, 60 mg / g, 70 mg / g, 80 mg / g, 90 mg / g, 100 mg / g, 110 mg / g, 120 mg / g, 130 mg / g, 140 mg / g, 150 mg / g, 160 mg / g, 180 mg / g, 200 mg / g, 220 mg / g, 250 mg / g, etc.; the content of loaded biological enzyme is closely related to the surface properties of the carrier. Too low content per unit specific surface area will lead to insufficient driving force of the nanomotor, affecting its movement in a physiological environment; too high content per unit specific surface area will cause enzyme aggregation, which will reduce enzyme activity and may also cause biological toxicity.

[0022] In a second aspect, the present invention provides a method for preparing a nanomotor with enhanced targeting behavior, comprising the following steps: A carrier, glutaraldehyde and two or more biological enzymes are used as raw materials to carry out a glutaraldehyde chemical cross-linking reaction to obtain a nanomotor.

[0023] Preferably, the glutaraldehyde chemical cross-linking reaction specifically includes: dispersing the carrier in deionized water by ultrasonic treatment to obtain a carrier dispersion; The carrier dispersion liquid and the glutaraldehyde aqueous solution are mixed to perform an aldehyde reaction to obtain an aldehyde reaction product; The product of the aldehyde reaction is dispersed in an aqueous solution of a biological enzyme, and an enzyme immobilization reaction is performed to obtain a nanomotor.

[0024] Further preferably, the ratio between the carrier and glutaraldehyde is 1 mg: (20-25) μg; the ratio between the mass of the carrier and the total mass of the biological enzyme is 1: (2.5-3.5).

[0025] More preferably, the concentration of the carrier dispersion is 0.5-1.5 mg / mL, including but not limited to 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, etc.; the mass concentration of the glutaraldehyde aqueous solution is 20-25%, including but not limited to 20%, 22%, 24%, 25%, etc.

[0026] More preferably, the total concentration of the biological enzyme in the aqueous solution of the biological enzyme is 1 to 5 mg / mL, including but not limited to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, etc.; when there are two biological enzymes, their molar ratio includes (0.2 to 5):1, including but not limited to 0.2:1, 0.3:1, 0.5:1, 1:1, 3:1, 5:1, etc.

[0027] More preferably, the aldehyde reaction is carried out by shaking at 20-28°C for 1.5-2.5 h; and the enzyme immobilization reaction is carried out by shaking at 20-28°C for 11-13 h.

[0028] In a third aspect, the present invention provides a nanomotor with enhanced targeting behavior for use in anti-tumor drugs and / or cancer diagnostic agents.

[0029] Preferably, the cancer comprises breast cancer or liver cancer.

[0030] In a fourth aspect, the present invention provides a nanomotor with enhanced targeting behavior as a targeted active drug delivery carrier and / or a bioimaging agent.

[0031] The main mechanism of action of the present invention is: (1) The present invention uses micro-nanoparticles with asymmetric structures or intrinsic asymmetry and rich surface amino groups and / or hydroxy groups as carriers, which have more surface active sites and are conducive to the loading of biological enzymes; (2) Two or more biological enzymes are linked to the surface of the carrier, which can be driven synergistically using multiple substrates released by physiological lesions as "fuel" to construct a self-driving micro-nano motor with enhanced mobility compared to a single fuel drive; (3) Using dual-fuel synergistic chemotaxis, in which at least one biological enzyme can catalyze cancer site-specific substrates, enhance the chemotactic selectivity of nanomotors for cancer cells, and achieve efficient targeted enrichment of nanomotors in cancer cell areas; (4) The nanomotor obtained by the present invention is modulatable. According to the differences in the multiple microenvironments of the target site and the non-target area, different combinations of biological enzymes can be selected to construct a nanomotor that can enhance the drive and chemotaxis in a specific microenvironment, thereby enhancing targeted drug delivery.

[0032] (5) The nanomotor of the present invention is prepared by the glutaraldehyde chemical cross-linking method. One side is connected to the carrier through the aldehyde groups at both ends of glutaraldehyde, and the other side is connected to the biological enzyme. The preparation steps are simple and easy to operate, which is conducive to industrialization.

