A biohybrid nanorobot, its preparation method and application

Abstract

The present invention discloses a biohybrid nanorobot and its preparation method and application. The biohybrid nanorobot is composed of interconnected gold-palladium nanozymes and bacterial outer membrane vesicles containing D-lactate dehydrogenase; the bacterial outer membrane vesicles are loaded with L-lactate oxidase. In the present invention, bacterial outer membrane vesicles containing D-lactate dehydrogenase are extracted, loaded with L-lactate oxidase by electroporation technology to obtain double-enzyme-loaded bacterial outer membrane vesicles, and then gold-palladium nanozymes are connected to the tail thereof as a chassis to obtain a biohybrid nanorobot with precise positioning, autonomous propulsion, deep penetration, and effective tumor inhibition system.

Description

Technical Field

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

[0002] Nanocatalytic medicine utilizes the unique properties of nanocatalysts to trigger chemical reactions in the tumor microenvironment, thereby potentially overcoming the limitations of traditional therapies. It has the advantages of improving treatment efficacy, reducing side effects, and dynamically regulating the tumor microenvironment, and has now become one of the highly regarded research hotspots in biomedicine. Researchers are committed to designing and preparing specific or multifunctional nanocatalysts to initiate catalytic reactions and dynamically regulate the tumor microenvironment, so as to achieve tumor treatment effects in a more efficient and lasting manner. This treatment strategy uses endogenous substances in tumors as treatment substrates, and generates toxins such as reactive oxygen species (ROS) or other therapeutic products in situ, avoiding the use of high-dose exogenous toxic chemicals, and has become a frontier method for anti-tumor treatment. At present, this field is still in the early stage, and the challenges include controlling the ROS level to avoid damage to normal tissues, and ensuring that the nanocatalysts can be safely metabolized or cleared out of the body. Future research needs to further optimize these technologies for clinical application, with the focus on improving targeting and reducing side effects.

[0003] Due to the complex tumor microenvironment and high tumor interstitial pressure, currently only about 0.7% of the administered dose can accurately reach the target position in the body. A large amount of drugs are forced to accumulate at the tumor edge or blood vessels, and it is difficult to penetrate into the tumor interior to achieve effective killing. Self-propelled nanosystems show great potential in overcoming the biological barriers existing in current tumor nanomedicine. In this context, micro- and nanomotors exhibit excellent targeting properties, and their drug delivery efficiency is significantly higher than that of traditional passive particles. In addition, in terms of penetrating biological barriers such as mucus, cells, or spheres, micro- and nanomotors also perform far better than traditional nanoparticles. Designing self-propelled particles using tumor endogenous substances as biocatalysts, because it can utilize endogenous fuels to achieve in-situ activation of nanomotors and design completely biocompatible motor-fuel complexes, is becoming a promising strategy. However, self-propelled motor systems have significant limitations that restrict their overall performance: lack of control over the direction of movement (i.e., random non-directional movement) and poor biocompatibility (cleared by the immune system).

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a biohybrid nanorobot and a preparation method and application thereof. The present invention extracts bacterial outer membrane vesicles containing D-lactate dehydrogenase, loads L-lactate oxidase through electroporation technology, obtains dual-enzyme-loaded bacterial outer membrane vesicles, and then connects gold palladium nanozyme at the tail as a chassis to obtain a biohybrid nanorobot with precise positioning, autonomous propulsion, deep penetration, and an effective tumor inhibition system.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The first aspect of the present invention provides a biohybrid nanorobot, which is composed of interconnected gold palladium nanozymes and bacterial outer membrane vesicles containing D-lactate dehydrogenase;

[0008] The bacterial outer membrane vesicles are loaded with L-lactate oxidase.

[0009] Preferably, the bacterial outer membrane vesicles containing D-lactate dehydrogenase are secreted by Lactobacillus crispatus.

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

[0011] (a) adding a gold nanoparticle solution to isopropanol, then adding a 4-mercaptobenzoic acid aqueous solution and a polyacrylic acid ethanol solution to react, after the reaction is completed, adding a TEOS solution and ammonia water to the reaction system to stir the reaction, centrifuge, and wash to obtain an Au-SiO2 heterojunction;

[0012] CTAB, H2PdCl4 and AA are sequentially added to the Au-SiO2 heterojunction solution for stirring reaction and centrifugation to obtain the Au-mSiO2-Pd heterojunction, and then the Au-mSiO2-Pd heterojunction is added to ammonia water overnight, centrifuged and washed with water to obtain the gold-palladium nanozyme;

[0013] (b) mixing a bacterial outer membrane vesicle solution containing D-lactate dehydrogenase, a glycerol solution, PBS and an L-lactate oxidase solution, and then performing an electroporation treatment, and then concentrating and purifying to obtain bacterial outer membrane vesicles containing D-lactate dehydrogenase loaded with L-lactate oxidase;

[0014] (c) adding EDC / NHS to the gold palladium nanozyme solution for activation reaction, and after the reaction is completed, adding a bacterial outer membrane vesicle solution containing D-lactate dehydrogenase loaded with L-lactate oxidase for incubation and centrifugation to obtain the biohybrid nanorobot.

