An anti-tumor Escherichia coli preparation and its preparation method and application

Abstract

The present invention relates to an anti-tumor Escherichia coli preparation, a preparation method and application thereof, and belongs to the technical field of drug preparation. A chitosan oligosaccharide nanoparticle emulsion and dopamine-encapsulated Escherichia coli are prepared separately, and then Poloxamer 188 is added to the dopamine-encapsulated Escherichia coli, and then mixed with the chitosan oligosaccharide nanoparticle emulsion, CTAB is added to form a mixed solution, vortexed for 2 to 3 minutes, and then centrifuged and washed to obtain an Escherichia coli preparation. The present invention modifies the surface of Escherichia coli with PDA, which is enriched on the tumor surface, completing the simulated camouflage of tumor cells; at the same time, in the tumor microenvironment, COS nanoparticles are responsively controlled to release, in situ activate the immune activity of macrophages and promote M1 polarization; through photothermal induction of the ICD effect of tumor cells, the stimulating effect of the immune adjuvant is further created, the overall immune biological effect is expanded, and finally the immune-photothermal synergistic enhancement is achieved to ablate breast cancer tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to an anti-tumor Escherichia coli preparation and a preparation method and application thereof. Background Art

[0002] Breast cancer (BC) is one of the most common malignancies worldwide, with the highest incidence among women. Traditional treatment options primarily include surgical resection, radiotherapy, and chemotherapy, all of which offer dismal clinical outcomes. Triple-negative breast cancer (TNBC) is the most aggressive type of BC. Immunohistochemical results for estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene HER-2 are negative, making it poorly responsive to conventional treatments and associated with a high risk of invasion and metastasis. Currently, treatment options for TNBC are limited, with chemotherapy remaining the primary adjuvant therapy. However, responses to chemotherapy vary among patients, and chemoresistance can easily develop, leading to treatment failure. Therefore, developing safe and effective treatments and strategies is a critical and pressing challenge in the clinical treatment of TNBC.

[0003] Conventional anti-cancer drugs are unable to reach the hypoxic zones of tumor tissue, contributing to poor prognosis, high recurrence, and metastasis. Bacterial-based tumor immunotherapy has attracted considerable attention due to its unique mechanism and diverse applications in triggering host anti-tumor immunity. Inadequate blood supply to the center of solid tumors causes extensive tumor cell necrosis, creating a hypoxic microenvironment. Bacteria, for example, can target tumors and preferentially colonize the core of the tumor. Photosynthetic bacteria, such as Salmonella and Escherichia coli, can actively migrate to hypoxic regions of the tumor after intravenous injection. Therefore, integrating nanomedicines with bacteria to create engineered bacteria can significantly increase drug concentrations in hypoxic tumor zones, thereby inhibiting tumor growth. Furthermore, because bacteria are rich in pathogen-associated molecular patterns, they can effectively activate immune cells even within the immunosuppressive tumor microenvironment, enhancing specific immune recognition and elimination of tumor cells. Bacterial-mediated tumor-targeted immunotherapy is expected to become an important approach for the treatment of TNBC. Summary of the Invention

[0004] The present invention aims to provide an Escherichia coli preparation, a preparation method thereof and application thereof in the preparation of anti-breast cancer tumor drugs.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing an Escherichia coli preparation is characterized by: separately preparing a chitosan oligosaccharide nanoparticle emulsion and dopamine-entrapped Escherichia coli, and then functionalizing the dopamine-entrapped Escherichia coli with the chitosan oligosaccharide nanoparticles. The functionalization comprises adding poloxamer 188 to the dopamine-entrapped Escherichia coli suspension, then mixing the mixture with the chitosan oligosaccharide nanoparticle emulsion, adding cetyltrimethylammonium bromide (CTAB) to form a mixed solution, vortexing for 2-3 minutes, and then centrifuging and washing to obtain the Escherichia coli preparation.

[0007] Furthermore, the volume ratio of the dopamine-encapsulated Escherichia coli used in the functionalization to the chitosan oligosaccharide nanoparticle emulsion is 1:1.

[0008] Furthermore, the amount of Poloxamer 188 added to the mixed solution is 0.05-0.08% w / v, and the amount of CTAB added is 0.02-0.05 mmol / L.

[0009] Furthermore, the pH of the mixed solution is adjusted to 7.2-7.6, preferably 7.5.

