Optically-driven heterojunction nano-robot and preparation method and application thereof

Abstract

The invention discloses a light-driven heterojunction nano-robot as well as a preparation method and application thereof. The light-driven heterojunction nano-robot takes palladium (Pd) nanoparticles as a carrier and is loaded with copper sulfide (CuS); according to the light-driven heterojunction nano-robot, Pd nano-particles serve as a chassis, CuS is loaded on the tail portions of the Pd nano-particles, heterojunction nano-enzyme is formed, H2O2 is efficiently catalyzed to generate. OH, cancer cell immunogenicity death is mediated, meanwhile, the light-driven heterojunction nano-robot is provided with a power system, and the light-driven heterojunction nano-robot can move under light driving; in addition, the head of the Pd nanoparticle is loaded with a neutrophil exosome modified by triphenylphosphine and loaded with lactate oxidase, and the light-driven heterojunction nano-robot with the navigation effect is obtained.

Description

Technical Field

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

[0002] Tumor catalytic therapy has become a highly promising alternative strategy for clinical tumor treatment due to its advantages of high targeting, wide applicability, and lack of drug resistance. Tumor catalytic therapy, that is, replacing traditional drugs with nanocatalysts, triggers in situ catalytic reactions in the tumor by responding to the specific internal microenvironment or exogenous stimulation of the tumor site, converts substrates in the tumor microenvironment into effective therapeutic drugs, and achieves tumor cell-specific oxidative damage and death without significantly affecting normal tissues. Obviously, the performance of the catalyst is the most critical factor in determining catalytic therapy. Therefore, most of the current research is focused on the development of high-performance nanocatalysts. Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, which have both catalytic and unique physical and chemical properties of nanomaterials, good biosafety, and broad application prospects in the biomedical field. So far, a variety of iron-based nanozymes, carbon-based nanozymes, metal nanozymes, polyoxometalate nanozymes, etc. have been used in tumor catalytic therapy. Since the superanion free radical dismutation induced by superoxide dismutase in tumors leads to the accumulation of hydrogen peroxide (H2O2) in the tumor microenvironment, nanozymes with peroxidase-like (POD) activity can catalyze H2O2 to generate highly oxidizing hydroxyl free radicals (·OH), which plays an important role in tumor treatment. Therefore, it is of great research value to construct nanozymes with high catalytic efficiency for tumor catalytic treatment.

[0003] Since tumor tissue has a dense extracellular matrix 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 penetrate tumor blood vessels and accumulate at the edge of the tumor, and cannot effectively kill cancer cells in the deep area 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) 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;

[0012] (b) Pd-SiO2 nanoparticles are resuspended in a polyvinyl pyrrolidone solution, and then a copper nitrate hydrate solution is added to react. After a period of reaction, hydrazine hydrate is added to continue the reaction. After the reaction is completed, a sodium sulfide aqueous solution is added to the reaction system to stir the reaction and centrifuge. Ammonia water is used to resuspend the precipitate and react and centrifuge to obtain CuS-Pd nanoparticles.

[0013] Preferably, the preparation method further comprises:

[0014] (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;

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

[0016] Preferably, in 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);

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

[0018] Preferably, in 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);

[0019] The concentration of copper nitrate hydrate solution is 0.08-0.12M;

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

[0021] Preferably, in 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 to CuS-Pd nanoparticles is (1-1.2):1.

[0023] A third aspect of the present invention provides a use 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 comprises colon cancer.

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

[0026] The light-driven heterojunction nanorobot of the present invention uses Pd nanoparticles as a chassis, and copper sulfide (CuS) is loaded at the tail thereof to form a heterojunction nanozyme, which efficiently catalyzes H2O2 to generate ·OH, mediates the immunogenic death of cancer cells, and at the same time enables it to have a power system and move under light drive; in addition, by loading triphenylphosphine-modified and lactate oxidase-loaded neutrophil exosomes (TPP-Exo@LOX) on the head of the Pd nanoparticles, a light-driven heterojunction nanorobot with a navigation effect is obtained, and its functions are as follows: (1) the functionally modified neutrophil exosomes endow the nanorobot with navigation effect. (1) The nanorobot has the ability to navigate tumors, enabling it to target the tumor site; (2) NIR-II laser irradiation causes a temperature gradient from the CuS at the tail to the exosomes at the head, giving the nanorobot a power system that allows 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, mediating the immunogenic death of cancer cells; (4) LOX catalyzes the consumption of tumor lactate, completely breaking the tumor high-lactate immunosuppressive microenvironment, inhibiting the activity of regulatory T cells, and enhancing 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 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 is a brief introduction to the drawings required for the specific embodiments or the description of 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 according to the actual scale.

