Nano-robot for tumor starvation treatment and application of nano-robot

Abstract

The invention discloses a nano robot for tumor hunger treatment and application thereof.The nano robot comprises nano particles, driving enzyme and a hunger treatment part, the driving enzyme and the hunger treatment part are connected to the nano particles, and the driving enzyme drives the nano robot with chemical substances in a tumor microenvironment as fuel; the hunger treatment part can consume nutrient substances in the tumor microenvironment. The nano-robot provided by the invention can break through the limitation of high compactness and high interstitial fluid pressure of tumor tissues, and has better infiltration capacity in tumors, so that the consumption of key nutrient substances in the tumors is enhanced in combination with a hunger treatment part, and a remarkable tumor inhibition effect is realized. A new tool and a new idea are provided for tumor starvation treatment, the treatment effect is excellent, the safety is high, and therefore the good clinical application potential is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of micro-nano devices, and particularly relates to a nano-robot for tumor starvation therapy, its preparation method and application. Background Art

[0002] Starvation therapy is a treatment method that inhibits tumor growth by consuming the nutrients required by tumors. In the past decade, by combining catalytic enzymes (such as glucose oxidase) with nanoparticles, a variety of nano-drug delivery systems for tumor starvation therapy have been developed. These nano-drug delivery systems based on passive diffusion are difficult to deeply penetrate and internalize into tumor tissues with high density and high interstitial fluid pressure, resulting in limited anti-tumor effects.

[0003] A nano-robot is an artificial micro-nano device that can convert other forms of energy in the surrounding environment into kinetic energy to achieve autonomous movement. As a drug delivery carrier, nano-robots are expected to significantly improve the penetration depth and cellular internalization efficiency of anti-tumor drugs in tumor tissues. In recent years, by combining glucose oxidase / catalase with nanoparticles, nano-robots using glucose as fuel have been developed, and they can be effectively driven in a high-concentration glucose solution (>25 mM). However, due to the usually low glucose concentration (0.1-0.8 mM) in the tumor microenvironment, the existing glucose-fueled nano-robots cannot move in the actual tumor microenvironment, and their anti-tumor effects have not been significantly improved compared with the nano-drug delivery systems based on passive diffusion. Summary of the Invention

[0004] In view of this, the present invention aims to develop a nano-robot that can break through the limitations of high density and high interstitial fluid pressure of tumor tissues, improve the penetration and uptake efficiency of nano-robots inside tumors, and then conduct effective tumor starvation therapy by promoting large-scale nutrient deprivation.

[0005] The technical solution of the present invention is specifically as follows: In the first aspect of the present invention, a nano-robot for tumor starvation therapy is provided, including nanoparticles, a driving enzyme and a starvation therapy component connected to the nanoparticles; wherein, the driving enzyme drives the nano-robot with a chemical substance in the tumor microenvironment as fuel, and the starvation therapy component is used to consume the nutrients in the tumor microenvironment.

[0006] In the nanorobot of the present invention, the chemical substances that can serve as fuels in the tumor microenvironment include, but are not limited to, urea, lactic acid, glutathione, etc. Therefore, the driving enzymes can correspondingly be urease, lactate oxidase, glutamyl transpeptidase, etc. Driven by the driving enzymes, the nanorobot effectively improves its diffusion range within the tumor, and then enhances the ability to consume key nutrients in the tumor through the starvation therapy component. However, it should be noted that due to the high density and high interstitial fluid pressure of tumor tissues, not all chemical substances in the tumor microenvironment and their corresponding driving enzymes can effectively drive the nanorobot within the tumor.

[0007] Preferably, in the nanorobot of the present invention, the driving enzyme is urease, which catalyzes a reaction with urea as the substrate, thereby effectively driving the diffusion of the nanorobot within the tumor.

[0008] It can be understood that the key nutrients in the tumor include, but are not limited to, glucose, lactic acid, lipids, amino acids, etc. In some embodiments of the present invention, for example, the starvation therapy component includes glucose oxidase, and glucose oxidase inhibits the growth and proliferation of tumors by competitively consuming glucose. Based on the change of the target nutrient, glucose oxidase can be replaced with other starvation therapy components, such as lactate oxidase, arginase, glucose oxidase-like inorganic catalysts, lactate oxidase-like inorganic catalysts, glutamine inhibitors, etc. Additionally, when the starvation therapy component contains glucose oxidase, an enzyme that provides oxygen can also be included. The inherent hypoxic environment of tumor tissues hinders the effectiveness of oxygen-dependent starvation therapy. By introducing peroxidase (such as catalase) or peroxidase-like inorganic catalysts (such as Prussian blue nanoparticles, platinum nanoparticles, etc.), the disproportionation reaction of hydrogen peroxide can be triggered to generate oxygen, accelerating the consumption of glucose.