[0010] Furthermore, the preparation of chitosan oligosaccharide nanoparticle emulsion is to take chitosan oligosaccharide (COS) and add distilled water to prepare COS solution, then take polyacrylic acid (PAA) and add distilled water to prepare PAA solution, dropwise add the PAA solution to the COS solution, and ultrasonically treat to obtain COS nanoparticle emulsion.

[0011] Furthermore, the concentration of the COS solution is 1.5-2.5 mg / mL, the concentration of the PAA solution is 0.8-1.2 mg / mL, and the volume ratio of the COS solution to the PAA solution is 4-6:1.

[0012] Furthermore, the ultrasonic temperature is room temperature, the ultrasonic power is 35-45 kHz, and the ultrasonic time is 3-5 min.

[0013] During the composite process, due to the difference in surface charge matching between dopamine-loaded Escherichia coli and COS nanoparticles, it is difficult for COS nanoparticles to achieve uniform attachment to the surface of dopamine-loaded Escherichia coli, and they are prone to fall off after attachment, resulting in unsatisfactory final effect.

[0014] Due to the hydrogen bond and covalent bond between E. coli and PDA to enhance adhesion stability and hydrophobic effect, PDA with high cell adhesion is evenly coated on the surface of E. coli to form PDA In the process of preparing COS nanoparticles, firstly, the present invention uses PAA as a carrier of COS, adjusts the concentration and dosage of PAA and COS, and regulates the ultrasonic effect so that the COS nanoparticles finally prepared are positively charged nanoparticles; secondly, in the composite process,PDA Poloxamer 188 was added to the EcN resuspension, mixed with the COS nanoparticle emulsion, and then CTAB was added. After mixing, the pH of the reaction system was adjusted to make the COS nanoparticles evenly attached to the PDA surface. PDA The EcN / COS surface is still electronegative.

[0015] During the compounding process, under a specific pH environment, Poloxamer 188 is adsorbed on the surface of PDA particles through hydrophobic interaction, forming a hydration layer on the particle surface, reducing the aggregation tendency, lowering the interfacial tension of the aqueous phase, and promoting the collision probability between EcN / PDA and COS nanoparticles. PDA An interfacial bridging effect is generated between EcN and COS nanoparticles. Under this condition, CTAB promotes the PDA The electrostatic attraction between EcN (negatively charged) and COS nanoparticles (positively charged) synergistically enhances adsorption between the two, improving composite efficiency and ensuring more uniform COS nanoparticle adhesion on the PDA surface. Furthermore, the hydration layer reduces the shear forces generated by vortexing that can damage the EcN / PDA complex. Furthermore, the hydration layer on the PDA surface effectively isolates CTAB, inhibiting its insertion into PDA and damaging the EcN, thereby ensuring the activity of the EcN in the final formulation.

[0016] Poloxamer 188 plays a major role in interfacial tension regulation and interfacial bridging in nanocomposites through its unique amphiphilic structure, complementing the cationic surfactant CTAB and effectively improving the performance of nanocomposites through balancing charge and steric stabilization. PDA Composite efficiency of EcN and COS nanoparticles.

[0017] Furthermore, the centrifugation is performed at 4000-5000 rpm for 7-10 min.

[0018] Furthermore, the preparation of dopamine-encapsulated Escherichia coli is to take dopamine hydrochloride and add it to Tris-HCl buffer to form PDA Tris-HCl buffer, resuspend the Escherichia coli, and then centrifuge to obtain dopamine-encapsulated Escherichia coli. PDA EcN.

[0019] Furthermore, the PDA Tris-HCl buffer concentration is 550-650 μL / mL, the Tris-HCl buffer concentration is 8-12 mmol / L, and the pH is 8.5.

[0020] Further, the resuspending step is to add 8-10 mL OD 600The E. coli bacterial suspension with a pH of 0.8 was centrifuged and resuspended in PDATris-HCl buffer to 10 mL.

[0021] Further, the centrifugation is carried out at 4000-5000 rpm for 7-10 min to obtain PDA EcN, resuspend again and refrigerate at 4℃ for later use.