[0015] Preferably, in the step (a), the volume ratio of the gold nanoparticle solution, isopropanol, 4-mercaptobenzoic acid aqueous solution, and polyacrylic acid ethanol solution is 5:(20-30):(0.1-0.3):(0.7-0.8);

[0016] The concentration of the gold nanoparticle solution is 0.2-0.5 mM; the concentration of the 4-mercaptobenzoic acid aqueous solution is 0.6-0.7 mM; the concentration of the polyacrylic acid ethanol solution is 4-6 mM.

[0017] Preferably, in the step (a), the volume ratio of the gold nanoparticle solution, TEOS solution, and ammonia water is 5:(8-10):(0.7-1.2);

[0018] The concentration of the TEOS solution is 8.5-10 mM; the concentration of the ammonia water is 25%-30% (w / w).

[0019] Preferably, in the step (a), the mass ratio of CTAB, H2PdCl4, AA, and Au-SiO2 heterojunction is (2-10):(1-4):(1-6):1.

[0020] Preferably, in the step (a), the reaction time for adding the 4-mercaptobenzoic acid aqueous solution and the polyacrylic acid ethanol solution is 40-50 min; the stirring reaction time for adding the TEOS solution and ammonia water to the reaction system is 3-5 h; the stirring reaction time for adding CTAB, H2PdCl4, and AA to the Au-SiO2 heterojunction solution in sequence is 20-40 min, and the temperature is room temperature.

[0021] In the present invention, conventional gold nanoparticles in the art can be used. Preferably, the gold nanoparticles can be prepared by the following method:

[0022] 17 nm Au NPs were prepared by the sodium citrate reduction method; then, HAuCl4 was dissolved in water and heated with vigorous stirring for 10 min; subsequently, the 17 nm Au NPs and sodium citrate dihydrate solution were added to the mixture, and the reaction was continued for 15 min to obtain 50 nm Au NPs.

[0023] Preferably, in the step (b), the volume ratio of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase, glycerol solution, PBS, and L-lactate oxidase solution is (300-350):(80-120):(40-50):(20-30);

[0024] The concentration of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase is 1.2-1.8 mg / ml;

[0025] The concentration of the glycerol solution is 40% - 60%; the concentration of the L-lactate oxidase solution is 10 - 20 μg / ml.

[0026] Preferably, the bacterial outer membrane vesicles containing D-lactate dehydrogenase are prepared by the following method:

[0027] Centrifuge 500 mL of Lactobacillus crispatus culture solution at 5000 g for 30 min to remove bacteria, and then filter through a 0.45 μm polyethersulfone membrane; concentrate the filtered liquid through a 100 kDa molecular weight cut-off centrifugal filter; ultra-centrifuge the concentrated culture medium at 150000 g for 3 h at 4°C, collect the precipitate and suspend it in sterile PBS, and then ultra-centrifuge again under the same conditions. Finally, resuspend the precipitate in PBS to obtain the bacterial outer membrane vesicles containing D-lactate dehydrogenase.

[0028] Preferably, in the step (b), the mixing time is 15 - 30 min; the number of electroporation treatments is 2 - 3 times; the voltage of the electroporation treatment is 500 V, the pulse width is 50 μs, and the number of pulses is infinite.

[0029] Preferably, in the step (c), the volume ratio of the gold-palladium nanozyme solution to the solution of bacterial outer membrane vesicles containing D-lactate dehydrogenase loaded with L-lactate oxidase is (5 - 10):1;

[0030] The concentration of the gold-palladium nanozyme solution is 0.8 - 1.2 mg / ml;

[0031] The concentration of the solution of bacterial outer membrane vesicles containing D-lactate dehydrogenase is 1.2 - 1.8 mg / ml.

[0032] Preferably, in the step (c), the activation reaction temperature is room temperature, the time is 25 - 40 min; the incubation time is 1 - 2 h; the centrifugation speed is 6500 - 7500 g.

