A light-driven heterojunction nanorobot and its preparation method and application

Abstract

The present invention discloses a light-driven heterojunction nanorobot and its preparation method and application. The light-driven heterojunction nanorobot uses palladium (Pd) nanoparticles as a carrier and is loaded with copper sulfide (CuS). The light-driven heterojunction nanorobot of the present invention uses Pd nanoparticles as a chassis, and loads CuS at its tail to form a heterojunction nanozyme, which can efficiently catalyze H2O2 to generate ·OH, mediate immunogenic death of cancer cells, and at the same time endow it with a power system, enabling it to move under light driving. In addition, by loading neutrophil extracellular vesicles modified with triphenylphosphine and loaded with lactate oxidase at the head of Pd nanoparticles, a light-driven heterojunction nanorobot with a navigation effect is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and particularly relates to a light-driven heterojunction nanorobot, a preparation method thereof, and an application thereof. Background Art

[0002] Tumor catalytic therapy has become a highly potential alternative strategy for clinical tumor treatment due to its advantages such as high targeting, wide applicability, and no drug resistance. Tumor catalytic therapy means using a nanocatalyst instead of a traditional drug, and by responding to the specific internal field microenvironment or external stimuli at the tumor site, triggering an in-situ catalytic reaction in the tumor, converting the substrate in the tumor microenvironment into an effective therapeutic drug, and achieving specific oxidative damage and death of tumor cells without significantly affecting normal tissues. Obviously, the performance of the catalyst is the most critical factor determining catalytic therapy. Therefore, currently, the vast majority of research focuses on the development of high-performance nanocatalysts. Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, combining catalytic and unique physical and chemical properties of nanomaterials, having good biosafety, and having broad application prospects in the biomedical field. So far, various iron-based nanozymes, carbon-based nanozymes, metal nanozymes, polyoxometalate nanozymes, etc. have been used in tumor catalytic therapy. Due to the accumulation of hydrogen peroxide (H2O2) in the tumor microenvironment caused by the disproportionation of superoxide radicals induced by superoxide dismutase in tumors, nanozymes with peroxidase (POD)-like activity can catalyze H2O2 to generate highly oxidative hydroxyl radicals (·OH), playing a crucial role in tumor treatment. Therefore, constructing nanozymes with high catalytic efficiency for tumor catalytic therapy has important research value.

[0003] Due to the dense extracellular matrix in tumor tissues, which is filled with interconnected collagen and glycosaminoglycans, the diffusion of nanozymes into deep solid tumors is greatly hindered. Therefore, another problem restricting the application of nanozyme catalytic therapy is that a large number of nanozymes can only aggregate at the tumor edge after penetrating tumor blood vessels and cannot effectively kill cancer cells in the deep regions of the tumor.

[0004] Although efficient nanozyme catalytic therapy has a good therapeutic effect on primary tumors, it cannot effectively prevent tumor recurrence and metastasis. Tumor immunotherapy is an emerging and promising treatment method after surgery, chemoradiotherapy and targeted therapy, and it is also the most effective strategy for treating tumor recurrence and metastasis. In 2021, the Hillman Cancer Center and Ludwig Collaborative and Swim Across laboratories in the United States published consecutive studies in Nature, showing that tumor cells form a high-lactic acid microenvironment through their unique glycolysis metabolic pathway. This microenvironment can "feed" regulatory T cells (Treg) with lactic acid, and at the same time "revolt" cytotoxic T cells (CTL) that attack cancer cells, reduce CTL cell secretion of cytokines, perforin and granzyme B, etc., and inhibit their cytotoxicity. Treg cells are an important type of immune T cell that can inhibit the activation and division of CTLs, maintain tolerance to self-antigens, and prevent autoimmune diseases. The high-lactic acid microenvironment produced by tumor cells can attract Treg cells and promote their functional specialization, thereby strongly inhibiting the tumor killing and patrol functions of CTLs. Therefore, how to break the high lactate microenvironment created by tumor glycolysis is the key to relieving tumor immunosuppression.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a light-driven heterojunction nanorobot and a preparation method and application thereof. The light-driven heterojunction nanorobot of the present invention uses Pd nanoparticles as a chassis and loads copper sulfide (CuS) at its tail to form a heterojunction nanozyme, which efficiently catalyzes H2O2 to generate ·OH, mediates the immunogenic death of cancer cells, and at the same time has a power system so that it can move under light drive.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] A first aspect of the present invention provides a light-driven heterojunction nanorobot, wherein the light-driven heterojunction nanorobot uses Pd nanoparticles as a carrier and is loaded with CuS.

