Biological hybrid nano-robot as well as preparation method and application thereof

Abstract

The invention discloses a biological hybrid nano-robot as well as a preparation method and application thereof. The biological hybrid nano-robot is composed of gold-palladium nano-enzyme and bacterial outer membrane vesicles containing D-lactic dehydrogenase which are connected with each other, the bacterial outer membrane vesicles are loaded with L-lactate oxidase; bacterial outer membrane vesicles containing D-lactate dehydrogenase are extracted, L-lactate oxidase is loaded through an electroporation technology, double-enzyme-loaded bacterial outer membrane vesicles are obtained, gold-palladium nano-enzyme is connected to the tail of the bacterial outer membrane vesicles to serve as a base plate, and the biological hybrid nano-robot with an accurate positioning, autonomous propulsion, deep penetration and effective tumor suppression system is obtained.

Description

Technical Field

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

[0002] Nanocatalytic medicine uses the unique properties of nanocatalysts to trigger chemical reactions in the tumor microenvironment, which may overcome the limitations of traditional therapies. It has the advantages of improving treatment effects, reducing side effects, and dynamically regulating the tumor microenvironment, and has now become one of the most popular 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, thereby achieving tumor therapeutic effects in a more efficient and lasting manner. This therapeutic strategy uses endogenous substances in the tumor as therapeutic substrates and avoids the use of high-dose exogenous toxic chemicals by in situ production of toxins such as reactive oxygen species (ROS) or other therapeutic products, becoming a cutting-edge method for anti-tumor treatment. At present, this field is still in its early stages, and the challenges it faces include controlling ROS levels to avoid damage to normal tissues and ensuring that nanocatalysts can be safely metabolized or cleared from the body. Future research needs to further optimize these technologies for clinical application, with a focus on improving targeting and reducing side effects.

[0003] Due to the complex tumor microenvironment and high tumor interstitial pressure, only about 0.7% of the administered dose can accurately reach the target location in vivo. A large amount of drugs are forced to accumulate at the edge of the tumor or in the blood vessels, making it difficult to penetrate into the tumor to achieve effective killing. Self-driven nanosystems have shown great potential in overcoming the biological barriers existing in current tumor nanomedicine treatment. 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, micro- and nanomotors also perform much better than traditional nanoparticles in penetrating biological barriers such as mucus, cells or spheres. Using tumor endogenous substances as biocatalysts to design self-propelled particles is becoming a promising strategy because it can use endogenous fuels to achieve on-site activation of nanomotors and design fully biocompatible motor-fuel complexes. However, self-driven motor systems have significant limitations that limit their overall performance: lack of motion direction control (i.e., random non-directional motion) and poor biocompatibility (immune system clearance).

[0004] In view of this, the present invention is 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 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; and the concentration of the polyacrylic acid ethanol solution is 4-6 mM.

[0017] Preferably, in 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);

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

[0019] Preferably, in 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), an aqueous 4-mercaptobenzoic acid solution and an ethanol solution of polyacrylic acid are added for a reaction time of 40 to 50 minutes; TEOS solution and aqueous ammonia are added to the reaction system for a stirring reaction time of 3 to 5 hours; CTAB, H2PdCl4 and AA are added to the Au-SiO2 heterojunction solution in sequence for a stirring reaction time of 20 to 40 minutes at room temperature.

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

[0022] 17 nm Au NPs were prepared by sodium citrate reduction method; then, HAuCl4 was dissolved in water and heated to react for 10 min under vigorous stirring; 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.

[0023] Preferably, in 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);

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

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

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

[0027] 500 mL of Lactobacillus crispatus culture was centrifuged at 5000 g for 30 min to remove bacteria, and then filtered through a 0.45 μm polyethersulfone filter membrane; the filtered liquid was concentrated through a centrifugal filter with a molecular weight cutoff of 100 kDa; 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.

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

[0029] Preferably, in 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;

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

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

[0032] Preferably, in step (c), the activation reaction temperature is room temperature, the time is 25 to 40 minutes, the incubation time is 1 to 2 hours, and the centrifugal speed is 6500 to 7500g.

[0033] The third aspect of the present invention provides a use of the biohybrid nanorobot or the biohybrid nanorobot prepared by the preparation method in the preparation of anti-tumor products.

[0034] Preferably, the tumor comprises colorectal cancer.