[0009] More preferably, in the nanorobot of the present invention, the starvation therapy component is composed of catalase and glucose oxidase, and the cascade reaction of the two can accelerate the consumption of nutrients within the tumor.

[0010] In the nanorobot of the present invention, the driving enzyme and the starvation therapy component can be connected to the nanoparticle by means of sulfur-metal bonds, the bridging effect between streptavidin and biotin, chemical coupling, electrostatic adsorption, etc.

[0011] In the nanorobot of the present invention, the connection methods of urease, catalase, and glucose oxidase include the following situations: Urease, catalase, and glucose oxidase are connected in the same layer or in different layers on the same side of the nanoparticle, forming an asymmetric structure; Urease, catalase, and glucose oxidase are connected in different layers or in the same layer. Urease is connected to one side of the nanoparticle, and catalase and glucose oxidase are connected to the entire surface of the nanoparticle; Urease, catalase, and glucose oxidase are connected in layers or in the same layer on the entire surface of the nanoparticles, forming a relatively symmetrical structure.

[0012] In the above situation, when urease, catalase, and glucose oxidase are connected in layers, the connection order is not limited.

[0013] Preferably, in the nanorobot of the present invention, urease is asymmetrically connected to one side of the nanoparticle; this method is more conducive to the diffusion of the nanorobot in tumors.

[0014] In the nanorobot of the present invention, the nanoparticles include but are not limited to nanogold, nanosilica (such as mesoporous silica, etc.), nanoliposomes, nanomagnetic particles (such as magnetite, ferric oxide, etc.), nanopolydopamine, etc. with different particle sizes. In some embodiments of the present invention, the nanoparticles are nanogold; the nanogold particles not only have adjustable sizes (from ten to several hundred nanometers), but also have unique optoelectronic, physical and chemical properties and good biocompatibility.

[0015] Preferably, in the nanorobot of the present invention, the size of the nanorobot is 20 - 200 nm; the nanorobot with this size has better diffusion performance in tumors and can be taken up into the interior of tumor cells, and then interfere with the metabolism of tumor cells by competitively consuming the nutrients of tumor cells, achieving a better therapeutic effect.

[0016] The second aspect of the present invention provides a method for preparing a nanorobot for tumor starvation therapy, wherein the nanorobot uses nanogold as the matrix material, and urease, catalase, and glucose oxidase are asymmetrically connected to the surface of the nanogold, specifically including the following steps: S1. Prepare gold - polystyrene eccentric nanoparticles; S2. Biotinylate urease, catalase, and glucose oxidase respectively to obtain biotinylated urease, biotinylated catalase, and biotinylated glucose oxidase; S3. Use streptavidin to couple biotinylated urease, biotinylated catalase, and biotinylated glucose oxidase on the same side of the gold - polystyrene eccentric nanoparticles.

[0017] Preferably, in the above method, step S1 includes the following process: S11. Prepare nanogold by the seed - mediated growth method, and different - sized nanogold particles can be obtained by controlling the number of growth times; S12. Mix potassium persulfate, sodium 4 - styrenesulfonate, water, and ethanol and pre - heat them, then add the mixture of styrene and divinylbenzene, and then add nanogold for reaction to obtain gold - polystyrene eccentric nanoparticles.

[0018] Preferably, step S3 is specifically: S31 disperses gold-polystyrene eccentric nanoparticles in water, adds SH-PEG-Biotin (mercapto-polyethylene glycol-biotin), then adds tetrahydrofuran and an mPEG-SH (methoxy-polyethylene glycol-mercapto) solution. After mixing, it is mixed with streptavidin and dispersed in a PB solution; S32: Take the above mixed solution, and gradually or simultaneously add biotinylated glucose oxidase, biotinylated catalase, and biotinylated urease for reaction to obtain a nano-robot with three enzymes assembled in layers or in the same layer.

[0019] Preferably, the molar ratio of biotinylated urease, biotinylated catalase, and biotinylated glucose oxidase coupled in step S3 is 1:1:1.

[0020] The third aspect of the present invention provides the application of the nano-robot for tumor starvation therapy of the present invention in the preparation of anti-tumor drugs or medical devices.

[0021] The fourth aspect of the present invention provides an anti-tumor drug, which uses the nano-robot for tumor starvation therapy of the present invention as an active ingredient. It can be understood that the anti-tumor drug may further include pharmaceutically acceptable excipients.

