A method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles

Abstract

The present invention relates to the field of immunology and biomedicine technology, and more particularly to a method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles. The preparation method specifically includes: first continuously subculturing Escherichia coli until growth is stable, then inducing the Escherichia coli cultured to the logarithmic phase after activation to produce bacterial outer membrane vesicles with IPTG; then the bacterial outer membrane vesicles are incubated with pentahydroxydopamine and ferrous sulfate in a one-pot method to obtain polydopamine iron-coated bacterial outer membrane vesicles (OMV-DF); finally, the polydopamine iron-coated bacterial outer membrane vesicles are incubated in the dark with HAuCl4, and then reduced to obtain nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles (OMV-DFA). The prepared nanoparticles have the advantages of both glucose oxidase and catalase activity, non-toxicity, and activation of autoimmunity.

Description

Technical Field

[0001] The present invention relates to the field of immunology and biomedicine technology, and in particular to a method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles. Background Art

[0002] Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. It has broad application prospects, but the tumor immunosuppressive microenvironment has become a key issue that hinders the effectiveness of tumor immunotherapy. Using nanoparticles to deliver immune drugs to tumor sites is a classic method to improve the tumor immunosuppressive microenvironment, but immunotherapy systems developed based on synthetic particles still face a series of challenges, such as complex preparation processes, low drug loading rates, and poor batch-to-batch reproducibility. In recent years, through continuous research, people have discovered that simple bacterial outer membrane vesicles (OMVs) naturally secreted by bacteria have certain potential in modulating the tumor immunosuppressive microenvironment. Bacterial outer membrane vesicles (OMVs) are vesicle-like nanoparticles secreted by the outer membrane of Gram-negative bacteria. The vesicles contain PAMP molecules (such as lipopolysaccharides and outer membrane proteins) of the Gram-negative bacteria's outer membrane and can effectively activate immune cells through Toll-like receptors (TLRs) of immune cells. Furthermore, OMVs, with diameters ranging from 20 to 200 nm, are the ideal size for nanomedicines, enabling effective accumulation at the tumor site through the enhanced permeability and retention (EPR) effect of solid tumors. However, direct intravenous injection of OMVs has strong toxic side effects, such as causing systemic cytokine storms and scurvy, which greatly limits their application.

[0003] Dopamine is the most abundant catecholamine neurotransmitter in the human brain. Studies have shown that it can adsorb onto any surface in a slightly alkaline environment (pH 7.5-8.0) and regulate various physiological functions of the central nervous system. It is widely used clinically to treat conditions such as myocardial infarction, renal failure, and endotoxin sepsis. Furthermore, its excellent biocompatibility allows it to be used as a coating or surface modifier for certain materials and drugs in the treatment of tumors and cardiovascular diseases.

[0004] Nanozymes are synthetic enzymes that mimic the activity of natural enzymes. They are prepared by modifying some highly active precious metals (such as Au and Pt), and their catalytic activity is inversely proportional to their particle size. Furthermore, compared to the protein components of natural enzymes, the reaction between nanozymes and substrates is less constrained by the environment. In recent years, gold nanoparticles have demonstrated excellent enzyme-mimicking activity, similar to peroxidases, oxidases, catalases, superoxide dismutases, or reductases. Moreover, compared to biological enzymes, gold nanozymes have advantages such as ease of synthesis, good tunability, good biocompatibility, and low cost. They are of great significance for competing for nutrients (glucose) in the tumor microenvironment, regulating tumor sugar metabolism, producing hydrogen peroxide, changing the redox environment of tumor tissue, and treating tumors. However, the delivery of nanozymes limits their biomedical applications. If bacterial outer membrane vesicles and nanozymes can be used to design nanoparticles that can also efficiently activate immune cells in the tumor microenvironment, it will be beneficial to the application of nanomedicines in medicine. Summary of the Invention