[0029] Figure 1 This is a schematic diagram of 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 This is the XPS characterization diagram of TPP-Exo@LOX-CuS-Pd in ​​Experimental Example 1 of the present invention;

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

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

[0034] Figure 6 This is a diagram showing the penetration of TPP-Exo@LOX-Pd-CuS in 3D tumor cell spheres under NIR-II light driving in Experimental Example 2 of the present invention;

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

[0036] Figure 8 The toxicity test results of different materials on CT26 colorectal cancer cells in Experimental Example 4 of the present invention are as follows;

[0037] Fig. 9 CD8 is a different material in Experimental Example 5 of the present invention. + Flow cytometry of T cell and Treg cell expression. DETAILED DESCRIPTION

[0038] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as 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 common 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. 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 copper sulfide (CuS) is loaded 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 enables it to have a power system and move under light drive;

[0042] In one embodiment, the Pd nanoparticles are further loaded with neutrophil exosomes 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 triphenylphosphine-modified and lactate oxidase-loaded neutrophil exosomes (TPP-Exo@LOX) on the head of the Pd nanoparticles. The functions of the light-driven heterojunction nanorobot are as follows: (1) the functionally modified neutrophil exosomes give the nanorobot the ability to navigate the tumor, so that it can target the tumor site; (2) NIR-II laser irradiation causes the nanorobot to generate a temperature gradient from the tail CuS to the head exosomes, which gives the nanorobot a power system, so that it can 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, mediating the immunogenic death of cancer cells; (4) LOX catalyzes the consumption of tumor lactate, completely breaks the tumor high-lactate immunosuppressive microenvironment, 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 method of Pd nanoparticles in the art. In one embodiment, Pd nanoparticles are prepared by the following method:

[0045] Palladium chloride is dissolved in dilute hydrochloric acid (HCl) and stirred and heated to 40-60°C for reaction for 20-40 minutes to obtain a tetrachloropalladic acid (H2PdCl4) aqueous solution; then, the H2PdCl4 solution is slowly added to a hexadecyltrimethylammonium bromide (CTAB) aqueous solution, and ascorbic acid (AA) solution is added dropwise under stirring conditions, and stirring is maintained at room temperature for 20-40 minutes; after the reaction is completed, Pd NPs nanoparticles are separated by centrifugation, washed with deionized water, and the precipitate is resuspended in water for later use.

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

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

[0048] (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;

[0049] (b) Pd-SiO2 nanoparticles are resuspended in a polyvinyl pyrrolidone solution, and then a copper nitrate hydrate solution is added to react. After a period of reaction, hydrazine hydrate is added to continue the reaction. After the reaction is completed, a sodium sulfide aqueous solution is added to the reaction system to stir the reaction and centrifuge. Ammonia water is used to resuspend the precipitate and react and centrifuge to obtain CuS-Pd nanoparticles.

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

[0051] (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;

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

[0053] In some embodiments, in 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);

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

[0055] In some embodiments, in 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);

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

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

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

[0059] Neutrophils were extracted from mouse peripheral blood, and neutrophil exosomes were extracted by ultrahigh-speed centrifugation.

[0060] In some embodiments, in 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 step (c), the concentration of the lactate oxidase solution is 0.8-1.2 mg / ml.

[0062] In some embodiments, in step (c), the liposomes are extruded 21 to 25 times.

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

[0064] Yet another embodiment of the present invention provides a use 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 comprises colon cancer.

[0066] The technical solution of the present invention is further described in detail below through specific embodiments.