[0022] For the anti-tumor drug provided by the present invention, its preferred administration method is in-situ injection.

[0023] The beneficial effects of the present invention are as follows: The nano-robot provided by the present invention can break through the limitations of high tumor tissue density and high interstitial fluid pressure, be effectively driven in the tumor by urease, improve its intratumoral distribution, increase the tumor cell uptake efficiency, and thus significantly improve the tumor suppression effect; the nano-robot also carries a tumor starvation therapy component that competitively consumes or inhibits nutrients in the tumor. When the tumor starvation therapy component includes glucose oxidase and catalase, the cascade reaction between the two can accelerate the consumption of nutrients. Animal experiments of the present invention show that the tumor inhibition rate can be increased to 66.7%, which is significantly higher than that of ordinary single-enzyme or double-enzyme nano-robots.

[0024] The nano-robot provided by the present invention can replace the types of carriers and functional molecules according to actual application needs without affecting its function, and has extremely high plasticity; moreover, since the nano-robot realizes tumor treatment through the combined action of enhancing intratumoral penetration and consuming key nutrients, its scope of application is wide, including but not limited to skin cancer, gastric cancer, liver cancer, breast cancer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic structural diagram of the nano-robot for tumor starvation therapy provided by the present invention.

[0026] Figure 2 It is the morphological structure characterization diagram of the nano-robot prepared in Example 2 of the present invention.

[0027] Figure 3 It is the diagram of the diffusion coefficient of the nano-robot prepared in Example 2 of the present invention varying with the fuel.

[0028] Figure 4 It is the detection result diagram of the penetration ability of the nano-robot for tumor starvation therapy in the present invention within 3D tumor spheres.

[0029] Figure 5 It is the treatment effect diagram of the nano-robot for tumor starvation therapy in the present invention in biological experiments.

[0030] Figure 6 It is the biological safety characterization diagram of the nano-robot for tumor starvation therapy in the present invention in biological experiments. Detailed implementation manners

[0031] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments and the accompanying drawings. It should be understood that the embodiments described herein are exemplary only and are used to illustrate and explain the present invention, but not to limit the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms "comprising" and "having" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusion.

[0033] For those not specifying specific technologies or conditions in the following embodiments, they are all carried out according to the technologies or conditions described in the literature in this field or according to the product specifications; for the reagents or instruments not indicating the manufacturers, they are all conventional products that can be obtained through commercial purchase.

[0034] Example 1 This example provides a nano-robot for tumor starvation therapy, and its structure and mechanism are as Figure 1 shown. This nano-robot uses nano-gold as the main body, and on one side of the nano-gold, biotinylated glucose oxidase, biotinylated catalase, and biotinylated urease are assembled in layers using biotinylated polyethylene glycol and streptavidin. Among them, glucose oxidase catalyzes the decomposition of glucose into gluconic acid and hydrogen peroxide, and catalase further decomposes hydrogen peroxide into water and oxygen. The two are cascaded to accelerate the consumption of glucose in tumors, thereby achieving the inhibition of tumor growth; urease is the driving enzyme, which is used to increase the penetration efficiency of the nano-robot in the tumor microenvironment, improve the dispersibility and uptake efficiency of the nano-robot in tumor tissues, and improve the tumor inhibition efficiency.

[0035] Example 2 This example provides a method for preparing the nanorobot with the structure shown in Example 1, specifically including the following steps: (1) Preparation of nanogolds with different sizes.

[0036] Synthesis of 13 nm nanogold seeds: Mix 100 mL of deionized water and a certain amount of 100 mM chloroauric acid aqueous solution, stir and heat in an oil bath at 150 °C until boiling, quickly add a certain amount of 38.8 mM sodium citrate aqueous solution, and react to obtain 13 nm nanogold seeds.

[0037] Primary growth of nanogold: Take 142.5 mL of deionized water, add a certain amount of 13 nm nanogold seed solution and a certain amount of 60 mM sodium citrate solution, stir and heat in an oil bath at 90 °C. After the system is stable, add a certain amount of 25 mM chloroauric acid solution in two portions and stir in the oil bath for 30 min to complete the primary growth of nanogold.

[0038] Subsequent growth of nanogold: Take out about 1 / 3 of the sample solution, add an equal amount of deionized water and a certain amount of 60 mM sodium citrate solution to the original three-necked flask, add a certain amount of 25 mM chloroauric acid solution in two portions, add a certain amount of 25 mM chloroauric acid solution in two portions and stir in the oil bath for 30 min. By controlling the growth times, nanogold particles with different sizes can be obtained.

[0039] (2) Preparation of gold–polystyrene eccentric nanoparticles.