[0022] Most specifically, a method for preparing an Escherichia coli preparation is characterized by comprising the following steps:

[0023] (I) Preparation of dopamine-loaded Escherichia coli

[0024] (1) Take a single EcN colony on solid LB medium and culture it in liquid LB medium for 8 h. Collect the bacteria, centrifuge at 3000 rpm for 5 min, and dilute with physiological saline to OD 600 The value is 0.8, and it is refrigerated at 4℃ for later use;

[0025] (2) Add 4-6 mg of dopamine hydrochloride to 10 mL of Tris-HCl buffer (10 mM, pH 8.5) to prepare a PDA Tris-HCl buffer solution with a concentration of 400-600 μL / mL;

[0026] (3) Take 8~10 mL of the bacterial solution in step (1) and centrifuge it. Resuspend it to 10 mL with the PDA Tris-HCl buffer prepared in step (2). Stir vigorously at room temperature for 2 h. Centrifuge at 4000~5000 rpm for 7~10 min to obtain PDA EcN, resuspend to 10 mL and refrigerate at 4 °C for later use;

[0027] (2) Preparation of COS nanoparticles

[0028] (1) Add chitosan oligosaccharide (COS) to distilled water to prepare a 1.5-2.5 mg / mL COS solution for later use. Weigh polyacrylic acid solution (PAA) to prepare a 0.8-1.2 mg / mL PAA solution for later use.

[0029] (2) Use a pipette to transfer 4-6 mL of COS solution into a small beaker. Under ultrasound, use a sterile syringe to dropwise add 1 mL of PAA solution. The transparent light yellow solution becomes milky and turbid, thus obtaining a COS nanoparticle emulsion.

[0030] (3) Functionalized Escherichia coli

[0031] Take 1 mL PDAThe EcN resuspension was added with Poloxamer 188, and then mixed with 1 mL of COS nanoparticle emulsion, and then CTAB was added, and the pH was adjusted to 7.2-7.6 to obtain a mixed solution, vortexed for 2 min, centrifuged at 4000-5000 rpm for 7-10 min, washed once with physiological saline, and resuspended to 1 mL to obtain PDA EcN / COS, the concentration of Poloxamer 188 in the mixed solution is 0.05-0.08% w / v, and the concentration of CTAB is 0.02-0.05 mmol / L.

[0032] The present invention completes the simulated camouflage of breast cancer tumor cells by modifying the surface of Escherichia coli with PDA with high tissue cell adhesion and then enriching it on the tumor surface; at the same time, in the tumor microenvironment, through the charge reversal of PDA, COS nanoparticles are responsively controlled to release, in situ activate the immune activity of macrophages and promote the polarization of M2 to M1; the activated macrophages recognize and phagocytize tumor cells that simulate the disguise of bacteria, and at the same time, induce the ICD effect of tumor cells through the photothermal effect, further creating an immune stimulating microenvironment, promoting the recruitment of macrophages to the tumor microenvironment, expanding the overall immune biological effect, and ultimately achieving the ablation of tumor cells through the immune-photothermal synergistic enhancement.

[0033] The Escherichia coli preparation prepared by the method and its application in preparing anti-breast cancer tumor drugs.

[0034] Furthermore, the E. coli preparation is used in the preparation of anti-triple-negative breast cancer tumor drugs.

[0035] The present invention has the following technical effects:

[0036] The present invention utilizes the excellent carrying and targeted colonization ability of anaerobic extracellular bacteria, modifies the surface of Escherichia coli with PDA with high tissue cell adhesion, and then compounds it with COS to form PDA EcN / COS is enriched on the tumor surface, completing the simulated camouflage of tumor cells; at the same time, in the tumor microenvironment, COS nanoparticles are responsively controlled to release, in situ activating the immune activity of macrophages and promoting M1 polarization; through photothermal induction of the ICD effect of tumor cells, the stimulating effect of the immune adjuvant is further created, the overall immune biological effect is expanded, and ultimately the immune-photothermal synergy is achieved to ablate breast cancer tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Dopamine encapsulates Escherichia coli PDA Transmission electron micrograph of EcN.

[0038] Figure 2: Transmission electron microscopy images of chitosan oligosaccharide nanoparticles COS NPs.

[0039] Figure 3 :Functionalized Escherichia coli PDA Transmission electron micrograph of EcN / COS.

[0040] Figure 4 : In the present invention, PDA, EcN and PDA Infrared spectrum of EcN.

[0041] Figure 5 :Functionalized Escherichia coli PDA In vitro photothermal images of EcN / COS.

[0042] Figure 6 :Functionalized Escherichia coli PDA Figure 3. In vitro antitumor activity of EcN / COS.

[0043] Figure 7 :Functionalized Escherichia coli PDA Live-dead staining images of tumor cells after EcN / COS treatment.

[0044] Figure 8 :Functionalized Escherichia coli PDA EcN / COS tumor sphere penetration diagram.