[0005] In order to solve the problems of toxic side effects of bacterial outer membrane vesicles and difficulty in delivering nanozymes, the present invention designs a method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles. The method mainly comprises reacting bacterial outer membrane vesicles with pentahydroxydopamine and ferrous sulfate in a one-pot method to obtain polydopamine iron-coated bacterial outer membrane vesicles, which are then incubated with HAuCl4 and reduced to obtain the obtained nanoparticles. The obtained nanoparticles can kill tumors through a combined immune-starvation-chemodynamic approach.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles, the preparation method specifically comprising the following steps:

[0008] S1, after activation, E. coli was cultured continuously for generations until it reached the logarithmic phase and then IPTG was used to induce the production of bacterial outer membrane vesicles;

[0009] S2, the bacterial outer membrane vesicles extracted in step S1 were mixed with dopamine, ferrous sulfate, PEG 2000 Polydopamine iron-coated bacterial outer membrane vesicles (OMV-DF) were prepared by a one-pot method.

[0010] S3. Incubate the bacterial outer membrane vesicles coated with polydopamine iron obtained in step S2 with HAuCl4 in the dark, and then reduce them to obtain nanoparticles OMV-DFA of nanogold@polydopamine iron coated bacterial outer membrane vesicles.

[0011] In some embodiments, step S1 specifically comprises: activating Escherichia coli with a culture medium and culturing to the logarithmic phase, then inoculating the culture medium into a new culture medium, and culturing at 100-300 r / min and 37° C. for 1-3 hours; then adding IPTG inducer for induction for 3-5 hours, collecting the bacterial liquid, and centrifuging at 6000-8000 r / min to collect the supernatant, filtering and concentrating the supernatant to remove bacterial debris, and finally extracting the bacterial outer membrane vesicles by ultrahigh-speed centrifugation at 130,000-150,000 g.

[0012] In some embodiments, the final concentration of the IPTG inducer is 10-30 μg / mL.

[0013] In some embodiments, the step S2 is specifically as follows: first, the bacterial outer membrane vesicles extracted in step S1 are resuspended in Tris-HCl-HEPES buffer to a final concentration of 1-2 mg / mL, and then dopamine, ferrous sulfate, PEG 2000 The mixture was reacted at 20-30 r / min for 1-2 hours, and then centrifuged at 14000-15000 g for 10-15 minutes to obtain the precipitate of bacterial outer membrane vesicles OMV-DF coated with polydopamine iron.

[0014] In some embodiments, the bacterial outer membrane vesicles are combined with dopamine, ferrous sulfate, PEG 2000 The mass ratio is 1~3:0.2~0.6:0.01~0.03:0.001~0.002.

[0015] In some embodiments, the bacterial outer membrane vesicles are combined with dopamine, ferrous sulfate, PEG 2000 The mass ratio is 2:0.5:0.01:0.001.

[0016] In some embodiments, step S3 is specifically as follows: resuspending the precipitated OMV-DF of step S2 in PBS buffer and ultrasonicating in an ice water bath for 5 to 15 minutes, then adding a HAuCl4 solution with a concentration of 130 to 160 μm / mL and incubating at 4°C in the dark for 1 to 3 hours, then adjusting the pH to 8 to 9 with an alkaline solution, and finally adding NaBH4 and rapidly stirring for 5 minutes to obtain nanoparticles OMV-DFA of bacterial outer membrane vesicles coated with nanogold@polydopamine iron.

[0017] The second aspect of the present invention provides nanoparticles of bacterial outer membrane vesicles coated with nanogold@polydopamine iron prepared by the preparation method described in the first aspect.

[0018] The third aspect of the present invention provides the use of the nanoparticles of the nanogold@polydopamine iron-coated bacterial outer membrane vesicles described in the second aspect in the preparation of anti-tumor drugs.