[0040] First, add 2.5 mL of water, 16.5 mL of ethanol, 1 mL of 50 mg / mL potassium persulfate solution, and 1 mL of 6 mg / mL sodium 4-styrenesulfonate solution to a three-necked flask, stir and heat in an oil bath at 70 °C for 10 min. Secondly, add 200 μL of the mixed solution (V styrene :V DVB = 200:1) and react for a period of time. Then add 3 mL of nanogold (90 nm), continue to stir and heat in the oil bath for 4 hours, and collect by centrifugation to obtain asymmetric gold–polystyrene eccentric nanoparticles.

[0041] (3) Preparation of biotinylated urease, biotinylated catalase, and biotinylated glucose oxidase.

[0042] Respectively mix urease solution, catalase solution, and glucose oxidase solution with biotinylation reagent, and shake evenly at room temperature. Wash 5 times with deionized water, dissolve in PB solution (10 mM, pH = 7.4), and store at 4 °C for later use.

[0043] (4) Preparation of nanorobots.

[0044] First, the obtained asymmetric gold–polystyrene eccentric nanoparticles were dispersed in 1 mL of ultrapure water, and 0.1 mL of mPEG-SH solution (1 μg / mL) was added and mixed with oscillation. Secondly, tetrahydrofuran was added to the sample to remove polystyrene, and it was dispersed in 1 mL of ultrapure water. Then 0.1 mL of SH-PEG-Biotin solution (1 μg / mL) was added, and after oscillation, it was dispersed in PB solution (10 mM, pH = 7.4). Then, 100 μL of the above solution was taken and added to 100 μL of streptavidin solution (0.01 μg / mL) and reacted with oscillation. After that, 100 μL of the mixed solution was taken and added to 100 μL of biotinylated glucose oxidase solution and reacted with oscillation. Subsequently, following the same method, streptavidin and biotinylated catalase were first coupled, and then streptavidin and biotinylated urease were continuously coupled. After the reaction was completed, the nanorobots for tumor starvation therapy were obtained by washing. The coupling amount ratio of glucose oxidase, hydrogen peroxide, and urease was 1:1:1.

[0045] The morphological structure of the nanorobots prepared in this example was characterized, and the results are as Figure 2 shown. Figures 2A and 2B are the transmission electron microscopy image and elemental analysis image of the nanorobots respectively (the scale bars are both 100 nm). According to the images, glucose oxidase, catalase, and urease are asymmetrically distributed on one side of the nanorobots, forming an asymmetric structure.

[0046] The motility of the nanorobots prepared in this example in urea solutions and glucose solutions with different concentrations was detected. The specific method was as follows: The nanorobots were placed in different solutions, and their diffusion coefficients were measured. The results are as Figure 3 shown: The nanorobots showed enhanced motility behavior dependent on urea concentration in urea aqueous solutions, and the nanorobots could be effectively driven at the urea concentration in the human body environment (2.5 - 7.5 mM) ( Figure 3 A); at the same time, the nanorobots showed enhanced motility behavior dependent on glucose concentration in glucose aqueous solutions, and they could be driven at the glucose concentration in the human blood circulation system (~7.5 mM), but it was difficult to move effectively at the low glucose concentration in the tumor microenvironment (0.1 - 0.8 mM) ( Figure 3 B). The above results indicate that it is difficult for the nanorobots to break through the limitations of the high density and high interstitial fluid pressure of tumor tissues on their motility relying on glucose oxidase, but the use of urease provides the possibility to solve the above problems.

[0047] Example 3 To verify the penetration and diffusion ability of the nanorobot for tumor starvation therapy of the present invention in tumor tissues, the present invention conducted a simulation experiment using 3D tumor spheres, specifically as follows: MCF-7 cells were dispersed in a complete medium containing methylcellulose and inoculated into a cell culture dish at a density of 1×10 5 cells / mL, and incubated inverted at 37 °C to grow into 3D tumor spheres with a diameter of about 500 μm; the nanorobots prepared in Example 2 were surface-modified with fluorescent groups and placed in a sugar-free medium with different urea concentrations (0 or 10 mM), and co-incubated with the 3D tumor spheres for 3 hours, and their penetration in the 3D tumor spheres was tested using a laser confocal microscope.

[0048] The detection results are as Figure 4 shown: in the presence of urea, the scanning results of different layers all showed stronger red fluorescence signals, indicating that the nanorobots have improved tumor penetration ability and cell uptake efficiency in the presence of urea ( Figure 4 A); at the same time, by reading the radial fluorescence intensity distribution of the central layer of the 3D tumor spheres, it can be seen that the nanorobots have stronger penetration ability (81 µm) in the urea-containing environment, which is 2.79 times the penetration depth (29 µm) of the nanorobots in the urea-free environment ( Figure 4 B).