[0033] The present invention is further described in detail below through specific examples. Among them, the raw materials not specifically specified are all commercially available raw materials; the preparation steps of the amino-functionalized Au / SiO2 nanocarrier include: dispersing 5 mg of Au / SiO2 nanoparticles in 10 mL of anhydrous ethanol, and then adding 125 μL of APTES to the above Au / SiO2 dispersion while ultrasonicating, and then refluxing and magnetically stirring at 110 °C for 20 h (speed 3). Then, the product is collected by centrifugation, and then washed alternately with water and ethanol three times to remove the unreacted APTES to obtain an amino-functionalized Au / SiO2 nanocarrier. Among them, the Au / SiO2 nanoparticles are Janus-structured Au / SiO2 nanoparticles prepared by depositing silica on the surface of gold nanospheres; the specific preparation method thereof refers to Chemical Science, 2022, 13(44): 13060-13067. The polydopamine carrier is a mesoporous bowl-shaped polydopamine nanoparticle prepared by a dual template method, and specific reference is made to the literature Angew. Chemie Int. Ed. 2018, 57, 6176–6180.

[0034] Example 1 A method for preparing a nanomotor with enhanced targeting behavior, wherein an amino-functionalized Au / SiO2 nanoparticle carrier, glutaraldehyde, catalase and urease are mixed and reacted to load catalase and urease on the surface of Au / SiO2, and the method specifically comprises the following steps: Take 1 mg of amino-functionalized Au / SiO2 nanoparticle carrier and disperse it in 0.9 mL of deionized water. After ultrasonic treatment for 10 minutes, the carrier dispersion was obtained; then 100 μL of 25% glutaraldehyde aqueous solution was added and placed in an oscillator at a temperature of 25°C and started to oscillate. After 2 hours of oscillation, the aldehyde formation process was completed.

[0035] The product of the aldehyde reaction was taken out and washed three times with deionized water, and then redispersed in 1 mL of enzyme-water solution (total concentration was 3 mg / mL, and included catalase and urease in a molar ratio of 1:3), and the enzyme was immobilized after oscillation at 25 °C for 12 h. The enzyme immobilization reaction product was repeatedly washed three times with water to obtain a nanomotor. The average particle size of the nanomotor was about 63 nm. The content of the loaded biological enzyme was measured by a protein detection kit and was 203 mg / g.

[0036] Example 2 The only difference from Example 1 is that the molar ratio of catalase and urease (n cat :n ure) was 5:1, 3:1, 1:1, 1:5, and the other steps and conditions were the same as in Example 1. The contents of the loaded biological enzyme were 76 mg / g, 92 mg / g, 156 mg / g and 166 mg / g, respectively.

[0037] This shows that the ratio between biological enzymes will affect their final loading capacity; and the ratio of catalase to urease in the prepared nanomotor can be controlled by regulating the ratio of enzymes in the solution.

[0038] Example 3 The only difference from Example 1 is that the concentration of the enzyme-water solution was adjusted to 1 mg / mL, 2 mg / mL, 4 mg / mL, and 5 mg / mL, respectively. The other steps and conditions were the same as those in Example 1, and the contents of the loaded biological enzyme were 43, 126, 221, and 234 mg / g, respectively.

[0039] This shows that the concentration of the biological enzyme will affect the loading capacity. As the concentration of the biological enzyme increases, the loading capacity first increases significantly. After reaching a certain concentration, the loading capacity does not increase much. Therefore, the total concentration of the biological enzyme in the aqueous solution of the preferred biological enzyme of the present invention is 1 to 5 mg / mL, and more preferably 2 to 4 mg / mL.

[0040] Structural characterization and performance testing 1. Characterization of amino functional carriers and nanomotors: The amino-functionalized Au / SiO2 nanoparticle carrier (Janus Au / SiO2-NH2) and the prepared nanomotors (JASNMs) used in Example 1 were characterized. Figure 1 and Figure 2 shown.

[0041] Figure 1 In the figure, (A) the infrared test results show that the surface of the carrier is rich in amino and hydroxyl groups and the presence of biological enzymes in the motor; (B) the Zeta potential characterization shows that the surface of the amino-supported carrier is positively charged, and the surface is negatively charged after the enzyme is linked, further proving the successful loading of the biological enzyme.

[0042] Figure 2 In the figure, (A) TEM characterizes the asymmetric structure of the nanomotor obtained by the present invention; (B) the distribution of Fe element in the surface scanning EDS basically coincides with the distribution of Si, indicating that the loaded biological enzyme is mainly distributed on the SiO2 side.

[0043] 2. Motility of nanomotors in urea solution: The motion characteristics of the prepared nanomotor were characterized using an inverted optical microscope (Leica DMI 3000 M). Specifically, 10 μL of urea solution of different concentrations (0-100 mM) was added to the ultrasonically cleaned coverslip, and then 1 μL of 1 mg / mL aqueous suspension of the nanomotor was dripped in. After the system was stabilized, the motion behavior of the nanomotor was recorded using an inverted dark field microscope; the motion trajectory and corresponding coordinates of the nanomotor were recorded using commercial software (Video Spot Tracker), and then the MSD and diffusion coefficient of the nanomotor were obtained. The results are shown in Figure 2. Figure 3 shown.