[0019] In some embodiments, the tumor is breast cancer.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention discloses nanoparticles of bacterial outer membrane vesicles coated with nanogold@polydopamine iron and a preparation method thereof. The invention comprises the following steps: firstly, Escherichia coli is activated and then continuously subcultured; after being cultured to the logarithmic phase, IPTG is used to induce the production of bacterial outer membrane vesicles; then, the bacterial outer membrane vesicles are reacted with pentahydroxydopamine and ferrous sulfate in a one-pot method to obtain bacterial outer membrane vesicles OMV-DF coated with polydopamine iron; finally, the bacterial outer membrane vesicles coated with polydopamine iron are incubated with HAuCl4 in the dark, and then reduced to obtain nanoparticles OMV-DFA coated with nanogold@polydopamine iron. The nanoparticle OMV-DFA prepared by the present invention has an EPR effect and accumulates at the tumor site. The dopamine shell can shield the toxicity of bacterial outer membrane vesicles, making the nanoparticle OMV-DFA non-toxic to normal tissues such as blood and organs; the nanoparticle OMV-DFA can also depolymerize the dopamine layer on the surface in the slightly acidic environment of the tumor. At this time, the OMVs exposed to the tumor microenvironment can efficiently activate immune cells, induce autoimmune reactions and kill tumors; at the same time, gold nanozymes can consume glucose in the tumor microenvironment to produce hydrogen peroxide, and produce hydroxyl free radicals through a Fenton-like reaction with iron ions to kill tumors, thereby achieving the purpose of combined immune-starvation-chemical dynamics to kill tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Transmission electron microscopy images of OMV (left) and OMV-DFA (right);

[0023] Figure 2 is the particle size distribution diagram of OMV and OMV-DFA;

[0024] Figure 3 Characterization of gold nanozyme activity of OMV-DFA, left: glucose consumption over time, right: gluconic acid production;

[0025] Figure 4 The iron ion release of OMV-DFA in different pH buffers;

[0026] Figure 5 is the level of free radicals generated by OMV-DFA in different pH buffers;

[0027] Figure 6 The toxicity of OMV-DFA to tumor cells at different pH values ​​(left: pH 7.4, right: pH 6.5). DETAILED DESCRIPTION

[0028] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0030] The sources of raw materials and instruments involved in the following examples or experimental examples are shown in the following table:

[0031] Raw materials or instruments Manufacturer Escherichia coli BL21 China Microorganism Culture Collection Center (CGMCC) Pentahydroxydopamine Sigma, United States <![CDATA[HAuCl4]]> Sigma, United States <![CDATA[NaBH4]]> Sinopharm Group, China Tris-HCl Solarbio, Beijing HEPES Solarbio, Beijing Phosphate buffered saline (PBS) Solarbio, Beijing Methylene blue Aladdin Reagents (Shanghai) Co., Ltd. Complete cell culture medium ThermoFisherScientific, USA LB (Luria-Bertani) medium Solarbio, Beijing IPTG inducer Solarbio, Beijing Glucose detection kit (micro method) Solarbio, Beijing BCA Reagent A Solarbio, Beijing BCA Reagent B Solarbio, Beijing ELISA kits ThermoFisherScientific, USA CCK-8 Cell Viability Detection Kit Solarbio, Beijing Live / Died Kit ThermoFisherScientific, USA Multifunctional microplate reader Tecan Infinite M200, USA Transmission electron microscope JEM-2100F JEOL Corporation of Japan

[0032] Example 1 Preparation Method of Nanoparticles of Nanogold@Polydopamine Iron Coated Bacterial Outer Membrane Vesicles

[0033] The preparation method specifically comprises the following steps:

[0034] 1. Extraction of bacterial outer membrane vesicles: Prepare LB liquid medium, sterilize the medium at 121 degrees Celsius for 20 minutes, then take out the BL21 strain from the -80 refrigerator, quickly dissolve it in 37 degrees Celsius water, and then inoculate it completely into the sterilized medium. After culturing for 12 hours, inoculate the activated strain into the newly prepared LB medium (1% inoculum volume) again. After culturing for 12 hours, take 50 mL and culture it to the logarithmic phase (OD 600 =0.5) were inoculated into 2L of freshly prepared LB medium and cultured at 200 rpm at 37 degrees Celsius for 2 hours. IPTG inducer (final concentration 20 μg / mL) was then added to induce the bacteria to produce bacterial outer membrane vesicles (OMVs). After further shaking for 4 hours, the bacterial liquid was collected and centrifuged at 8000 rpm to collect the supernatant. The supernatant was then filtered through a 0.45 μm filter, concentrated to remove bacterial debris, and then ultracentrifuged at 140,000 g for 3 hours. The light brown substance at the bottom of the centrifuge tube was OMVs. 2 mL of PBS was added to the centrifuge tube and washed three times. Then, 200 μL of PBS was added to the centrifuge tube and the OMVs were resuspended to obtain an OMV solution (protein concentration was 40 mg / mL as measured by BCA assay). The prepared OMV solution was used in subsequent experiments.

[0035] The bacterial outer membrane vesicles (OMV) extracted in step 1 were subjected to characterization tests. The specific operations included:

[0036] 1) BCA reagent A and BCA reagent B were thoroughly mixed at a volume ratio of 50:1 to prepare a BCA working solution; 10 μL of the resuspended OMVs solution from step 1 of Example 1 was taken and diluted to 100 μL with PBS buffer; bovine serum albumin (0.5 mg / mL) was added to the standard wells of a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μL, respectively, and then the BCA standard diluent was added to 20 μL. 10 μL of OMVs sample and 10 μL of standard diluent were added to the sample wells of the 96-well plate, 200 μL of BCA working solution were added to each well, and the plates were incubated at 37°C for 30 min. The absorbance was measured at 562 nm using a microplate reader to draw a standard curve.

[0037] 2) The bacterial outer membrane vesicles extracted from step 1 of Example 1 were placed in a tissue fixative (4% paraformaldehyde) for 30 minutes, and then 10 μL of the diluted (400 μg / mL) OMVs solution was dropped onto a carbon-supported copper mesh. After staining and drying, the OMVs were observed using a transmission electron microscope. The results are shown in FIG. Figure 1 As shown (left), bacterial outer membrane vesicles (OMVs) are uniformly granular.

[0038] 2. Preparation of OMV-DFA

[0039] (1) The light brown OMVs at the bottom of the centrifuge tube in step 1 were resuspended in Tris-HCl-HEPES buffer (prepared with 0.25 mM Tris-HCl and 25 mM HEPES at pH 8) to a final concentration of 2 mg / mL (protein concentration measured by BCA method). 500 μg / mL dopamine, 10 μg / mL ferrous sulfate, and 1 μg / mL PEG were then added in sequence. 2000 The mixture was placed in a suspension instrument and reacted at a speed of 20 r / min for 1 hour. After the reaction was completed, the mixture was centrifuged at 14000g for 10 minutes, and the precipitate was obtained to obtain polydopamine iron-coated bacterial outer membrane vesicles (OMV-DF).

[0040] (2) The OMV-DF collected by centrifugation was resuspended in PBS buffer (2 mL, 0.2 mg / mL) and ultrasonicated in an ice-water bath at an ultrasonic power of 80 W for 10 min. Then, 150 μm / mL of HAuCl4 solution was added and incubated at 4 degrees in the dark for 1 h. The pH of the solution was adjusted to 8.0 with NaOH, and pre-cooled NaBH4 (1 mL, 1 mM) was added. After rapid stirring for 5 min, OMV-DFA was obtained. The collected OMV-DFA was centrifuged at 14000 g for 10 min. After centrifugation, the precipitate was dissolved and then ultrasonicated in an ice-water bath for 10 min (power of 80 W) to obtain dispersed OMV-DFA (1.2 mg / mL).