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

[0036] The present invention extracts bacterial outer membrane vesicles containing D-lactate dehydrogenase, loads L-lactate oxidase through electroporation technology, obtains bacterial outer membrane vesicles loaded with dual enzymes, and then connects gold palladium nanozymes at the 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 membrane vesicles gives the biohybrid nanorobot the ability to actively target tumor tissues; (2) Autonomous driving and deep penetration: The catalase-like properties of asymmetric palladium nanoparticles catalyze the overexpressed H2O2 in the tumor microenvironment to produce O2, alleviate the tumor hypoxic microenvironment, and provide autonomous driving force for the nanorobot's deep tumor penetration; (3) Catalytic treatment and immunogenic death of primary tumors: palladium nanoparticles In the acidic environment of tumor cells, the peroxidase-like properties catalyze H2O2 to produce ·OH, inducing immunogenic death of tumor cells; (4) Immune microenvironment regulation and immunotherapy: bacterial extracellular vesicles are loaded with a dual enzyme system of L-lactate oxidase and D-lactate dehydrogenase, which converts immunosuppressive L-lactate into immunopromoting D-lactate through a two-step cascade reaction, reversing the immunosuppressive microenvironment and mediating efficient tumor immunotherapy; (5) Synergistic mechanism: The byproduct H2O2 of L-lactate oxidase catalyzing L-lactic acid provides a continuous substrate supplement for the driving and catalytic treatment of nanorobots; gold nanoparticles as a bridge not only provide assembly sites for the navigation and driving systems of nanorobots, but also serve as an excellent electron transfer 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 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 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.

[0039] Figure 1 This is a schematic diagram of the preparation of a biohybrid nanorobot in an 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 images of E@CMV-AP in Experimental Example 1 of the present invention;

[0042] Figure 4 It is the motion trajectory test diagram of AP and E@CMV-AP in Experimental Example 2 of the present invention;

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

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

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

[0046] Figure 8 The results of the toxicity test on CT26 colorectal cancer cells after being treated with different materials in Experimental Example 5 of the present invention;

[0047] Fig. 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 DESCRIPTION

[0048] 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.

[0049] 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.

[0050] The embodiment 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;

[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 through electroporation technology, obtains bacterial outer membrane vesicles loaded with dual enzymes, and then connects gold palladium nanozymes at the 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 membrane vesicles gives the biohybrid nanorobot the ability to actively target tumor tissues; (2) Autonomous driving and deep penetration: The catalase-like properties of asymmetric palladium nanoparticles catalyze the overexpressed H2O2 in the tumor microenvironment to produce O2, alleviate the tumor hypoxic microenvironment, and provide autonomous driving force for the nanorobot's deep tumor penetration; (3) Catalytic treatment and immunogenic death of primary tumors: palladium nanoparticles In the acidic environment of tumor cells, the peroxidase-like properties catalyze H2O2 to produce ·OH, inducing immunogenic death of tumor cells; (4) Immune microenvironment regulation and immunotherapy: bacterial extracellular vesicles are loaded with a dual enzyme system of L-lactate oxidase and D-lactate dehydrogenase, which converts immunosuppressive L-lactate into immunopromoting D-lactate through a two-step cascade reaction, reversing the immunosuppressive microenvironment and mediating efficient tumor immunotherapy; (5) Synergistic mechanism: The byproduct H2O2 of L-lactate oxidase catalyzing L-lactic acid provides a continuous substrate supplement for the driving and catalytic treatment of nanorobots; gold nanoparticles as a bridge not only provide assembly sites for the navigation and driving systems of nanorobots, but also serve as an excellent electron transfer 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 method for preparing the above-mentioned biohybrid nanorobot, the preparation method comprising the following steps:

[0055] (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;

[0056] 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;

[0057] (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;

[0058] (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.

[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; and 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, the TEOS solution, and the ammonia solution is 5:(8-10):(0.7-1.2);

[0062] The concentration of TEOS solution is 8.5-10 mM; the concentration of 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 the step (a), 4-mercaptobenzoic acid aqueous solution and polyacrylic acid ethanol solution are added for a reaction time of 40 to 50 minutes; TEOS solution and ammonia water are added to the reaction system and stirred for a reaction time of 3 to 5 hours; CTAB, H2PdCl4 and AA are added to the Au-SiO2 heterojunction solution in sequence and stirred for a reaction time of 20 to 40 minutes at room temperature.

[0065] The present invention does not specifically define the source of the gold nanoparticles, and commercially available gold nanoparticles in the art may be used. In one embodiment, the gold nanoparticles may be prepared by the following method:

[0066] 17 nm Au NPs were prepared by sodium citrate reduction method; then, HAuCl4 was dissolved in water and heated to react for 10 min under vigorous stirring; 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, the glycerol solution, PBS and the 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 to 1.8 mg / ml;

[0069] The concentration of the glycerol solution is 40% to 60%; the concentration of the L-lactate oxidase solution is 10 to 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 was centrifuged at 5000 g for 30 min to remove bacteria, and then filtered through a 0.45 μm polyethersulfone filter membrane; the filtered liquid was concentrated through a centrifugal filter with a molecular weight cutoff of 100 kDa; 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.