[0009] Preferably, the Pd nanoparticles are also loaded with neutrophil exosomes modified with triphenylphosphine and loaded with lactate oxidase.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned light-driven heterojunction nanorobot, the preparation method comprising the following steps:

[0011] (a) Mix the Pd nanoparticle dispersion with isopropanol, then add 4-mercaptobenzoic acid and polyacrylic acid and react under stirring; after the reaction is completed, add tetraethyl orthosilicate and ammonia water to the reaction system and continue stirring and reacting, then centrifuge to obtain Pd-SiO2 nanoparticles;

[0012] (b) Resuspend the Pd-SiO2 nanoparticles in a polyvinylpyrrolidone solution, then add a copper(II) nitrate trihydrate solution to react. After reacting for a period of time, add hydrazine hydrate and continue reacting. After the reaction is completed, add an aqueous sodium sulfide solution to the reaction system for stirring and reacting, then centrifuge. Resuspend the precipitate with ammonia water and react and centrifuge to obtain CuS-Pd nanoparticles.

[0013] Preferably, the preparation method further includes:

[0014] (c) React DSPE-PEG-TPP with neutrophil extracellular vesicles. After the reaction is completed, add a lactate oxidase solution and mix for liposome extrusion and centrifugal purification to obtain TPP-Exo@LOX;

[0015] (d) Perform click chemistry connection on TPP-Exo@LOX and CuS-Pd nanoparticles to obtain the light-driven heterojunction nanorobot.

[0016] Preferably, in the step (a), the concentration of the Pd nanoparticle dispersion is 1.5 - 2.5 mM; the volume ratio of the Pd nanoparticle dispersion, isopropanol, 4-mercaptobenzoic acid, polyacrylic acid, tetraethyl orthosilicate and ammonia water is (4 - 6):(20 - 30):(0.1 - 0.3):(0.6 - 1):(4 - 6):(0.6 - 1);

[0017] The concentration of ammonia water is 25% - 28%.

[0018] Preferably, in the step (b), the mass-volume ratio of the Pd-SiO2 nanoparticles to the polyvinylpyrrolidone solution is (1 - 3) mg:1 mL; the concentration of the polyvinylpyrrolidone solution is 0.8% - 1.2%; the volume ratio of the polyvinylpyrrolidone solution, copper(II) nitrate trihydrate solution, hydrazine hydrate, aqueous sodium sulfide solution and ammonia water is (4 - 6):(0.02 - 0.05):(0.06 - 0.1):(0.8 - 1.2);

[0019] The concentration of the copper(II) nitrate trihydrate solution is 0.08 - 0.12 M;

[0020] The concentration of the aqueous sodium sulfide solution is 8 - 12 mM.

[0021] Preferably, in the step (c), the mass ratio of DSPE-PEG-TPP, neutrophil exosomes and lactate oxidase is (1.5-2):1:(6-10).

[0022] Preferably, in the step (d), the mass ratio of TPP-Exo@LOX and CuS-Pd nanoparticles is (1-1.2):1.

[0023] The third aspect of the present invention provides an application of the above-mentioned light-driven heterojunction nanorobot or the light-driven heterojunction nanorobot prepared by the preparation method in the preparation of anti-tumor drugs.

[0024] Preferably, the tumor includes colon cancer.