[0041] The prepared nanogold@polydopamine iron coated bacterial outer membrane vesicle nanoparticles (OMV-DFA) were characterized and tested. The specific operations included:

[0042] 1) 1 μL of the nanoparticle OMV-DFA solution prepared in Example 1 was added to 10 mL of deionized water to obtain a 10,000-fold diluted OMV-DFA solution, and the particle size was measured using a particle size analyzer (NTA, Germany) (the results are shown in FIG. Figure 2 The average particle size of OMV-DFA is 100 nm.

[0043] 2) Take 1 μL of the nanoparticle OMV-DFA solution prepared in Example 1 and add it to 1 mL of deionized water to obtain a 1000-fold diluted OMV-DFA solution. Then take 10 μL of the diluted solution with an average particle size of 100 nm and drop it on a carbon support film copper grid. After staining and drying, the size and morphology of the solution were observed using a transmission electron microscope. The results are shown in FIG. Figure 1 As shown (right), the surface of OMV-DFA is coated with dopamine iron and distributed with gold nanoparticles.

[0044] Example 2 Preparation Method of Nanoparticles of Nanogold@Polydopamine Iron Coated Bacterial Outer Membrane Vesicles

[0045] The preparation method specifically comprises the following steps:

[0046] 1. The method for extracting bacterial outer membrane vesicles is the same as that in Example 1, and the obtained bacterial outer membrane vesicles OMVs are also uniform in particles;

[0047] 2. Preparation of OMV-DFA:

[0048] (1) The light brown OMVs at the bottom of the centrifuge tube in step 1 were resuspended in Tris-HCl-HEPES buffer (prepared with 0.25 mM Tris-HCl and 25 mM HEPES at pH 8) to a final concentration of 1 mg / mL (protein concentration measured by the BCA method). 600 μg / mL dopamine, 10 μg / mL ferrous sulfate, and 1 μg / mL PEG were then added in sequence. 2000 The mixture was placed in a suspension instrument and reacted at a speed of 20 r / min for 1 hour. After the reaction was completed, the mixture was centrifuged at 14000g for 10 minutes, and the precipitate was obtained to obtain polydopamine iron-coated bacterial outer membrane vesicles (OMV-DF).

[0049] (2) The OMV-DF collected by centrifugation was resuspended in PBS buffer and ultrasonicated in an ice-water bath at an ultrasonic power of 80W for 10 minutes. Then, 160μm / mL HAuCl4 solution was added and incubated at 4 degrees in the dark for 3 hours. The pH of the solution was adjusted to 8.0 with NaOH, and pre-cooled NaBH4 was added. After rapid stirring for 5 minutes, OMV-DFA was obtained. The collected OMV-DFA was centrifuged at 14000g for 10 minutes. After centrifugation, the precipitate was dissolved and then placed in an ice-water bath for 10 minutes (power of 80W) to obtain dispersed OMV-DFA (1.2 mg / mL). Similarly, the surface of OMV-DFA was coated with dopamine iron and distributed with gold nanoparticles.

[0050] Example 3 Preparation Method of Nanoparticles of Nanogold@Polydopamine Iron Coated Bacterial Outer Membrane Vesicles

[0051] The preparation method specifically comprises the following steps:

[0052] 1. The method for extracting bacterial outer membrane vesicles is the same as that in Example 1, and the obtained bacterial outer membrane vesicles OMVs are also uniform in particles;

[0053] 2. Preparation of OMV-DFA:

[0054] (1) The light brown OMVs at the bottom of the centrifuge tube in step 1 were resuspended in Tris-HCl-HEPES buffer (prepared with 0.25 mM Tris-HCl and 25 mM HEPES at pH 8) to a final concentration of 2 mg / mL (protein concentration measured by the BCA method). 200 μg / mL dopamine, 10 μg / mL ferrous sulfate, and 1 μg / mL PEG were then added in sequence. 2000 The mixture was placed in a suspension instrument and reacted at a speed of 30 r / min for 1 hour. After the reaction was completed, the mixture was centrifuged at 14000 g for 10 minutes, and the precipitate was obtained to obtain polydopamine iron-coated bacterial outer membrane vesicles (OMV-DF).