[0033] The third aspect of the present invention provides an application of the biological hybrid nanorobot or the biological hybrid nanorobot prepared by the preparation method in the preparation of anti-tumor products.

[0034] Preferably, the tumor includes colorectal cancer.

[0035] Compared with the prior art, the beneficial effects of the present invention at least include:

[0036] The present invention extracts bacterial outer membrane vesicles containing D-lactate dehydrogenase, loads L-lactate oxidase by electroporation technology to obtain double-enzyme-loaded bacterial outer membrane vesicles, and then connects gold-palladium nanozyme at its tail as a chassis to obtain a biohybrid nanorobot with precise positioning, autonomous propulsion, deep penetration, and effective tumor suppression system. Specifically, (1) Precise targeting: The targeting effect of bacterial outer vesicles endows the biohybrid nanorobot with the ability of active targeting to tumor tissues; (2) Autonomous driving and deep penetration: The catalase-like property of asymmetric palladium nanoparticles catalyzes H2O2 overexpressed in the tumor microenvironment to produce O2, providing an autonomous driving force for the deep penetration of the nanorobot into the tumor while alleviating the tumor hypoxic microenvironment; (3) Primary tumor catalytic therapy and immunogenic cell death: The peroxidase-like property of palladium nanoparticles in the acidic environment of tumor cells catalyzes H2O2 to produce ·OH, inducing immunogenic cell death of tumor cells; (4) Immune microenvironment regulation and immunotherapy: Bacterial outer vesicles are loaded with a double-enzyme system of L-lactate oxidase and D-lactate dehydrogenase, and convert immunosuppressive L-lactate into immunostimulatory D-lactate through a two-step cascade reaction, reversing the immunosuppressive microenvironment and mediating efficient tumor immunotherapy; (5) Synergistic mechanism: The by-product H2O2 of L-lactate oxidase catalyzing L-lactate provides a continuous substrate supplement for the driving and catalytic therapy of the nanorobot; Gold nanoparticles, as a bridge, not only endow the nanorobot with the assembly sites of the navigation and driving systems, but also are an excellent electron transport medium, synergistically enhancing the catalytic efficiency of bioenzymes and nanozymes.

[0037] The biohybrid 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

[0038] 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 denoted by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale.

[0039] Figure 1 Schematic diagram of the preparation of the biohybrid nanorobot in the embodiment of the present invention;

[0040] Figure 2 TEM images of AP, E@CMV, and E@CMV-AP in Experimental Example 1 of the present invention;

[0041] Figure 3 High-resolution XPS and XRD characterization diagrams of E@CMV-AP in Experimental Example 1 of the present invention;

[0042] Figure 4 This is the test diagram of the movement trajectories of AP and E@CMV-AP in Experimental Example 2 of the present invention;

[0043] Figure 5 This is the diagram of the penetration of E@CMV-AP in 3D tumor cell spheroids in Experimental Example 2 of the present invention;

[0044] Figure 6 This is the test diagram of the catalytic performance of different materials in Experimental Example 3 of the present invention;

[0045] Figure 7 This is the diagram of the mediated lactic acid configuration conversion of E@CMV-AP in Experimental Example 4 of the present invention;

[0046] Figure 8 This is the test result of the toxicity of CT26 colorectal cancer cells after treatment with different materials in Experimental Example 5 of the present invention;

[0047] Figure 9 This is the flow cytometry analysis result of the regulatory effect of different nanoformulations on T cells in Experimental Example 6 of the present invention. Detailed implementation manners

[0048] Hereinafter, embodiments of the technical solution of the present invention will be 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 therefore are only examples and cannot be used to limit the protection scope of the present invention.

[0049] 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.

[0050] The embodiment of the present invention provides a biohybrid nanorobot, and the biohybrid nanorobot is composed of interconnected gold-palladium nanozymes and bacterial outer membrane vesicles containing D-lactate dehydrogenase;

[0051] The bacterial outer membrane vesicles are loaded with L-lactate oxidase.