[0025] Compared with the prior art, the beneficial effects of the present invention at least include:

[0026] The light-driven heterojunction nanorobot of the present invention uses Pd nanoparticles as the chassis, and loads copper sulfide (CuS) at its tail to form a heterojunction nanozyme, which efficiently catalyzes H2O2 to generate ·OH, mediates immunogenic death of cancer cells, and at the same time enables it to have a power system and can move under light driving; in addition, by loading neutrophil exosomes modified with triphenylphosphine and loaded with lactate oxidase (TPP-Exo@LOX) at the head of Pd nanoparticles, a light-driven heterojunction nanorobot with a navigation effect is obtained, and its functions are as follows: (1) The functionalized neutrophil exosomes endow the nanorobot with the ability to navigate to tumors and target tumor sites; (2) Laser irradiation in the NIR-II region causes a temperature gradient from the tail CuS to the head exosomes of the nanorobot, endowing the nanorobot with a power system, enabling it to penetrate deep into the tumor; (3) The heterojunction nanozyme (CuS-Pd) constructed by two nanozymes Pd and CuS with POD enzyme activity efficiently catalyzes H2O2 to generate ·OH, mediates immunogenic death of cancer cells; (4) LOX catalyzes the consumption of tumor lactic acid, completely breaks the tumor high-lactic acid immunosuppressive microenvironment, inhibits the activity of regulatory T cells, and enhances the activity of cytotoxic T cells.

[0027] The light-driven heterojunction nanorobot provided by the present invention exhibits excellent anti-cancer effects through multimodal treatment methods, and has significant application potential, especially in the fields of tumor catalysis and immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale.

[0029] Figure 1 Schematic diagram for the preparation of the light-driven heterojunction nanorobot of the present invention;

[0030] Figure 2 TEM images of different materials in Experimental Example 1 of the present invention;

[0031] Figure 3 XPS characterization diagram of TPP-Exo@LOX-CuS-Pd in Experimental Example 1 of the present invention;

[0032] Figure 4 UV-vis diagrams of different materials in Experimental Example 1 of the present invention;

[0033] Figure 5 Motion trajectory test diagram of TPP-Exo@LOX-Pd-CuS in Experimental Example 2 of the present invention;

[0034] Figure 6 Penetration diagram of TPP-Exo@LOX-Pd-CuS in 3D tumor cell spheroids under NIR-II light driving in Experimental Example 2 of the present invention;

[0035] Figure 7 Test results of the ability of TPP-Exo@LOX-Pd-CuS to generate hydroxyl radicals and superoxide anions under near-infrared light irradiation in Experimental Example 3 of the present invention;

[0036] Figure 8 Test results of the toxicity of different materials to CT26 colorectal cancer cells in Experimental Example 4 of the present invention;

[0037] Figure 9 For CD8 of different materials in Experimental Example 5 of the present invention + Flow cytometry diagrams of the expression of T cells and Treg cells. Detailed implementation manners

[0038] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the embodiments. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0039] It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meanings understood by those skilled in the art to which the present invention belongs.

[0040] The embodiment of the present invention provides a light-driven heterojunction nanorobot, and the light-driven heterojunction nanorobot uses Pd nanoparticles as a carrier and is loaded with CuS.

[0041] The light-driven heterojunction nanorobot of the present invention uses Pd nanoparticles as the chassis, and loads copper sulfide (CuS) at its tail to form a heterojunction nanozyme, which efficiently catalyzes H2O2 to generate ·OH, mediates immunogenic death of cancer cells, and at the same time enables it to have a power system and be able to move under light driving;

[0042] In one embodiment, the Pd nanoparticles are further loaded with neutrophil extracellular vesicles modified with triphenylphosphine and loaded with lactate oxidase.

[0043] The present invention also obtains a light-driven heterojunction nanorobot with a navigation effect by loading neutrophil extracellular vesicles modified with triphenylphosphine and loaded with lactate oxidase (TPP-Exo@LOX) at the head of Pd nanoparticles. Its functions are as follows: (1) The functionalized neutrophil extracellular vesicles endow the nanorobot with the ability to navigate tumors and target tumor sites; (2) Laser irradiation in the NIR-II region generates a temperature gradient from the tail CuS to the head extracellular vesicles of the nanorobot, endowing the nanorobot with a power system and enabling it to penetrate deep into the tumor; (3) A heterojunction nanozyme (CuS-Pd) constructed by two nanozymes Pd and CuS with POD enzyme activity efficiently catalyzes H2O2 to generate ·OH, mediating immunogenic death of cancer cells; (4) LOX catalyzes the consumption of tumor lactic acid, completely breaks the high-lactic acid immunosuppressive microenvironment of tumors, inhibits the activity of regulatory T cells, and enhances the activity of cytotoxic T cells.