[0055] (2) The OMV-DF collected by centrifugation was resuspended in PBS buffer and ultrasonicated in an ice-water bath at an ultrasonic power of 80W for 10 minutes. Then, 130μm / mL HAuCl4 solution was added and incubated at 4 degrees in the dark for 1 hour. The pH of the solution was adjusted to 9.0 with NaOH, and pre-cooled NaBH4 was added. After rapid stirring for 5 minutes, OMV-DFA was obtained. The collected OMV-DFA was centrifuged at 14000g for 10 minutes. After centrifugation, the precipitate was dissolved and then placed in an ice-water bath for 10 minutes (power of 80W) to obtain dispersed OMV-DFA (1.2mg / mL). Similarly, the surface of OMV-DFA was coated with dopamine iron and distributed with gold nanoparticles.

[0056] Experimental Example 1 Performance Verification of Nanoparticles of Nanogold@Polydopamine Iron Coated Bacterial Outer Membrane Vesicles

[0057] Taking the nanoparticles of gold@polydopamine iron coated bacterial outer membrane vesicles (OMV-DFA) prepared in Example 1 as an example, the following performance verification was performed:

[0058] (1) Characterization of the nanozyme performance of OMV-DFA nanoparticles: 20 μL of OMV-DFA was added to 2 mL of freshly prepared glucose solution (10 mmol / mL). The glucose and gluconic acid in the supernatant of the solution were detected at different time points (0, 2, 4, 6, 8, 15, 30, and 60 min) using a glucose detection kit. Figure 3 As shown, the glucose in the solution is gradually consumed over time, and gluconic acid is gradually produced.

[0059] (2) Characterize the iron ion release level of OMV-DFA nanoparticles: Take 0.5 mL of OMV-DFA and add it into 5 mL of PBS buffer with pH 6.5. Take the supernatant after 24 and 48 hours respectively, and then measure the iron ion content in the solution by o-phenanthroline method (the maximum absorption wavelength of o-phenanthroline and divalent iron complex is 510 nm). Figure 4 As shown, OMV-DFA nanoparticles can release iron ions in a buffer solution with a pH of 6.5.

[0060] (3) Determination of oxygen free radical content: 20 μL of OMV-DFA nanoparticles were resuspended in 2 mL of 400 μmol / mL H2O2 buffer at pH 6.5, and then 50 μL of 24 mmol / L 3,3',5,5'-tetramethylbenzidine (TMB) was added to produce a color reaction. The oxygen free radical content at 652 nm was measured using a multifunctional microplate reader at 0, 2, 4, 6, 8, 10, 15, and 30 min of reaction, as shown in Figure 2. Figure 5 As shown, OMV-DFA nanoparticles can catalyze H2O2 to produce hydroxyl radicals in a buffer solution with a pH of 6.5.

[0061] (4) The prepared OMV-DFA can kill tumor cells: the mouse triple-negative breast cancer (4T1) cell line was cultured in a constant temperature incubator at 37°C and 5% CO2. When the cells grew to 80% of the culture dish, the cells were seeded in a 96-well plate and cultured with complete cell culture medium for 24 hours. The culture medium was replaced (replaced with pH 7.4 and 6.5 culture medium respectively, wherein the slightly acidic complete culture medium of pH 6.5 was prepared in advance with concentrated hydrochloric acid). Then the drug was added, and the dosage was 0, 10, 20, 40, 80, 120, 160, 200 μg / mL, respectively. After the cells were cultured for another 24 hours, the supernatant was discarded, and the cells were washed three times with PBS. The activity was tested with a CCK-8 cell activity detection kit (incubated in a constant temperature incubator at 37°C and 5% CO2 for 2 hours in the dark). The absorbance at 450 nm was measured with an enzyme marker. The results are as follows: Figure 6 As shown (right), the killing effect of OMV-DFA nanoparticles on tumor cells can reach up to 70% under the condition of pH 6.5; while the cytotoxicity under normal pH conditions is negligible ( Figure 6 Left).