[0044] Depend on Figure 3 It can be seen that the nanomotor prepared in Example 1 exhibits significantly enhanced diffusion in a physiological level of urea (2.5-11.8 mM) solution compared to Brownian motion (i.e., the group with a urea solution concentration of 0 mM), and its diffusion coefficient has a fuel concentration-dependent characteristic, increasing with the increase of urea concentration. The test results show that the prepared nanomotor can be driven at a physiological urea concentration.

[0045] 3. Motility of nanomotors in hydrogen peroxide solution: An inverted optical microscope (Leica DMI 3000 M) was used to characterize the motion characteristics of the prepared nanomotor. Specifically, 10 μL of hydrogen peroxide solution of different concentrations (0-500 μM) was added to the ultrasonically cleaned coverslip, and then 1 μL of 1 mg / mL nanomotor aqueous suspension was dripped in. After the system was stabilized, the motion behavior of the nanomotor was recorded using an inverted dark field microscope; the motion trajectory and corresponding coordinates of the nanomotor were recorded using commercial software (Video Spot Tracker), and then the MSD and diffusion coefficient of the nanomotor were obtained. The results are shown in Figure 2. Figure 4 shown.

[0046] Depend on Figure 4 It can be seen that the nanomotor prepared in Example 1 exhibits significantly enhanced diffusion compared to Brownian motion in a physiological level of hydrogen peroxide (10-100 µM) solution, and its diffusion coefficient has a fuel concentration-dependent characteristic, increasing with the increase of hydrogen peroxide concentration. This indicates that the prepared nanomotor can be driven at a physiological hydrogen peroxide concentration.

[0047] 4. Mobility of nanomotors in dual-fuel solutions: The motion characteristics of the prepared nanomotor were characterized using an inverted optical microscope (Leica DMI 3000 M). Specifically, 10 μL of hydrogen peroxide and urea solutions of different concentrations were added to the ultrasonically cleaned coverslip, including 10 μM H2O2 + 5 mM urea, 50 μM H2O2 + 5 mM urea, and 50 μM H2O2 + 10 mM urea; then 1 μL of 1 mg / mL aqueous suspension of the nanomotor was dropped in respectively, and after the system was stabilized, the motion behavior of the nanomotor was recorded using an inverted dark field microscope; the motion trajectory and corresponding coordinates of the nanomotor were recorded using commercial software (Video Spot Tracker), and the MSD and diffusion coefficient were subsequently obtained. The results are shown in Figure 2. Figure 5 shown.

[0048] Depend on Figure 5 It can be seen that the nanomotors prepared in Example 1 and Example 2 showed an increased diffusion coefficient compared to the single fuel (hydrogen peroxide or urea) system under the condition of the co-existence of hydrogen peroxide and urea, indicating that their mobility was improved. Figure 5 The results showed that the optimal ratio of catalase to urease in the construction of the nanomotor was 1:3, which depended on the motor's motility in dependence on hydrogen peroxide and urea concentrations, as well as the concentrations of hydrogen peroxide and urea at physiological levels.

[0049] 5. Chemotaxis of nanomotors under different fuel gradient conditions: Schematic diagram of the chemotaxis assay mechanism Figure 6 As shown, the specific operation is to pre-add 10 µM H2O2 (or 5 mM urea) aqueous solution into the microfluidic device, add the nanomotor obtained in Example 1 into the left reservoir of the microfluidic device; place an agar block fully swollen in 100 µM H2O2 (or 10 mM urea) aqueous solution in the right reservoir, and the chemotaxis enrichment observation position is basically at the center of the microfluidic device. An inverted optical microscope (Leica DMI 3000 M) is used to observe the movement behavior of the nanomotor between the left reservoir and the center of the microfluidic device, and examine the chemotaxis behavior of the nanomotor under a physiological H2O2 (urea) concentration gradient. The results are shown in FIG. Figure 7 shown.

[0050] Depend on Figure 7 It can be seen that the prepared nanomotor exhibits obvious directional movement under physiological urea or H2O2 gradient, indicating that the nanomotor has chemotactic behavior of directional migration towards high-concentration fuel in a single-fuel system.