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

[0073] In some embodiments, in 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;

[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 minutes, the incubation time is 1 to 2 hours, and the centrifugal speed is 6500 to 7500g.

[0077] Yet another embodiment of the present invention provides a use 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 and has significant application potential, especially in the fields of tumor catalysis and immunotherapy.

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

[0080] The technical solution of the present invention is further described in detail below through specific embodiments.

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

[0082] Gold nanoparticles were prepared by the following method:

[0083] 17 nm Au NPs were prepared by sodium citrate reduction method; then, HAuCl4 was dissolved in water and heated to react for 10 min under vigorous stirring; 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 fluid was centrifuged at 5000 g for 30 min to remove bacteria, and then filtered through a 0.45 μm polyethersulfone filter membrane; the filtered liquid was concentrated through a centrifugal filter with a molecular weight cutoff of 100 kDa; the concentrated culture medium was ultracentrifuged at 150,000 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 (denoted as CMV) containing D-lactate dehydrogenase.

[0086] Example

[0087] This embodiment is a method for preparing a biohybrid nanorobot, such as Figure 1 As shown, the preparation method comprises the following steps:

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

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

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

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

[0092] Bacterial outer membrane vesicles (330 μL, 1.5 mg / mL) containing D-lactate dehydrogenase 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; then, electroporation was performed twice using Bio-Rad Gene Pulser Xcell (500 V, 50, ∞) to obtain CMVs loaded with L-LOX (E@CMV), which were concentrated and purified by using a 100-kDa filter (5,000×g, 1 h, 4°C), and then stored in PBS (400 μL, 1×, pH 7.4) and stored at -20°C for future use;

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

[0094] At room temperature, 1 mg / mL AP nanozyme solution and EDC / NHS were mixed and reacted at room temperature for 30 min. 1.5 mg / mL E@CMV solution was added to the activated nanozyme solution at a volume ratio of 8:1. After the reaction, centrifugation was performed at 7000 g and washing was repeated 3 times to obtain the biohybrid nanorobot (E@CMV-AP).

[0095] Experimental Example 1

[0096] Transmission electron microscopy scanning of AP, E@CMV, and E@CMV-AP;

[0097] The scan results are as follows Figure 2 As shown, Figure 2 In the figure, the left picture is AP, the middle picture is E@CMV, and the right picture is E@CMV-AP. Figure 2 As shown, the "head" of E@CMV-AP is modified with bacterial extracellular vesicles, and the "tail" is modified with gold palladium. This asymmetric heterojunction structure ensures that it can be targeted by the head and driven by the tail, proving the successful preparation of the E@CMV-AP morphology.

[0098] High-resolution X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis of E@CMV-AP;

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

[0100] Experimental Example 2

[0101] This experimental example is E@CMV-AP motion representation:

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

[0103] E@CMV-AP was placed in a PBS solution, and its movement trajectory was observed and recorded using a dark-field microscope and a CUDA spot tracker with or without the addition of 1 mM H2O2. The tracking test time was 15 s, and the E@CMV-AP movement curve was drawn.

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

[0105] Add 50 μL of 1% agarose to each well of a 96-well plate and irradiate under UV light for 30 minutes. Then, add 200 μL of 1×10 3 cells / mL of U14 cell suspension, after 10 days of culture, formed 3D cell spheres. Subsequently, E@CMV-AP labeled with FITC dye was added. The cell spheres were removed from the well plate and washed with PBS three times, and the penetration of nanorobots in the cell spheres was observed by laser confocal microscopy.

[0106] Experimental results:

[0107] The motion trajectory test diagram of AP and E@CMV-AP is shown in the figure Figure 4 As shown by Figure 4 It can be seen that under the recruitment of tumor cells, E@CMV-AP showed excellent motion performance, and the motion trajectory was approximately linear, while the AP group without E@CMV showed a spiral motion trajectory without an obvious forward motion trend.

[0108] 3D tumor cell cluster penetration diagram Figure 5 As shown by Figure 5 It can be seen that E@CMV-AP can move deeply into the center of the tumor cell sphere, which shows that the active motion performance of E@CMV-AP can greatly promote its penetration into the tumor.

[0109] Experimental Example 3

[0110] This experimental example is a catalytic performance test:

[0111] The ability of E@CMV-AP to generate hydroxyl radicals was determined in the presence of H2O2 using TMB as a substrate. 0.3 mL of 8 mM TMB and 90 μL of 10 mM H2O2 were added to 2.57 mL of PBS solution at pH 5.4, and then E@CMV-AP was added to the reaction system. In addition, the generation of hydroxyl radicals was detected using electron spin resonance (ESR), and the catalase-like properties were detected using a dissolved oxygen meter.