[0049] Example 4 This example further demonstrated the significant advantages of the nanorobot for tumor starvation therapy of the present invention in tumor treatment through animal experiments. The specific experimental process and detection results are as follows: 2×10 6 4T1 tumor cells were directly subcutaneously implanted into BALB / c mice to establish a 4T1 tumor-bearing mouse model. The nanorobots prepared in Example 2 were resuspended with PBS and injected into the tumors of 4T1 breast cancer-bearing mice in situ, and the operation was repeated every two days for six times. The mice were sacrificed two days later. The tumor size and body weight of the mice were recorded every day, and hematoxylin & eosin staining was performed on the main organs (heart, liver, spleen, lung, kidney, etc.).

[0050] The control treatments (G1-G5) in this example only differed in the injected nanorobots. The preparation methods of the urease-driven nanorobots, glucose oxidase-driven nanorobots, glucose oxidase / urease-driven nanorobots, and glucose oxidase / catalase-driven nanorobots refer to Example 2, and different biocatalysts were assembled on one side of the gold nanoparticles through the bridging action of biotin and streptavidin.

[0051] The tumor suppression effect is as Figure 5 shown, where Figure 5 A shows the change of the mouse tumor over time, Figure 5 B is a tumor picture,Figure 5 C is the tumor weight. The treatment group with urease-driven nanorobots (G2) showed antitumor effects, and the tumor growth inhibition rate reached 28.3%. Although triggering the GOx / CAT cascade reaction, the treatment group with glucose oxidase / catalase-driven nanorobots (G5) only achieved a tumor growth inhibition rate of 18.3%. After introducing urease, the glucose oxidase / catalase / urease-driven nanorobots (G6) deeply penetrated and were efficiently internalized in tumors, triggering the effective consumption of glucose in tumors, and the tumor growth inhibition rate increased to 66.7%, which was 3.6 times that of the glucose oxidase / urease-driven nanorobots. Among them, the tumor growth inhibition rate = (1 - tumor volume of the treatment group / tumor volume of the control group) × 100%.

[0052] The biosafety is as Figure 6 shown, where Figure 6 A shows the change in the body weight of mice over time, Figure 6 B is the tissue section image of mice, Figure 5 C is the blood biochemical indexes of mice (glucose, urea, alkaline phosphatase, alanine aminotransferase, and total protein). The results show that the nanorobots provided by the present invention for tumor starvation therapy have good biosafety.

[0053] In summary, the nanorobots provided by the present invention can be effectively driven by urea in the tumor microenvironment, thereby significantly enhancing the penetration and uptake efficiency of the nanorobots inside the tumor. At the same time, the starvation therapy component is used to achieve efficient treatment of tumors by depriving a large range of nutrients.

[0054] It should be noted that the above embodiments are only a part of the embodiments of the present invention rather than all of them, and are only used to illustrate the technical solutions of the present invention rather than to limit them; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

Claims

1. A nanorobot for starvation therapy of tumors, characterized in that: It includes nanoparticles, a driving enzyme connected to the nanoparticles, and a starvation treatment component. The driving enzyme uses chemicals in the tumor microenvironment as fuel to drive the nanorobot, and the starvation treatment component can consume nutrients in the tumor microenvironment.

2. The nanorobot according to claim 1, characterized in that: The driving enzyme is urease, and the starvation treatment component is composed of catalase and glucose oxidase.

3. The nanorobot according to claim 2, characterized in that: The urease is asymmetrically distributed on one side of the nanoparticle.

4. The nanorobot according to claim 2, characterized in that: The urease, catalase and glucose oxidase are coupled on the surface of the nanoparticles in the same layer or in layers.

5. The nanorobot according to claim 2, characterized in that: The urease, catalase and glucose oxidase are linked to the nanoparticles via the bridging action between streptavidin and biotin.

6. The nanorobot according to claim 1, characterized in that: The nanoparticles are any one of nanogold, nanosilicon dioxide, nanoliposomes, nanomagnetic particles and nanopolydopamine.

7. Use of the nanorobot for tumor starvation therapy as claimed in any one of claims 1 to 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The anti-tumor drug is administered by in situ injection.

9. An anti-tumor drug, characterized in that: The nanorobot for tumor starvation treatment according to any one of claims 1 to 6 is used as the active ingredient.

10. The anti-tumor drug according to claim 9, characterized in that: Contains pharmaceutically acceptable excipients.

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