[0067] Example 1

[0068] This embodiment is a method for preparing Pd nanoparticles, which comprises the following steps:

[0069] A 10 mM aqueous solution of tetrachloropalladic acid was prepared by adding 17.8 mg of palladium chloride (PdCl2) to HCl (10 mL, 20 mM), and the solution was stirred at 50 ° C for 30 min; under stirring, 2 mL of H2PdCl4 solution was added to 40 mL of 3 mM aqueous hexadecyltrimethylammonium bromide solution, and then 1 mL of 0.1 M ascorbic acid (AA) as a reducing agent was 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 embodiment is a method for preparing a light-driven heterojunction nanorobot (CuS-Pd nanoparticles), and the preparation method comprises the following steps:

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

[0073] Pd-SiO2 nanoparticles are dispersed in 5mL of PVP solution (1%), and 35μL of copper nitrate hydrate solution (0.1M) is added to the above system under stirring at room temperature to react for 10 minutes, and then 80μL of hydrazine hydrate is quickly added to the above system to continue the reaction for 10 minutes to obtain Cu2O-Pd-SiO2; then, 1mL of Na2S (10mM) aqueous solution is added to the above system and stirred at room temperature for 20 minutes to form CuS-Pd-SiO2 nanoparticles (referred to as CuS-Pd-SiO2); the prepared synthesized nanoparticles are collected by centrifugation (6000rpm, 6min), and dispersed in 1mL of ammonia water (26%), and after reacting overnight under stirring at room temperature, the reaction is centrifuged (6000rpm, 6min) and the precipitate is collected to obtain CuS-Pd nanoparticles (referred to as CuS-Pd or Pd-CuS).

[0074] Example 3

[0075] This embodiment is a method for preparing a light-driven heterojunction nanorobot (TPP-Exo@LOX-Pd-CuS), such as Figure 1 As shown, the preparation method comprises the following steps:

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

[0077] The mitochondrial-targeted DSPE-PEG-TPP was reacted with neutrophil exosomes in a 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, extruded 21 times through a liposome extruder, and centrifuged to obtain TPP-Exo@LOX, wherein the mass ratio of DSPE-PEG-TPP, neutrophil exosomes, and lactate oxidase was 1.8:1:8;

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

[0079] Experimental Example 1

[0080] The different materials prepared in Examples 2 to 3 were scanned by a transmission electron microscope;

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

[0082] like Figure 2 As 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 of TPP-Exo@LOX-CuS-Pd;

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

[0085] The CuS-Pd-SiO2, CuS-Pd and TPP-Exo@LOX-Pd-CuS were analyzed by UV-Vis absorption test;

[0086] The analysis results are as follows Figure 4 As shown, Figure 4The optical absorption properties of three materials, Pd, CuS and TPP-Exo@LOX-CuS-Pd, were demonstrated. The TPP-Exo@LOX-CuS-Pd heterojunction material exhibited better optical absorption in the second near-infrared band (1064nm).

[0087] Experimental Example 2

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

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

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

[0091] Motion trajectory test diagram Figure 5 As shown, Figure 5 The motion principle diagram of TPP-Exo@LOX-Pd-CuS is shown in Figure 5 As shown in figure a: 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 head exosome, thereby generating an asymmetric thermal gradient field and a forward thermophoretic force to drive the nanorobot forward. The movement trajectory of TPP-Exo@LOX-Pd-CuS with and without NIR-Ⅱ light irradiation is shown in Figure 2. Figure 5 As shown in b and c. Driven by NIR-Ⅱ light, TPP-Exo@LOX-CuS-Pd exhibited excellent motion performance, and the motion trajectory was linear, while the TPP-Exo@LOX-CuS-Pd group without near-infrared light irradiation showed a Brownian motion trajectory without an obvious forward motion trend.

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

[0093] 50 μL of 1% agarose was added to each well of a 96-well plate and irradiated with UV light for 30 min. 200 μL of 1×10 3 cells / mL of CT26 cell suspension, and 3D cell spheres were obtained after culturing for 10 days. Subsequently, TPP-Exo@LOX-Pd-CuS labeled with DiI dye was added to the well plate and irradiated with 1064nm laser for 10min. The cell spheres were removed from the well plate and washed with PBS three times, and then the penetration of TPP-Exo@LOX-Pd-CuS in the cell spheres was observed using laser confocal microscopy.