[0051] The operation steps of the chemotaxis experiment in the dual-fuel system are similar to those in the single-fuel system, except that a mixed solution of 10 µM H2O2 and 5 mM urea is added to the microfluidic device in advance, and the nanomotor obtained in Example 1 is added to the left reservoir of the microfluidic device; an agar block fully swollen in a mixed solution of 100 µM H2O2 and 10 mM urea is placed in the right reservoir, and the movement behavior of the nanomotor is observed using an inverted optical microscope (Leica DMI 3000 M) to examine the chemotaxis behavior of the nanomotor under the dual-fuel concentration gradient.

[0052] Figure 7 B and C are the mass displacement and directionality of the prepared nanomotor under the conditions of no gradient I (control), H2O2 gradient II, urea gradient III and dual fuel gradient IV. As can be seen from the figure, in the presence of H2O2 and urea gradients, the mass displacement and movement directionality of the prepared nanomotor per unit time have been greatly improved, and their values ​​are significantly higher than the sum of the two single fuel systems, indicating that its chemotactic behavior is significantly enhanced.

[0053] 6. Group chemotaxis of nanomotors: The prepared nanomotor was labeled with anthocyanin cy5 fluorescent dye. Specifically, 0.5 mg Cy5 was added to 1 mL of 1 mg / mL nanomotor water suspension, shaken overnight at room temperature, and then washed with deionized water until the supernatant became transparent.

[0054] The chemotactic behavior of the prepared nanomotors was investigated using Figure 8 The microfluidic device shown in the figure was tested. A mixed solution of 10 μM hydrogen peroxide and 5 mM urea was pre-added to the chemotaxis device to simulate the fuel concentration in a normal physiological environment. Agar blocks pre-soaked in 10 μM hydrogen peroxide and 5 mM urea, 50 μM hydrogen peroxide and 10 mM urea, and 10 μM hydrogen peroxide and 10 mM urea were added to the b, c, and d areas of the microfluidic device, respectively. The cy5-labeled nanomotor was added to area a, and the changes in fluorescence intensity in areas a, b, c, and d within 60 min under different gradient conditions were observed. The results are shown in FIG. Fig. 9 shown.

[0055] Depend on Fig. 9It can be seen that the fluorescence intensity of region a gradually decreases with time, indicating that the number of nanomotors in region a gradually decreases, while the increase in fluorescence intensity in regions b, c, and d indicates that the number of nanomotors migrating thereto gradually increases. Among them, the fuel concentration contained in the agar block placed in region b is consistent with that in the device, indicating that the migration of the motor to region b mainly depends on its Brownian diffusion, the migration to region c depends on its response to the hydrogen peroxide gradient, and the migration to region d represents its response to the hydrogen peroxide and urea gradients. The significantly increased fluorescence intensity in region d indicates that the prepared nanomotors have higher chemotactic efficiency in the presence of hydrogen peroxide and urea gradients, which can be used to achieve its selective targeting to specific regions.

[0056] 7. Selective targeting behavior of nanomotors on liver cancer cells and breast cancer cells: Xiang Ru Figure 8 Lysates (5 × 10 50 cells / mL) of normal breast epithelial cells (MCF-10), breast cancer cells (MCF-7), and liver cancer cells (HepG2) were added to the b, c, and d regions of the microfluidic device, respectively. 6 The agar blocks were immersed in 4% paraformaldehyde (P < 0.05) per mL for 12 h, and the cy5-labeled nanomotors were added to area a. The fluorescence intensity changes of areas b, c, and d at 30, 60, and 90 min, respectively, were observed using a fluorescence microscope.

[0057] The results are as follows Fig.10 and Fig.11 As shown, the prepared nanomotor exhibited efficient enrichment in the liver cancer cell area, with the enrichment rate being 4 times higher than that in the normal cell area and 1.5 times higher than that in the breast cancer cell area. This indicates that the nanomotor obtained by the present invention has selective targeting behavior for both liver cancer cells and breast cancer cells, especially liver cancer cells.

[0058] Comparative Example 1 The urease-driven pot-shaped micro-nano motor prepared in Example 1 of patent CN110182750A - A urease-driven pot-shaped micro-nano motor and its preparation method is used.

[0059] The results showed that the average size of the motor was 2.3 μm, the driving force came from the bubble recoil caused by the confinement effect of the can-shaped space, and it did not have physiological level of hydrogen peroxide responsiveness.

[0060] Comparative Example 2 The enzyme-driven nanomotor prepared in Example 1 of patent CN 118286458A - an enzyme-driven chemotactic self-navigating upconversion colloid motor and its preparation method and application is adopted.