[0045] Figure 9 : Diagram of macrophage activation by PBS, COS and EcN in vitro.

[0046] Figure 10 : Diagram of macrophage activation by PBS, COS and EcN in vitro.

[0047] Figure 11 : PDA EcN, PDA EcN / COS and PDA Diagram of EcN / COS+nir macrophage activation in vitro.

[0048] Figure 12 : PDA EcN, PDA EcN / COS and PDA Diagram of EcN / COS+nir macrophage activation in vitro.

[0049] Figure 13 :Functionalized Escherichia coli PDA In vivo photothermal images of EcN / COS.

[0050] Figure 14 :Functionalized Escherichia coli PDA Diagram of the tumor inhibition effect of EcN / COS in vivo.

[0051] Figure 15 :Poloxamer 188 and CTAB for PDA Effect of EcN / COS on tumor inhibition in vitro.

[0052] Figure 16 : Comparative Example 1-(3) prepared PDA Transmission electron micrograph of EcN / COS. DETAILED DESCRIPTION

[0053] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention based on the contents of the present invention.

[0054] The tumor cells used for testing in the present invention are specifically 4T1 cells of the triple-negative breast cancer cell line.

[0055] Example 1

[0056] A method for preparing an Escherichia coli preparation comprises the following steps:

[0057] (I) Preparation of dopamine-loaded Escherichia coli

[0058] (1) Take a single EcN colony on solid LB medium and culture it in liquid LB medium for 8 h. Collect the bacteria, centrifuge at 3000 rpm for 5 min, and dilute with physiological saline to OD 600 The value is 0.8, and it is refrigerated at 4℃ for later use;

[0059] (2) Add 5 mg of dopamine hydrochloride to 10 mL of Tris-HCl buffer (10 mM, pH 8.5) to prepare a PDA Tris-HCl buffer solution with a concentration of 500 μL / mL;

[0060] (3) Centrifuge 9 mL of the bacterial solution from step (1) and resuspend it to 10 mL with the PDA Tris-HCl buffer prepared in step (2). Stir vigorously at room temperature for 2 h and centrifuge at 4500 rpm for 8 min to obtain PDA EcN, resuspend to 10 mL and store at 4°C until use;

[0061] (II) Preparation of COS nanoparticles

[0062] (1) Chitosan oligosaccharide (COS) was added to distilled water to prepare a 2.5 mg / mL COS solution for later use. Polyacrylic acid solution (PAA) was weighed and prepared to prepare a 1.2 mg / mL PAA solution for later use.

[0063] (2) Use a pipette to transfer 6 mL of COS solution into a small beaker. Under ultrasound, add 1 mL of PAA solution dropwise using a sterile syringe. The transparent light yellow solution becomes milky and turbid, thus obtaining a COS nanoparticle emulsion.

[0064] (3) Functionalized Escherichia coli

[0065] Take 1 mL of the prepared PDA The EcN resuspension was added with Poloxamer 188, and then mixed with 1 mL of COS nanoparticle emulsion, and then CTAB was added, and the pH was adjusted to 7.2 to obtain a mixed solution, vortexed for 2 minutes, centrifuged at 40500 rpm for 8 minutes, washed once with physiological saline, and resuspended to 1 mL to obtain PDA EcN / COS preparation, the concentration of Poloxamer 188 in the mixed solution is 0.05% w / v, and the concentration of CTAB is 0.02 mmol / L.

[0066] Example 2

[0067] A method for preparing an Escherichia coli preparation, characterized by comprising the following steps:

[0068] (I) Preparation of dopamine-loaded Escherichia coli

[0069] (1) Take a single EcN colony on solid LB medium and culture it in liquid LB medium for 8 h. Collect the bacteria, centrifuge at 3000 rpm for 5 min, and dilute with physiological saline to OD 600 The value is 0.8, and it is refrigerated at 4℃ for later use;

[0070] (2) Add 4 mg of dopamine hydrochloride to 10 mL of Tris-HCl buffer (10 mM, pH 8.5) to prepare a PDA Tris-HCl buffer solution with a concentration of 400 μL / mL;

[0071] (3) Take 8 mL of the bacterial solution from step (1) and centrifuge it. Resuspend it to 10 mL with the PDA Tris-HCl buffer prepared in step (2). Stir vigorously at room temperature for 2 h. Centrifuge at 4000 rpm for 10 min to obtain PDA EcN, resuspend to 10 mL and refrigerate at 4 °C for later use;

[0072] (II) Preparation of COS nanoparticles

[0073] (1) Take chitosan oligosaccharide (COS), add distilled water, and prepare a 1.5 mg / mL COS solution for later use. Weigh polyacrylic acid solution (PAA) and prepare a 0.8 mg / mL PAA solution for later use.