[0052] The present invention extracts bacterial outer membrane vesicles containing D-lactate dehydrogenase, loads L-lactate oxidase by electroporation technology to obtain double-enzyme-loaded bacterial outer membrane vesicles, and then connects gold-palladium nanozymes to its tail as a chassis to obtain a biohybrid nanorobot with precise positioning, autonomous propulsion, deep penetration, and effective tumor inhibition system. Specifically, (1) Precise targeting: The targeting effect of bacterial outer vesicles endows the biohybrid nanorobot with the ability of active targeting to tumor tissues; (2) Autonomous driving and deep penetration: The catalase-like property of asymmetric palladium nanoparticles catalyzes H2O2 overexpressed in the tumor microenvironment to generate O2, relieving the tumor hypoxic microenvironment and providing an autonomous driving force for the deep penetration of the nanorobot into the tumor; (3) Primary tumor catalytic therapy and immunogenic cell death: The peroxidase-like property of palladium nanoparticles in the acidic environment of tumor cells catalyzes H2O2 to generate ·OH, inducing immunogenic cell death of tumor cells; (4) Immune microenvironment regulation and immunotherapy: Bacterial outer vesicles are loaded with a double-enzyme system of L-lactate oxidase and D-lactate dehydrogenase, and immunosuppressive L-lactate is converted into immunostimulatory D-lactate through a two-step cascade reaction, reversing the immunosuppressive microenvironment and mediating efficient tumor immunotherapy; (5) Synergistic mechanism: The by-product H2O2 of L-lactate oxidase catalyzing L-lactate provides a continuous substrate supplement for the driving and catalytic therapy of the nanorobot; Gold nanoparticles, as a bridge, not only endow the nanorobot with an assembly site for the navigation and driving system, but also are an excellent electron transport medium, synergistically enhancing the catalytic efficiency of bioenzymes and nanozymes.

[0053] In one embodiment, the bacterial outer membrane vesicles containing D-lactate dehydrogenase are secreted by Lactobacillus crispatus.

[0054] Another embodiment of the present invention provides a preparation method of the above biohybrid nanorobot, and the preparation method includes the following steps:

[0055] (a) Add a gold nanoparticle solution to isopropanol, then add an aqueous solution of 4-mercaptobenzoic acid and an ethanol solution of polyacrylic acid for reaction. After the reaction is completed, add a TEOS solution and ammonia water to the reaction system for stirring reaction, centrifugation, and washing to obtain an Au-SiO2 heterojunction;

[0056] Add CTAB, H2PdCl4, and AA to the Au-SiO2 heterojunction solution in sequence for stirring reaction and centrifugation to obtain an Au-mSiO2-Pd heterojunction, and then add the Au-mSiO2-Pd heterojunction to ammonia water overnight, centrifugation, and water washing to obtain gold-palladium nanozyme;

[0057] (b) Mix the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase, glycerol solution, PBS, and L-lactate oxidase solution, then perform electroporation treatment, and then concentrate and purify to obtain bacterial outer membrane vesicles containing D-lactate dehydrogenase loaded with L-lactate oxidase;

[0058] (c) Add EDC / NHS to the gold-palladium nanozyme solution for activation reaction. After the reaction ends, add the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase loaded with L-lactate oxidase for incubation and centrifugation to obtain the biohybrid nanorobot.

[0059] In some embodiments, in step (a), the volume ratio of the gold nanoparticle solution, isopropanol, 4-mercaptobenzoic acid aqueous solution, and polyacrylic acid ethanol solution is 5∶(20 - 30)∶(0.1 - 0.3)∶(0.7 - 0.8);

[0060] The concentration of the gold nanoparticle solution is 0.2 - 0.5 mM; the concentration of the 4-mercaptobenzoic acid aqueous solution is 0.6 - 0.7 mM; the concentration of the polyacrylic acid ethanol solution is 4 - 6 mM.

[0061] In some embodiments, in step (a), the volume ratio of the gold nanoparticle solution, TEOS solution, and ammonia water is 5∶(8 - 10)∶(0.7 - 1.2);

[0062] The concentration of the TEOS solution is 8.5 - 10 mM; the concentration of the ammonia water is 25% - 30% (w / w).

[0063] In some embodiments, in step (a), the mass ratio of CTAB, H2PdCl4, AA, and Au-SiO2 heterojunction is (2 - 10)∶(1 - 4)∶(1 - 6)∶1.

[0064] In some embodiments, in step (a), the reaction time for adding the 4-mercaptobenzoic acid aqueous solution and the polyacrylic acid ethanol solution is 40 - 50 min; the stirring reaction time for adding the TEOS solution and ammonia water to the reaction system is 3 - 5 h; the stirring reaction time for sequentially adding CTAB, H2PdCl4, and AA to the Au-SiO2 heterojunction solution is 20 - 40 min, and the temperature is room temperature.