[0112] The results of catalase-like and peroxidase-like properties of E@CMV-AP nanorobots are shown in Figure 6 As shown by Figure 6 It can be seen that Figure 6 a) The catalase-like properties of asymmetric palladium nanoparticles catalyze the H2O2 overexpressed in the tumor microenvironment to produce O2, indicating that the nanorobot has the ability to alleviate the tumor hypoxic microenvironment and provide an autonomous driving force for the nanorobot's deep tumor penetration; Figure 6 b Using TMB as a probe, the peroxidase-like properties of bio-palladium nanoparticles were evaluated to catalyze H2O2 to produce ·OH in the acidic environment of tumor cells. Figure 6 c Further verification was performed using electron spin resonance (ESR) spectroscopy.

[0113] Experimental Example 4

[0114] This experimental example is a study on the conversion of lactic acid mediated by biological nanorobots:

[0115] The purpose of this experiment was to convert L-lactic acid (L-LA) into pyruvate (PA) using L-lactate oxidase (L-LOX), convert PA into D-lactic acid using D-lactate oxidase (D-LDH), and analyze the reaction process by measuring the consumption of L-lactic acid and the production of D-lactic acid.

[0116] First, PBS, CMV, E@CMV, AP and E@CMV-AP were reacted with 5 mM lactic acid at 37 °C and pH 6.5. The consumption of L-lactic acid was detected by a lactic acid detection kit. L-LOX catalyzed the oxidation of L-lactic acid to produce pyruvic acid and hydrogen peroxide. The reaction progress was then monitored by the colorimetric reaction of peroxidase (POD) with aminoantipyrine (4-AAP) and N,N-dimethylaniline (DMA). The consumption of L-lactic acid was calculated by measuring the absorbance change at 565 nm using a spectrophotometer.

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

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

[0119] Depend on Figure 7 It can be seen that the dual enzyme system (L-LOX and D-LDH) in E@CMV works synergistically to achieve the conversion of L-lactic acid to D-lactic acid. As the reaction time increases, 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, while D-LDH further reduces pyruvate to D-lactic acid. This synergistic catalytic effect exhibits a high conversion efficiency, especially at high enzyme concentrations and suitable reaction conditions, showing the effectiveness of E@CMV as a dual enzyme carrier in the conversion of L-lactic acid to D-lactic acid. These results demonstrate the application potential of the E@CMV system in lactic acid conversion.

[0120] Experimental Example 5

[0121] This experimental example is a tumor cell killing toxicity test:

[0122] U14 cells were cultured in 96-well plates for 24 h, then the culture medium was gently aspirated and the cells were washed three times with PBS, and fresh culture medium containing different nanoformulations (CMV, AP, E@CMV, and E@CMV-AP) at a concentration of 100 μg / mL was added and cultured for another 24 h. PBS without materials was used as a control group. The activity of CT26 cells was detected using a CCK-8 kit.

[0123] Toxicity test of CT26 colorectal cancer cells after treatment with different materials Figure 8 As shown, the CCK-8 cytotoxicity experiment showed that after E@CMV-AP treatment, the survival rate of U14 cells was as low as 33.9%, which showed that E@CMV-AP had a good anti-tumor killing effect.

[0124] Experimental Example 6

[0125] This experimental example is an in vitro T cell immune activation study:

[0126] T cells were isolated from the spleen of 6-week-old healthy Balb / c female mice and cultured in a medium containing 20 mM lactate. After 24 h of culture, different nanoformulations (CMV, AP, E@CMV, and E@CMV-AP) were added to the culture medium and cultured for another 48 h. Subsequently, flow cytometry antibodies anti-CD3APC, anti-CD4 BV605, anti-CD8 FITC, anti-CD25 PE / Cy7h, and anti-Foxp3 BV421 were used to stain T cells and flow cytometry analysis was performed to evaluate the effects of the constructed nanorobots on cytotoxic T cells (CD8 + T cells) and regulatory T cells (Tregs).

[0127] Flow cytometry analysis results Fig. 9 As shown, after adding different nanoformulation treatment groups, CD8 + The highest proportion of T cells can effectively activate CD8 + The E@CMV-AP-treated group showed a decrease in the proportion of Tregs, which further demonstrated that it could 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 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 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.

2. The biohybrid nanorobot according to claim 1, characterized in that: The bacterial outer membrane vesicles containing D-lactate dehydrogenase are secreted by Lactobacillus crispatus.

3. The method for preparing the biohybrid nanorobot according to claim 1 or 2, characterized in that: The preparation method 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.

4. The preparation method according to claim 3, 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.

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

6. The preparation method according to claim 3, 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.

7. The preparation method according to claim 3, 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.

8. The preparation method according to claim 3, 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.

9. Use of the biohybrid nanorobot described in claim 1 or 2 or the biohybrid nanorobot prepared by the preparation method described in any one of claims 3 to 8 in the preparation of anti-tumor products.

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

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