[0062] In addition, the OMV-DFA of Examples 2 and 3 has the same or similar properties as Example 1, namely, it can produce gluconic acid, release iron ions, catalyze H2O2 to produce hydroxyl radicals, and has a killing effect on tumor cells.

[0063] Based on the above performance tests, it can be seen that the OMV-DFA nanoparticles prepared by the method of the present invention can kill tumors through the combined immune-starvation-chemodynamics, and have important potential application value.

[0064] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles, characterized in that: The preparation method specifically comprises the following steps: S1. Activating Escherichia coli and continuously subculturing the cells until the logarithmic phase, and then inducing the cells to produce bacterial outer membrane vesicles with IPTG, wherein the final concentration of the IPTG inducer is 10-30 μg / mL; S2, the bacterial outer membrane vesicles extracted in step S1 were mixed with dopamine, ferrous sulfate, PEG 2000 Polydopamine iron-coated bacterial outer membrane vesicles (OMV-DF) were prepared by a one-pot method, wherein the mass ratio of the bacterial outer membrane vesicles to dopamine, ferrous sulfate, and PEG2000 was 1-3:0.2-0.6:0.01-0.03:0.001-0.002; S3. The bacterial outer membrane vesicles coated with polydopamine iron obtained in step S2 were incubated with HAuCl4 in the dark, and then reduced to obtain nanoparticles OMV-DFA of nanogold@polydopamine iron-coated bacterial outer membrane vesicles: the precipitated OMV-DF of step S2 was resuspended in PBS buffer and sonicated in an ice water bath for 5 to 15 min, and then a HAuCl4 solution with a concentration of 130 to 160 μm / mL was added and incubated at 4°C in the dark for 1 to 3 h, and then the pH was adjusted to 8 to 9 with an alkaline solution, and finally NaBH4 was added and stirred rapidly for 5 min to obtain nanoparticles OMV-DFA of nanogold@polydopamine iron-coated bacterial outer membrane vesicles.

2. The method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles according to claim 1, characterized in that: The step S1 specifically comprises: activating Escherichia coli with a culture medium and culturing to the logarithmic phase, then inoculating the culture medium into a new culture medium, and culturing at 100-300 r / min and 37° C. for 1-3 hours; then adding an IPTG inducer for induction for 3-5 hours, collecting the bacterial liquid, and centrifuging at 6000-8000 r / min to collect the supernatant, filtering and concentrating the supernatant to remove bacterial debris, and finally extracting the bacterial outer membrane vesicles by ultrahigh-speed centrifugation at 130,000-150,000 g.

3. The method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles according to claim 1, characterized in that: The step S2 is specifically as follows: first, the bacterial outer membrane vesicles extracted in step S1 are resuspended in Tris-HCl-HEPES buffer to a final concentration of 1-2 mg / mL, and then dopamine, ferrous sulfate, PEG 2000 The mixture was reacted at 20-30 r / min for 1-2 h, and then centrifuged at 14,000-15,000 g for 10-15 min to obtain the precipitate of bacterial outer membrane vesicles (OMV-DF) coated with polydopamine iron.

4. The method for preparing nanoparticles of nanogold@polydopamine iron-coated bacterial outer membrane vesicles according to claim 1, characterized in that: The bacterial outer membrane vesicles are combined with dopamine, ferrous sulfate, PEG 2000 The mass ratio is 2:0.5:0.01:0.

001.

5. Nanoparticles of bacterial outer membrane vesicles coated with nanogold@polydopamine iron prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the nanoparticles of the nanogold@polydopamine iron-coated bacterial outer membrane vesicles according to claim 5 in the preparation of anti-breast cancer drugs.

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