[0065] The present invention does not make specific limitations on the source of the gold nanoparticles. Commercially available gold nanoparticles in the art can be used. In one embodiment, the gold nanoparticles can be prepared by the following method:

[0066] 17 nm Au NPs were prepared by the sodium citrate reduction method; then, HAuCl4 was dissolved in water and heated under vigorous stirring for 10 min; subsequently, 17 nm Au NPs and sodium citrate dihydrate solution were added to the mixture and the reaction was continued for 15 min to obtain 50 nm Au NPs.

[0067] In some embodiments, in step (b), the volume ratio of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase, glycerol solution, PBS, and L-lactate oxidase solution is (300 - 350)∶(80 - 120)∶(40 - 50)∶(20 - 30);

[0068] The concentration of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase is 1.2 - 1.8 mg / ml;

[0069] The concentration of the glycerol solution is 40% - 60%; the concentration of the L-lactate oxidase solution is 10 - 20 μg / ml.

[0070] In one embodiment, the bacterial outer membrane vesicles containing D-lactate dehydrogenase are prepared by the following method:

[0071] 500 mL of Lactobacillus crispatus culture solution was centrifuged at 5000 g for 30 min to remove bacteria, and then filtered through a 0.45 μm polyethersulfone membrane; the filtered liquid was concentrated by a 100 kDa molecular weight cut-off centrifugal filter; the concentrated culture medium was ultracentrifuged at 150000 g for 3 h at 4°C, the precipitate was collected and suspended in sterile PBS, and then ultracentrifuged again under the same conditions. Finally, the precipitate was resuspended in PBS to obtain the bacterial outer membrane vesicles containing D-lactate dehydrogenase.

[0072] In some embodiments, in step (b), the mixing time is 15 - 30 min; the number of electroporation treatments is 2 - 3 times; the voltage of the electroporation treatment is 500 V, the pulse width is 50 μs, and the number of pulses is infinite.

[0073] In some embodiments, in step (c), the volume ratio of the gold-palladium nanozyme solution and the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase loaded with L-lactate oxidase is (5 - 10)∶1;

[0074] The concentration of the gold-palladium nanozyme solution is 0.8 - 1.2 mg / ml;

[0075] The concentration of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase is 1.2 - 1.8 mg / ml.

[0076] In some embodiments, in step (c), the activation reaction temperature is room temperature, the time is 25 to 40 min; the incubation time is 1 to 2 h; the centrifugation speed is 6500 to 7500 g.

[0077] Another embodiment of the present invention provides an application of the above-mentioned biohybrid nanorobot or the biohybrid nanorobot prepared by the preparation method in the preparation of anti-tumor products.

[0078] The biohybrid 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.

[0079] In one embodiment, the tumor includes colorectal cancer.

[0080] The technical solutions of the present invention will be further described in detail below through specific examples.

[0081] The raw materials used in the following examples are as follows:

[0082] The gold nanoparticles were prepared by the following method:

[0083] 17 nm Au NPs were prepared by the sodium citrate reduction method; then, HAuCl4 was dissolved in water and heated under vigorous stirring for 10 min; subsequently, 17 nm Au NPs and sodium citrate dihydrate solution were added to the mixture and the reaction was continued for 15 min to obtain 50 nm Au NPs.

[0084] The bacterial outer membrane vesicles containing D-lactate dehydrogenase were prepared by the following method:

[0085] 500 mL of Lactobacillus crispatus culture solution was centrifuged at 5000 g for 30 min to remove bacteria, and then filtered through a 0.45 μm polyethersulfone membrane; the filtered liquid was concentrated by a 100 kDa molecular weight cut-off centrifugal filter; the concentrated culture medium was ultracentrifuged at 150000 g for 3 h at 4 °C, the precipitate was collected and suspended in sterile PBS, and then ultracentrifuged again under the same conditions. Finally, the precipitate was resuspended in PBS to obtain bacterial outer membrane vesicles containing D-lactate dehydrogenase (denoted as CMV).

[0086] Example

[0087] This example is a preparation method of a biohybrid nanorobot, as Figure 1 shown, the preparation method includes the following steps:

[0088] (a) Preparation of gold-palladium nanozyme:

[0089] Under vigorous stirring, 5 mL of gold nanoparticle solution (concentration: 0.5 mM) was transferred into 25 mL of isopropanol. Then, 4-mercaptobenzoic acid (200 μL, aqueous solution with a concentration of 0.645 mM) and polyacrylic acid (720 μL, ethanol solution with a concentration of 5 mM) were added, and the reaction was carried out for 45 min. After that, 9 mL of TEOS solution (8.9 mM) and 0.9 mL of NH₃·H₂O were added to the system, and the reaction continued under stirring for 4 h. Then, it was centrifuged and washed with isopropanol and water to collect the Au-SiO₂ heterojunction;