[0044] The present invention does not specifically limit the preparation method of Pd nanoparticles. Those skilled in the art can adopt the conventional preparation methods of Pd nanoparticles in the art. In one embodiment, the Pd nanoparticles are prepared by the following method:

[0045] Dissolve palladium chloride in dilute hydrochloric acid (HCl), stir and heat to 40-60 °C for reaction for 20-40 min to obtain an aqueous solution of tetrachloropalladic acid (H2PdCl4); subsequently, slowly add the H2PdCl4 solution to an aqueous solution of cetyltrimethylammonium bromide (CTAB), and dropwise add an ascorbic acid (AA) solution under stirring conditions, and keep stirring at room temperature for 20-40 min; after the reaction is completed, separate the Pd NPs nanoparticles by centrifugation, wash with deionized water, and resuspend the precipitate in water for later use.

[0046] Furthermore, the mass-volume ratio of palladium chloride to dilute hydrochloric acid is (1.5-2):1; the concentration of dilute hydrochloric acid is 18-22 mM; the volume ratio of the aqueous solution of tetrachloropalladic acid, the aqueous solution of cetyltrimethylammonium bromide and the ascorbic acid solution is 2:(35-45):1; the concentration of the aqueous solution of cetyltrimethylammonium bromide is 2-4 mM; the concentration of the ascorbic acid solution is 0.8-0.12 M.

[0047] Another embodiment of the present invention provides a preparation method of the above-mentioned light-driven heterojunction nanorobot, and the preparation method includes the following steps:

[0048] (a) Mix the Pd nanoparticle dispersion with isopropanol, then add 4-mercaptobenzoic acid and polyacrylic acid and react under stirring; after the reaction is completed, add tetraethyl orthosilicate and ammonia water to the reaction system and continue stirring and reacting, and then centrifuge to obtain Pd-SiO2 nanoparticles;

[0049] (b) Resuspend the Pd-SiO2 nanoparticles in a polyvinylpyrrolidone solution, then add a copper nitrate hydrate solution to react, add hydrazine hydrate to continue reacting after reacting for a period of time, after the reaction is completed, add an aqueous sodium sulfide solution to the reaction system for stirring and reacting, and then centrifuge, resuspend the precipitate with ammonia water and react and centrifuge to obtain CuS-Pd nanoparticles.

[0050] In some embodiments, the preparation method further includes:

[0051] (c) React DSPE-PEG-TPP with neutrophil extracellular vesicles, after the reaction is completed, then add a lactate oxidase solution for mixing, perform liposome extrusion and centrifugal purification to obtain TPP-Exo@LOX;

[0052] (d) Perform click chemistry connection on TPP-Exo@LOX and CuS-Pd nanoparticles to obtain the light-driven heterojunction nanorobot.

[0053] In some embodiments, in the step (a), the concentration of the Pd nanoparticle dispersion is 1.5-2.5 mM; the volume ratio of the Pd nanoparticle dispersion, isopropanol, 4-mercaptobenzoic acid, polyacrylic acid, tetraethyl orthosilicate and ammonia water is (4-6):(20-30):(0.1-0.3):(0.6-1):(4-6):(0.6-1);

[0054] The concentration of ammonia water is 25%-28%.

[0055] In some embodiments, in the step (b), the mass-volume ratio of the Pd-SiO2 nanoparticles to the polyvinylpyrrolidone solution is (1-3) mg:1 mL; the concentration of the polyvinylpyrrolidone solution is 0.8%-1.2%; the volume ratio of the polyvinylpyrrolidone solution, copper nitrate hydrate solution, hydrazine hydrate, aqueous sodium sulfide solution and ammonia water is (4-6):(0.02-0.05):(0.06-0.1):(0.8-1.2);

[0056] The concentration of the copper nitrate hydrate solution is 0.08-0.12 M;

[0057] The concentration of the aqueous sodium sulfide solution is 8-12 mM.