[0061] The results showed that the motor was a core-shell structure particle, and the construction of the motor asymmetry relied on physical encapsulation, which was cumbersome to operate and had poor uniformity. It could not achieve physiological levels of fuel drive and gradient response when moving in a 50 mM urea solution and performing chemotaxis under a 50 mM urea gradient.

[0062] In summary, the present invention provides a nanomotor with enhanced targeting behavior and its preparation method and application, which utilizes the specific catalysis of multiple biological enzymes-substrates to enhance the mobility of the nanomotor in a physiological environment, effectively improving the driving force and chemotaxis efficiency of the nanomotor in a physiological environment; utilizing the differences in multiple microenvironments between cancer cells and non-target areas (such as hydrogen peroxide and urea concentrations) and the chemotaxis synergy of multiple biological enzymes to enhance the targeting ability of the nanomotor to cancer cells. Therefore, the present invention combines the specific catalysis of biological enzymes-substrates and the chemotaxis of biological enzymes to enhance the mobility and autonomous targeting ability of the nanomotor in a physiological environment. Compared with the prior art, the present invention has the following advantages: First, it can enhance the driving force of the nanomotor in a physiological environment; second, it has modulatable chemotaxis; third, it can select a targeted multi-signal response area.

[0063] The present invention not only provides a nanomotor that can efficiently respond to the heterogeneity of cancers such as liver tumors, but also proposes a strategy for constructing nanomotors that can overcome the off-target problem caused by single signal interference in complex environments and improve the targeting rate, which is of great significance to the application of micro-nanomotors in the biomedical field.

[0064] 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 nanomotor with enhanced targeting behavior, characterized in that: It includes a carrier, and two or more biological enzymes modified on the surface of the carrier; the carrier is a micro-nano particle rich in active groups; the biological enzyme is an enzyme that uses a marker released by a physiological lesion as a substrate and has substrate concentration gradient chemotaxis.

2. The nanomotor with enhanced targeting behavior according to claim 1, characterized in that: The particle size of the carrier is 10 nm to 1000 nm.

3. The nanomotor with enhanced targeting behavior according to claim 1, characterized in that: The support includes amino-functionalized Au / SiO2 nanoparticles.

4. The nanomotor with enhanced targeting behavior according to claim 1, characterized in that: The markers include at least two of hydrogen peroxide, urea, superoxide anion, lactate and glutathione; The biological enzymes include at least two of catalase, urease, superoxide dismutase, lactate oxidase, and glutathionease.

5. The nanomotor with enhanced targeting behavior according to claim 1, characterized in that: The total content of the biological enzyme loaded on the surface of the nanomotor is 50-250 mg / g.

6. The method for preparing a nanomotor with enhanced targeting behavior according to any one of claims 1 to 5, characterized in that: The following steps are involved: The nano motor is prepared by using a carrier, glutaraldehyde and two or more biological enzymes as raw materials and carrying out a glutaraldehyde chemical cross-linking reaction.

7. The method for preparing a nanomotor with enhanced targeting behavior according to claim 6, characterized in that: The glutaraldehyde chemical cross-linking reaction specifically comprises: dispersing the carrier in deionized water by ultrasonic treatment to obtain a carrier dispersion; The carrier dispersion liquid and the glutaraldehyde aqueous solution are mixed to carry out an aldehyde reaction to obtain an aldehyde reaction product; The aldehyde reaction product is dispersed in an aqueous solution of a biological enzyme, and an enzyme immobilization reaction is performed to obtain a nanomotor.

8. The method for preparing a nanomotor with enhanced targeting behavior according to claim 7, characterized in that: The ratio between the carrier and glutaraldehyde is 1 mg: (20-25) μg; the ratio between the mass of the carrier and the total mass of the biological enzyme is 1: (2.5-3.5); The concentration of the carrier dispersion is 0.5 to 1.5 mg / mL; The mass concentration of the glutaraldehyde aqueous solution is 20-25%; The total concentration of the biological enzyme in the aqueous solution of the biological enzyme is 1 to 5 mg / mL; The aldehyde reaction is carried out by shaking at 20-28°C for 1.5-2.5 h; The enzyme immobilization reaction is carried out by shaking at 20-28°C for 11-13 hours.

9. Use of the nanomotor with enhanced targeting behavior as claimed in any one of claims 1 to 5 in the preparation of anti-tumor drugs and / or cancer diagnostic and therapeutic drugs, characterized in that: The cancer includes breast cancer or liver cancer.

10. Use of the nanomotor with enhanced targeting behavior as claimed in any one of claims 1 to 5 as a targeted active drug delivery carrier or a bioimaging agent.

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