[0090] According to the mass ratio of CTAB, H₂PdCl₄, AA, and Au-SiO₂ heterojunction being 6∶2∶4∶1, CTAB, H₂PdCl₄, and AA were successively added to the above Au-mSiO₂ heterojunction solution. After stirring at room temperature for 30 min, it was centrifuged to obtain the Au-mSiO₂-Pd heterojunction, which was then etched with 100 μL of ammonia water overnight, centrifuged, and washed with water to obtain the gold-palladium nanozyme (denoted as AP nanozyme, or AP);

[0091] (b) Preparation of bacterial outer membrane vesicles loaded with L-lactate oxidase and containing D-lactate dehydrogenase (denoted as E@CMV):

[0092] Bacterial outer membrane vesicles containing D-lactate dehydrogenase (330 μL, 1.5 mg / mL) were mixed with glycerol solution (100 μL, 50%), PBS (45 μL, 1×, pH 7.4), and L-LOX (25 μL, 15 μg / ml) for 20 min; Subsequently, two electro-poration treatments (500 V, 50, ∞) were carried out using Bio-Rad GenePulser Xcell to obtain CMVs loaded with L-LOX (E@CMV), which were concentrated and purified by using a 100-kDa filter membrane (5,000×g, 1 hour, 4 °C), and then stored in PBS (400 μL, 1×, pH 7.4) and preserved at -20 °C for standby;

[0093] (c) Preparation of biohybrid nanorobot (denoted as E@CMV-AP):

[0094] At room temperature, 1 mg / mL of AP nanozyme solution and EDC / NHS were mixed and reacted at room temperature for 30 min. In the activated nanozyme solution, 1.5 mg / mL of E@CMV solution was added with a volume ratio of 8∶1. After the reaction, it was centrifuged at 7,000 g and washed repeatedly 3 times to obtain the biohybrid nanorobot (E@CMV-AP).

[0095] Experimental Example 1

[0096] AP, E@CMV, and E@CMV-AP were scanned by transmission electron microscopy;

[0097] The scanning results are as follows Figure 2 shown Figure 2 In the figure, the left figure is AP, the middle figure is E@CMV, and the right figure is E@CMV-AP. As Figure 2 shown, the "head" of E@CMV-AP is modified with bacterial outer vesicles, and the "tail" is modified with gold-palladium. This asymmetric heterojunction structure ensures its ability to target through the head and drive through the tail, demonstrating the successful preparation of the morphology of E@CMV-AP.

[0098] Perform high-resolution X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analyses on E@CMV-AP;

[0099] The analysis results are as follows Figure 3 shown. It can be seen from Figure 3 that the elements Au, Pd, C, N, O, and P exist in E@CMV-AP.

[0100] Experimental Example 2

[0101] This experimental example is for the motion characterization of E@CMV-AP:

[0102] (1) E@CMV-AP motion trajectory test:

[0103] Place E@CMV-AP in PBS solution, and observe and record its motion trajectory using a dark-field microscope and a CUDA spot tracker with or without an additional 1 mM H2O2 treatment. The tracking test time is 15 s, and the E@CMV-AP motion curve is plotted.

[0104] (2) 3D tumor cell mass culture and penetration

[0105] Add 50 μL of 1% agarose to each well of a 96-well plate and irradiate it under ultraviolet light for 30 minutes. Then, add 200 μL of a U14 cell suspension with a concentration of 1×10 3 cells / mL, and 3D cell spheres are formed after 10 days of culture. Subsequently, add E@CMV-AP labeled with FITC dye. Take the cell spheres out of the well plate, wash them 3 times with PBS, and observe the penetration of the nanorobot in the cell spheres through a laser confocal microscope.

[0106] Experimental results:

[0107] The motion trajectory test diagrams of AP and E@CMV-AP are as follows Figure 4 shown. It can be seen from Figure 4 that under the recruitment of tumor cells, E@CMV-AP exhibits excellent motion performance, and its motion trajectory is approximately linear, while the AP group without E@CMV shows a spiral motion trajectory and has no obvious forward motion trend.

[0108] The 3D tumor cell mass penetration diagram is as follows Figure 5 shown. It can be seen from Figure 5 that E@CMV-AP can actively penetrate into the center of the tumor cell sphere, indicating that the active movement performance of E@CMV-AP can greatly promote its deep penetration into the tumor interior.