[0074] (2) Use a pipette to transfer 4 mL of COS solution into a small beaker. Under ultrasound, use a sterile syringe to add 1 mL of PAA solution. The transparent light yellow solution becomes milky and turbid, thus obtaining a COS nanoparticle emulsion.

[0075] (3) Functionalized Escherichia coli

[0076] Take 1 mL of the prepared PDA The EcN resuspension was added with Poloxamer 188, and then mixed with 1 mL of COS nanoparticle emulsion, and then CTAB was added, and the pH was adjusted to 7.6 to obtain a mixed solution, vortexed for 2 minutes, centrifuged at 4000 rpm for 10 minutes, washed once with physiological saline, and resuspended to 1 mL to obtain PDA EcN / COS preparation, the concentration of Poloxamer 188 in the mixed solution is 0.08% w / v, and the concentration of CTAB is 0.05 mmol / L.

[0077] Example 3

[0078] A method for preparing an Escherichia coli preparation, characterized by comprising the following steps:

[0079] (I) Preparation of dopamine-loaded Escherichia coli

[0080] (1) Take a single EcN colony on solid LB medium and culture it in liquid LB medium for 8 h. Collect the bacteria, centrifuge at 3000 rpm for 5 min, and dilute with physiological saline to OD 600 The value is 0.8, and it is refrigerated at 4℃ for later use;

[0081] (2) Add 6 mg of dopamine hydrochloride to 10 mL of Tris-HCl buffer (10 mM, pH 8.5) to prepare a PDA Tris-HCl buffer solution with a concentration of 600 μL / mL;

[0082] (3) Take 10 mL of the bacteria from step (1) and centrifuge, resuspend to 10 mL with the PDA Tris-HCl buffer prepared in step (2), vigorously stir at room temperature for 2 h, and centrifuge at 5000 rpm for 10 min to obtain PDA EcN, resuspend to 10 mL and refrigerate at 4 °C for later use;

[0083] Dopamine-loaded Escherichia coli prepared in this example PDA Transmission electron microscopy images of EcN are shown in Figure 2. Figure 1 As shown, it can be seen that the PDA nanoparticles are evenly attached to the surface of E. coli, forming a uniform wrapping of E. coli;

[0084] (II) Preparation of COS nanoparticles

[0085] (1) Take chitosan oligosaccharide (COS), add distilled water, and prepare a 2 mg / mL COS solution for later use. Weigh polyacrylic acid solution (PAA) and prepare a 1 mg / mL PAA solution for later use.

[0086] (2) Use a pipette to transfer 5 mL of COS solution into a small beaker. Under ultrasound, use a sterile syringe to dropwise add 1 mL of PAA solution. The transparent light yellow solution becomes milky and turbid, thus obtaining a COS nanoparticle emulsion.

[0087] Transmission electron microscopy images of the prepared COS nanoparticles are shown in Figure 2. Figure 2 As shown, it can be seen that the nanoparticles have uniform particle size and excellent dispersion;

[0088] (3) Functionalized Escherichia coli

[0089] Take 1 mL of the prepared PDA The EcN resuspension was added with Poloxamer 188 and then mixed with 1 mL of COS nanoparticle emulsion. CTAB was then added and the pH was adjusted to 7.5 to obtain a mixture, which was vortexed for 2 min and centrifuged at 5000 rpm for 7 min. The mixture was washed once with physiological saline and resuspended in 1 mL to obtain PDA EcN / COS, the concentration of Poloxamer 188 in the mixture is 0.06% w / v, and the concentration of CTAB is 0.03 mmol / L;

[0090] Figure 3 Functionalized Escherichia coli PDA The transmission electron microscopy image of EcN / COS shows that COS nanoparticles aggregated on the surface of dopamine-loaded Escherichia coli, and the morphology of COS nanoparticles also changed from the original spherical shape to a square structure.

[0091] The electrophoretic velocity (μ) of the particles in the electric field is measured by a Zeta potential meter and calculated using the formula:

[0092]

[0093] Where η is the viscosity of the medium and ε is the dielectric constant.