[0058] In one embodiment, in the step (c), neutrophil exosomes are prepared by the following method:

[0059] Neutrophils are extracted by extracting mouse peripheral blood, and neutrophil exosomes are extracted by ultra-high speed centrifugation.

[0060] In some embodiments, in the step (c), the mass ratio of DSPE-PEG-TPP, neutrophil exosomes and lactate oxidase is (1.5 - 2):1:(6 - 10).

[0061] In some embodiments, in the step (c), the concentration of the lactate oxidase solution is 0.8 - 1.2 mg / ml.

[0062] In some embodiments, in the step (c), the number of times of liposome extrusion is 21 - 25 times.

[0063] In some embodiments, in the step (d), the mass ratio of TPP-Exo@LOX and CuS-Pd nanoparticles is (1 - 1.2):1.

[0064] Another embodiment of the present invention provides an application of the above-mentioned light-driven heterojunction nanorobot or the light-driven heterojunction nanorobot prepared by the preparation method in the preparation of anti-tumor drugs.

[0065] In one embodiment, the tumor includes colon cancer.

[0066] The technical solution of the present invention will be further described in detail below through specific examples.

[0067] Example 1

[0068] This example is a preparation method of Pd nanoparticles, and the preparation method includes the following steps:

[0069] 17.8 mg of palladium chloride (PdCl2) is added to HCl (10 mL, 20 mM), and the solution is stirred at 50 °C for 30 min to obtain a 10 mM aqueous solution of tetrachloropalladic acid; under stirring, 2 mL of the H2PdCl4 solution is added to 40 mL of a 3 mM aqueous solution of cetyltrimethylammonium bromide, and then 1 mL of 0.1 M ascorbic acid (AA) as a reducing agent is added dropwise to the reaction system, stirred at room temperature for 30 min, and then centrifuged at 7000 rpm for 10 min to obtain Pd nanoparticles (denoted as Pd NPs).

[0070] Example 2

[0071] This example is a preparation method of a light-driven heterojunction nanorobot (CuS-Pd nanoparticles), and the preparation method includes the following steps:

[0072] Under vigorous stirring, 5 mL of Pd NPs dispersion was added to 25 mL of isopropanol solution; subsequently, 200 μL of 4-mercaptobenzoic acid and 800 μL of polyacrylic acid were successively added to the mixed solution and reacted for 60 min. Then, 5 mL of TEOS (8.9 mM) and 0.9 mL of ammonia water were successively added to the above system, and the reaction was continuously carried out for 4 h under slow stirring at room temperature; then Pd-SiO2 nanoparticles were collected by centrifugation (7500 rpm, 10 min).

[0073] The Pd-SiO2 nanoparticles were dispersed in 5 mL of PVP solution (1%). Under stirring at room temperature, 35 μL of copper nitrate hydrate solution (0.1 M) was added to the above system and reacted for 10 min. Then, 80 μL of hydrazine hydrate was quickly added to the above system and reacted for another 10 min to obtain Cu2O-Pd-SiO2; then, 1 mL of Na2S (10 mM) aqueous solution was added to the above system and stirred at room temperature for 20 min to form CuS-Pd-SiO2 nanoparticles (denoted as CuS-Pd-SiO2); the synthesized nanoparticles were collected by centrifugation (6000 rpm, 6 min) and dispersed in 1 mL of ammonia water (26%). After reacting overnight under stirring at room temperature, centrifugation (6000 rpm, 6 min) was carried out and the precipitate was collected to obtain CuS-Pd nanoparticles (denoted as CuS-Pd or Pd-CuS).

[0074] Example 3

[0075] This example is a preparation method of a light-driven heterojunction nanorobot (TPP-Exo@LOX-Pd-CuS), as Figure 1 shown, and the preparation method includes the following steps:

[0076] Neutrophils were extracted from mouse peripheral blood, and neutrophil exosomes were obtained by ultra-high speed centrifugation.