[0094] The penetration movement of TPP-Exo@LOX-CuS-Pd nanorobots modified with red fluorescent dye DiI in 3D tumor cell spheroids under near-infrared light irradiation is shown in Figure 2. Figure 6 As shown; under NIR-Ⅱ light irradiation, the nanorobot can move deeply into the center of the tumor cell sphere, which shows that the active motion performance of TPP-Exo@LOX-CuS-Pd can greatly promote its penetration into the tumor.

[0095] Experimental Example 3

[0096] This experimental example is a catalytic performance test:

[0097] The ability of TPP-Exo@LOX-Pd-CuS nanorobots to produce hydroxyl radicals was determined using TMB as a substrate in the presence of H2O2.

[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 thermally driven TPP-Exo@LOX-Pd-CuS to produce superoxide anions 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 produce hydroxyl radicals and superoxide anions under near-infrared light are as follows: Figure 7 As shown;

[0101] Depend on Figure 7 It can be seen that TMB and DPBF indicators prove that TPP-Exo@LOX-Pd-CuS can produce hydroxyl radicals and superoxide anions under NIR-Ⅱ light irradiation. At the same time, ESR test further proves that TPP-Exo@LOX-Pd-CuS can produce a large amount of hydroxyl radicals and superoxide anions under near-infrared irradiation.

[0102] Experimental Example 4

[0103] This experimental example is a tumor cell killing toxicity test:

[0104] CT26 cells were cultured in a 96-well plate for 24 h, then the culture medium was gently aspirated and the cells were washed three times with PBS, fresh culture medium containing 100 μg / mL TPP-Exo@LOX-Pd-CuS was added, and culture was continued for 24 h. PBS without material was used as a control group. The activity of CT26 cells was detected using a CCK-8 kit.

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

[0106] Experimental Example 5

[0107] This experimental example is an in vitro T cell immune activation study:

[0108] T cells were isolated from the spleen 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 nanoformulations (CuS-Pd-SiO2, CuS-Pd and TPP-Exo@LOX-Pd-CuS) were added to the culture medium and cultured for another 48 h. Subsequently, T cells were stained with flow cytometry antibodies anti-CD3 APC, anti-CD4 BV605, anti-CD8FITC, anti-CD25PE / Cy7h and anti-Foxp3 BV421 and flow cytometry analysis was performed to evaluate the effects of the constructed light-driven heterojunction nanorobot on cytotoxic T cells (CD8 + T cells) and regulatory T cells (Tregs).

[0109] The analysis results are as follows Fig. 9 As shown, CD8 + The proportion of T cells increased, while the CD8 + The proportion of T cells was the highest, indicating that NIR-Ⅱ illumination 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 treated group showed a decrease in the proportion of Tregs, which further demonstrated 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification 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.

2. The light-driven heterojunction nanorobot according to claim 1, characterized in that: The Pd nanoparticles are also loaded with triphenylphosphine-modified neutrophil exosomes loaded with lactate oxidase.

3. The method for preparing the light-driven heterojunction nanorobot according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (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) Pd-SiO2 nanoparticles are resuspended in a polyvinyl pyrrolidone solution, and then a copper nitrate hydrate solution is added to react. After a period of reaction, hydrazine hydrate is added to continue the reaction. After the reaction is completed, a sodium sulfide aqueous solution is added to the reaction system to stir the reaction and centrifuge. Ammonia water is used to resuspend the precipitate and react and centrifuge to obtain CuS-Pd nanoparticles.

4. The preparation method according to claim 3, characterized in that: The preparation method further comprises: (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.

5. The preparation method according to claim 3, 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).

6. The preparation method according to claim 3, 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 8-12 mM.

7. The preparation method according to claim 4, 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).

8. The preparation method according to claim 4, characterized in that: In the step (d), the mass ratio of TPP-Exo@LOX to CuS-Pd nanoparticles is (1-1.2):

1.

9. Use of the light-driven heterojunction nanorobot according to claim 1 or 2 or the light-driven heterojunction nanorobot prepared by the preparation method according to any one of claims 3 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumors include colon cancer.

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