[0109] Experimental Example 3

[0110] This experimental example is for the catalytic performance test:

[0111] In the presence of H2O2, using TMB as the substrate, the ability of E@CMV-AP to generate hydroxyl radicals was measured. 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 5.4, and then E@CMV-AP was added to the reaction system. In addition, electron spin resonance (ESR) was used to detect the generation of hydroxyl radicals, and a dissolved oxygen meter was used to detect the catalase-like properties.

[0112] The results of the catalase-like and peroxidase-like properties of the E@CMV-AP nanorobot are as follows Figure 6 shown. It can be seen from Figure 6 that Figure 6 a shows that the catalase-like property of the asymmetric palladium nanoparticles catalyzes the overexpressed H2O2 in the tumor microenvironment to produce O2, indicating that the nanorobot has the ability to relieve the tumor hypoxic microenvironment and provide an autonomous driving force for the deep penetration of the nanorobot into the tumor; Figure 6 b uses TMB as a probe to evaluate the peroxidase-like property of the biopalladium nanoparticles to catalyze H2O2 to produce ·OH in the acidic environment of tumor cells, Figure 6 c is further verified using electron spin resonance (ESR) spectroscopy.

[0113] Experimental Example 4

[0114] This experimental example is for the study of the mediated lactic acid configuration conversion of the bio-nanorobot:

[0115] This experiment aims to use L-lactate oxidase (L-LOX) to convert L-lactate (L-LA) into pyruvate (PA), use D-lactate dehydrogenase (D-LDH) to convert PA into D-lactic acid, and analyze the reaction process by measuring the consumption of L-lactic acid and the generation of D-lactic acid.

[0116] First, under the conditions of 37 °C and pH 6.5, PBS, CMV, E@CMV, AP, and E@CMV-AP were reacted with 5 mM lactic acid. The consumption of L-lactic acid was detected by a lactic acid detection kit. L-LOX catalyzes the oxidation of L-lactic acid to pyruvate and hydrogen peroxide. Subsequently, through the chromogenic reaction of peroxidase (POD) with 4-aminoantipyrine (4-AAP) and N,N-dimethylaniline (DMA), the reaction process was monitored. The absorbance change was measured at 565 nm by a spectrophotometer, and the consumption of L-lactic acid was calculated.

[0117] Next, D-LDH reduces pyruvate to D-lactic acid under the action of NADH. Since NADH has a characteristic absorption peak at 340 nm while NAD+ has no absorption, the change in absorbance of the reaction system at 340 nm can indirectly reflect the activity of D-LDH. The reaction solution was appropriately diluted in 1% bovine serum albumin solution (BSA) and added to a solution containing potassium phosphate buffer, β-NADH, sodium pyruvate solution, and DMA. Immediately invert and mix, and record the decrease in absorbance at 340 nm within 120 minutes. By calculating the enzyme activities of L-LOX and D-LDH, the conversion efficiency of L-lactic acid to D-lactic acid can be evaluated, thereby analyzing the combined catalytic effect of the enzymes.

[0118] The experimental results are as Figure 7 shown;

[0119] As Figure 7 can be seen: The dual-enzyme system (L-LOX and D-LDH) in E@CMV acts synergistically to achieve the conversion of L-lactic acid to D-lactic acid. As the reaction time extends, the concentration of L-lactic acid decreases significantly, while the concentration of D-lactic acid increases significantly, indicating that L-LOX first oxidizes L-lactic acid to pyruvate, and D-LDH further reduces pyruvate to D-lactic acid. This combined catalytic effect shows a relatively high conversion efficiency, especially under higher enzyme concentrations and appropriate reaction conditions, demonstrating the effectiveness of E@CMV as a dual-enzyme carrier in the conversion of L-lactic acid to D-lactic acid. These results prove the application potential of the E@CMV system in lactic acid conversion.

[0120] Experimental Example 5

[0121] This experimental example is for the cytotoxicity test of tumor cell killing:

[0122] U14 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 culture medium containing different nanopreparations (CMV, AP, E@CMV, and E@CMV-AP) 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.

[0123] The cytotoxicity test of CT26 colorectal cancer cells after treatment with different materials is as Figure 8 shown. The CCK-8 cytotoxicity experiment shows that after treatment with E@CMV-AP, the survival rate of U14 cells is as low as 33.9%, indicating that E@CMV-AP has good anti-tumor killing effect.