[0077] DSPE-PEG-TPP with mitochondrial targeting was reacted with neutrophil exosomes in PBS solution at room temperature for 12 h to obtain TPP-Exo; then, 1 mL of LOX solution (1 mg / mL) was mixed with the prepared TPP-Exo in water, and after extruding 21 times through a liposome extruder, centrifugal purification was carried out to obtain TPP-Exo@LOX, where the mass ratio of DSPE-PEG-TPP, neutrophil exosomes, and lactate oxidase is 1.8∶1∶8.

[0078] According to the mass ratio of TPP-Exo@LOX to CuS-Pd nanoparticles being 1.1:1, the above-prepared TPP-Exo@LOX was connected to CuS-Pd prepared in Example 2 through click chemistry to obtain a light-driven heterojunction nanorobot (denoted as TPP-Exo@LOX-Pd-CuS).

[0079] Experimental Example 1

[0080] Transmission electron microscopy scanning was performed on the different materials prepared in Examples 2 to 3;

[0081] The TEM images of CuS-Pd-SiO2, CuS-Pd, and TPP-Exo@LOX-Pd-CuS are as Figure 2 shown, Figure 2 where (a) is CuS-Pd-SiO2, (b) is CuS-Pd, and (c) is TPP-Exo@LOX-Pd-CuS.

[0082] As Figure 2 shown: The "head" of TPP-Exo@LOX-Pd-CuS is modified with exosomes, and the "tail" is modified with copper sulfide. This asymmetric heterojunction structure ensures that it can be targeted by the head and driven by the tail. The TEM image proves the successful preparation of the morphology of TPP-Exo@LOX-Pd-CuS.

[0083] X-ray photoelectron spectroscopy analysis was performed on TPP-Exo@LOX-CuS-Pd;

[0084] The analysis results are as Figure 3 shown, and the high-resolution X-ray photoelectron spectroscopy characterization of TPP-Exo@LOX-CuS-Pd is as Figure 3 (a) shows two main peaks of Cu 2p 2 / 3 and Cu 2p 1 / 2 located at approximately 932 eV and 952 eV respectively, accompanied by satellite peaks, indicating the presence of Cu 2+ oxidation state. Figure 3 (b) shows two main peaks of Pd 3d 5 / 2 and Pd 3d 3 / 2 located at approximately 335 eV and 340 eV respectively, and no obvious satellite peaks are seen, indicating that Pd is mainly in the metallic state or low oxidation state.

[0085] UV-Vis absorption test analysis was performed on CuS-Pd-SiO2, CuS-Pd, and TPP-Exo@LOX-Pd-CuS;

[0086] The analysis results are as Figure 4 shown, Figure 4The optical absorption characteristics of Pd, CuS, and TPP-Exo@LOX-CuS-Pd are shown. The TPP-Exo@LOX-CuS-Pd heterojunction material exhibits more excellent optical absorption in the second near-infrared band (1064 nm).

[0087] Experimental Example 2

[0088] This experimental example is for the motion characterization of the light-driven heterojunction nanorobot:

[0089] 1. Motion trajectory test of the light-driven heterojunction nanorobot (TPP-Exo@LOX-Pd-CuS)

[0090] Place TPP-Exo@LOX-Pd-CuS in PBS solution, and observe and record its motion trajectory using a dark-field microscope and a CUDA spot tracker under 1064 nm laser irradiation. The tracking test time is 15 s, and the motion curve of the nanorobot is plotted.

[0091] The motion trajectory test diagram is as Figure 5 shown, Figure 5 In, the motion principle diagram of TPP-Exo@LOX-Pd-CuS is as Figure 5 shown in a of : Under NIR-II light irradiation, copper sulfide converts light into heat, and the water molecules close to copper sulfide obtain more heat than the water molecules close to the exosome at the head, thus generating an asymmetric thermal gradient field and generating a forward thermophoretic force to drive the nanorobot forward. The motion trajectories of TPP-Exo@LOX-Pd-CuS with and without NIR-II light irradiation are as Figure 5 shown in b and c of. Under NIR-II light driving, TPP-Exo@LOX-CuS-Pd exhibits excellent motion performance, and the motion trajectory is linear, while the TPP-Exo@LOX-CuS-Pd group without near-infrared light irradiation shows a Brownian motion trajectory without an obvious forward motion trend.

[0092] 2. 3D tumor cell mass culture and penetration

[0093] Add 50 μL of 1% agarose to each well of a 96-well plate and irradiate with a UV lamp for 30 min. Add 200 μL of a CT26 cell suspension with a concentration of 1×10 3 cells / mL to each well, and 3D cell spheres are obtained after culturing for 10 days. Subsequently, add DiI dye-labeled TPP-Exo@LOX-Pd-CuS to the well plate and irradiate with 1064 nm laser for 10 min. Remove the cell spheres from the well plate, wash them 3 times with PBS, and then observe the penetration of TPP-Exo@LOX-Pd-CuS in the cell spheres using laser confocal microscopy.

[0094] The penetration movement of TPP-Exo@LOX-CuS-Pd nanorobots modified with the red fluorescent dye DiI in 3D tumor cell spheroids under near-infrared light irradiation is as follows Figure 6 shown; under NIR-II light irradiation, the nanorobots can move deep into the center of the tumor cell spheroids, indicating that the active movement performance of TPP-Exo@LOX-CuS-Pd can greatly promote its penetration into the tumor interior.

[0095] Experimental Example 3

[0096] This experimental example is for catalytic performance testing:

[0097] In the presence of H2O2, using TMB as the substrate, the ability of TPP-Exo@LOX-Pd-CuS nanorobots to generate hydroxyl radicals was measured.

[0098] 0.3 mL of 8 mM TMB and 90 μL of 10 mM H2O2 were added to 2.57 mL of PBS solution with a pH of 4.5, and then TPP-Exo@LOX-Pd-CuS was added to the reaction system.

[0099] The indicator used to test the generation of superoxide anions by thermally driven TPP-Exo@LOX-Pd-CuS in vitro was DPBF, and the test method was similar to the above method. In addition, electron spin resonance (ESR) was used to detect the generation of hydroxyl radicals and superoxide anions.

[0100] The test results of the ability of TPP-Exo@LOX-Pd-CuS nanorobots to generate hydroxyl radicals and superoxide anions under near-infrared light irradiation are as follows Figure 7 shown;

[0101] From Figure 7 it can be seen that through TMB and DPBF indicators, it was proved that under NIR-II light irradiation, TPP-Exo@LOX-Pd-CuS can generate hydroxyl radicals and superoxide anions. At the same time, ESR testing also further proved that TPP-Exo@LOX-Pd-CuS can generate a large amount of hydroxyl radicals and superoxide anions under near-infrared irradiation.

[0102] Experimental Example 4

[0103] This experimental example is for tumor cell killing toxicity testing:

[0104] CT26 cells were cultured in a 96-well plate for 24 h. Subsequently, the culture medium was gently aspirated and the cells were washed 3 times with PBS. Fresh medium containing TPP-Exo@LOX-Pd-CuS at a concentration of 100 μg / mL was added and the cells were cultured for another 24 h. PBS without the material was used as the control group. The CCK-8 kit was used to detect the activity of CT26 cells.

[0105] The cytotoxicity test of different materials on CT26 colorectal cancer cells is as Figure 8 shown. The CCK-8 cytotoxicity experiment showed that after treatment with TPP-Exo@LOX-Pd-CuS under NIR-II light irradiation, the survival rate of CT26 cells was as low as 22.9%, indicating that TPP-Exo@LOX-Pd-CuS has good anti-tumor killing effect under NIR-II light irradiation.

[0106] Experimental Example 5

[0107] This experimental example is for in vitro T cell immune activation research:

[0108] T cells were isolated from the spleens of 6-week-old healthy Balb / c female mice and cultured in a medium containing 20 mM lactic acid. After 24 h of culture, different nanomaterials (CuS-Pd-SiO2, CuS-Pd, and TPP-Exo@LOX-Pd-CuS) were added to the medium and the cells were cultured for another 48 h. Subsequently, the T cells were stained with flow antibodies anti-CD3 APC, anti-CD4 BV605, anti-CD8 FITC, anti-CD25 PE / Cy7h, and anti-Foxp3 BV421 and analyzed by flow cytometry to evaluate the regulatory effects of the constructed light-driven heterojunction nanorobots on cytotoxic T cells (CD8 + T cells) and regulatory T cells (Tregs).