[0124] Experimental Example 6

[0125] This experimental example is for in vitro T cell immune activation research:

[0126] 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 (CMV, AP, E@CMV, and E@CMV-AP) were added to the medium and the culture was continued for 48 h. Subsequently, the T cells were stained with flow antibodies anti-CD3APC, 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 nanorobots on cytotoxic T cells (CD8 + T cells) and regulatory T cells (Treg).

[0127] The results of flow cytometry analysis are as Figure 9 shown. The proportion of CD8 + T cells in the treatment groups with different nanomaterials added is the highest, which can effectively activate CD8 + T cells. The E@CMV-AP treatment group showed a decrease in the proportion of Treg, further indicating that it can improve the tumor immunosuppressive microenvironment and enhance the efficacy of anti-tumor immunotherapy.

[0128] 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 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 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 covered by the scope of the claims and the description of the present invention.

Claims

1. A biohybrid nanorobot, characterized in that: The biohybrid nanorobot is composed of interconnected gold palladium nanozymes and bacterial outer membrane vesicles containing D-lactate dehydrogenase; The bacterial outer membrane vesicles are loaded with L-lactate oxidase; The bacterial outer membrane vesicles containing D-lactate dehydrogenase are secreted by Lactobacillus crispatus; The preparation method of the biohybrid nanorobot comprises the following steps: (a) adding a gold nanoparticle solution to isopropanol, then adding a 4-mercaptobenzoic acid aqueous solution and a polyacrylic acid ethanol solution to react, after the reaction is completed, adding a TEOS solution and ammonia water to the reaction system to stir the reaction, centrifuge, and wash to obtain an Au-SiO2 heterojunction; CTAB, H2PdCl4 and AA are sequentially added to the Au-SiO2 heterojunction solution for stirring reaction and centrifugation to obtain the Au-mSiO2-Pd heterojunction, and then the Au-mSiO2-Pd heterojunction is added to ammonia water overnight, centrifuged and washed with water to obtain the gold-palladium nanozyme; (b) mixing a bacterial outer membrane vesicle solution containing D-lactate dehydrogenase, a glycerol solution, PBS and an L-lactate oxidase solution, and then performing an electroporation treatment, and then concentrating and purifying to obtain bacterial outer membrane vesicles containing D-lactate dehydrogenase loaded with L-lactate oxidase; (c) adding EDC / NHS to the gold palladium nanozyme solution for activation reaction, and after the reaction is completed, adding a bacterial outer membrane vesicle solution containing D-lactate dehydrogenase loaded with L-lactate oxidase for incubation and centrifugation to obtain the biohybrid nanorobot.

2. The biohybrid nanorobot according to claim 1, characterized in that: In the step (a), the volume ratio of the gold nanoparticle solution, isopropanol, 4-mercaptobenzoic acid aqueous solution and polyacrylic acid ethanol solution is 5: (20-30): (0.1-0.3): (0.7-0.8); The concentration of the gold nanoparticle solution is 0.2-0.5 mM; the concentration of the 4-mercaptobenzoic acid aqueous solution is 0.6-0.7 mM; and the concentration of the polyacrylic acid ethanol solution is 4-6 mM.

3. The biohybrid nanorobot according to claim 1, characterized in that: In the step (a), the volume ratio of the gold nanoparticle solution, the TEOS solution and the ammonia solution is 5:(8-10):(0.7-1.2); The concentration of TEOS solution is 8.5-10 mM; the concentration of ammonia water is 25%-30%.

4. The biohybrid nanorobot according to claim 1, characterized in that: In the step (a), the mass ratio of CTAB, H2PdCl4, AA and Au-SiO2 heterojunction is (2-10):(1-4):(1-6):

1.

5. The biohybrid nanorobot according to claim 1, characterized in that: In the step (b), the volume ratio of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase, the glycerol solution, PBS and the L-lactate oxidase solution is (300-350): (80-120): (40-50): (20-30); The concentration of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase is 1.2 to 1.8 mg / ml; The concentration of the glycerol solution is 40% to 60%; the concentration of the L-lactate oxidase solution is 10 to 20 μg / ml.

6. The biohybrid nanorobot according to claim 1, characterized in that: In the step (c), the volume ratio of the gold palladium nanozyme solution to the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase loaded with L-lactate oxidase is (5-10):1; The concentration of gold palladium nanozyme solution is 0.8-1.2 mg / ml; The concentration of the bacterial outer membrane vesicle solution containing D-lactate dehydrogenase is 1.2-1.8 mg / ml.

7. Use of the biohybrid nanorobot described in any one of claims 1 to 6 in the preparation of anti-colorectal cancer products.

Images