[0109] The analysis results are as Figure 9 shown. The proportion of CD8 + T cells in the CuS-Pd + NIR-II and TPP-Exo@LOX-Pd-CuS + NIR-II treatment groups increased, and the proportion of CD8 + T cells in the TPP-Exo@LOX-Pd-CuS + NIR-II treatment group was the highest, indicating that NIR-II light irradiation combined with TPP-Exo@LOX-Pd-CuS or CuS-Pd treatment can effectively activate CD8 + T cells. The TPP-Exo@LOX-Pd-CuS + NIR-II treatment group showed a decrease in the proportion of Tregs, further indicating that it can improve the tumor immunosuppressive microenvironment and enhance the efficacy of anti-tumor immunotherapy.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention, and they should all be covered within the scope of the claims and the description of the present invention.

Claims

1. A light-driven heterojunction nanorobot, characterized in that: The light-driven heterojunction nanorobot uses Pd nanoparticles as carriers and is loaded with CuS; The Pd nanoparticles are also loaded with neutrophil exosomes modified with triphenylphosphine and loaded with lactate oxidase; The light-driven heterojunction nanorobot is prepared by the following method: (a) mixing a Pd nanoparticle dispersion with isopropanol, and then adding 4-mercaptophenylacetic acid and polyacrylic acid to react under stirring; after the reaction is completed, adding tetraethyl orthosilicate and ammonia water to the reaction system, continuing to stir the reaction, and centrifuging to obtain Pd-SiO2 nanoparticles; (b) resuspending Pd-SiO2 nanoparticles in a polyvinyl pyrrolidone solution, adding a copper nitrate hydrate solution to react, adding hydrazine hydrate to continue the reaction after a period of reaction, adding a sodium sulfide aqueous solution to the reaction system after the reaction is completed, stirring the reaction, centrifuging, resuspending the precipitate with ammonia water, reacting, and centrifuging to obtain CuS-Pd nanoparticles; (c) reacting DSPE-PEG-TPP with neutrophil exosomes, and after the reaction is completed, adding lactate oxidase solution to mix, liposome extrusion, and centrifugal purification to obtain TPP-Exo@LOX; (d) TPP-Exo@LOX and CuS-Pd nanoparticles are connected by click chemistry to obtain the light-driven heterojunction nanorobot.

2. The light-driven heterojunction nanorobot according to claim 1, characterized in that: In the step (a), the concentration of the Pd nanoparticle dispersion is 1.5-2.5 mM; the volume ratio of the Pd nanoparticle dispersion, isopropanol, 4-mercaptophenylacetic acid, polyacrylic acid, tetraethyl orthosilicate and ammonia water is (4-6): (20-30): (0.1-0.3): (0.6-1): (4-6): (0.6-1).

3. The light-driven heterojunction nanorobot according to claim 1, characterized in that: In the step (b), the mass volume ratio of Pd-SiO2 nanoparticles and polyvinyl pyrrolidone solution is (1-3) mg: 1 mL; the concentration of polyvinyl pyrrolidone solution is 0.8%-1.2%; the volume ratio of polyvinyl pyrrolidone solution, copper nitrate hydrate solution, hydrazine hydrate, sodium sulfide aqueous solution and ammonia water is (4-6): (0.02-0.05): (0.06-0.1): (0.8-1.2); The concentration of copper nitrate hydrate solution is 0.08-0.12M; The concentration of the sodium sulfide aqueous solution is 8 to 12 mM.

4. The light-driven heterojunction nanorobot according to claim 1, characterized in that: In the step (c), the mass ratio of DSPE-PEG-TPP, neutrophil exosomes and lactate oxidase is (1.5-2):1:(6-10).

5. The light-driven heterojunction nanorobot according to claim 1, characterized in that: In the step (d), the mass ratio of TPP-Exo@LOX to CuS-Pd nanoparticles is (1-1.2):

1.

6. Use of the light-driven heterojunction nanorobot according to any one of claims 1 to 5 in the preparation of drugs for treating colon cancer.

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