[0094] The Zeta potential test shows that PDA The ζ potential of EcN is -35mV, and the ζ potential of COS nanoparticles is +20mV. PDAThe zeta potential of EcN / COS is -12mV. In the process of preparing COS nanoparticles, if the amount of PAA is further increased, the prepared COS nanoparticles will be negatively charged, resulting in a significant increase in the difficulty of compounding. PDA The ablation effect of EcN / COS on tumor cells was significantly reduced. PDA The activity of EcN in EcN / COS was 92.6% of that before functionalization.

[0095] Figure 4 Functionalized Escherichia coli PDA The infrared spectrum of EcN / COS shows that dopamine has a peak at 3028.658 cm -1 and 1282.913 cm -1 The characteristic peak of E. coli appears at 1499.865 cm -1 , dopamine-encapsulated E. coli exhibited both characteristic peaks. Therefore, dopamine was successfully encapsulated into E. coli.

[0096] Figure 5 Functionalized Escherichia coli PDA In vitro photothermal images of EcN / COS. From left to right, the images are taken every minute after the preparation was irradiated with laser for 5 minutes. It can be seen that the temperature of the preparation can rise to about 45°C within 5 minutes after laser irradiation.

[0097] Figure 6 Functionalized Escherichia coli PDA In vitro anti-tumor activity of EcN / COS. The PBS group was the control group, and the calculation formula was: Cell viability (%) = (A 制剂 – A 空白 ) / (A 对照 – A 空白 ) × 100%;

[0098] A 空白 The absorbance corresponding to the wells without cells, A 制剂 Groups are grouped by each preparation, A 对照 Corresponding to the control group.

[0099] from Figure 6 It can be seen that the preparation group killed more than 50% of tumor cells in vitro.

[0100] Figure 7 Calcein-AM / PI live cell / dead cell staining kit was used. Red represents dead cells and green represents live cells. Figure 6 and Figure 7 It can be found that the preparation has a strong killing effect on tumor cells in vitro.

[0101] Figure 8 Functionalized Escherichia coli PDA EcN / COS tumor sphere penetration image. It can be seen that bacteria containing green fluorescent protein successfully entered the interior of the tumor sphere containing red fluorescent protein, fully demonstrating the ability of the agent to penetrate tumor tissue.

[0102] Figures 9-12 It is PBS, COS, EcN, PDA EcN, PDA EcN / COS and PDA EcN / COS+nir in vitro macrophage activation diagram. CD80 is a specific antibody for the M1 type, CD206 is a characteristic antibody for the M2 type, and F4 / 80 is an antibody shared by all macrophages. Therefore, the second quadrant of the figure expresses M1 and M2 macrophages. It can be seen that compared with the PBS group, the M2 type of macrophages in the preparation group and the preparation plus light group was significantly reduced, and the M1 type was significantly increased.

[0103] Figure 13 Functionalized Escherichia coli PDA In vivo photothermal images of EcN / COS. Using an infrared thermal imager to capture images of laser-irradiated mouse tumor sites every minute, it can be seen that the temperature at the laser-irradiated site rises to approximately 45°C within 5 minutes.

[0104] Figure 14 Functionalized Escherichia coli PDA The in vivo tumor inhibition effect of EcN / COS is shown in the figure. The figure shows the dissected tumors of mice in different dosing groups, with six replicates in each group. It can be seen that the tumors in the mice in the final formulation group were significantly suppressed compared to the PBS group.

[0105] Comparative Example 1

[0106] Compared with Example 3, in the functional compounding process of step (iii), the compounding conditions were adjusted as follows:

[0107] (1) Poloxamer 188 and CTAB were not added during the functionalization process (Comparative Example 1-(1));

[0108] (2) Only Poloxamer 188 was added during the functionalization process (Comparative Examples 1-(2));

[0109] (3) Only CTAB was added during the functionalization process (Comparative Example 1-(3)).

[0110] By testing the Zeta potential, we can know that PDA The ζ potential of EcN is -35mV, and the ζ potential of COS nanoparticles is +20mV. The composites of Comparative Examples 1-(1), 1-(2), and 1-(3)PDA The zeta potentials of EcN / COS were -22mV, -20mV and -8mV respectively. PDA The activities of EcN in EcN / COS were 93.8%, 92.7% and 83.4% of those before functionalization, respectively. PDA The ζ potential of EcN / COS was not significantly affected, but the addition of CTAB alone significantly reduced the PDA The zeta potential of EcN / COS was calculated. On this basis, Poloxamer 188 and CTAB were added in Example 3. Under the influence of Poloxamer 188, the final PDA Zeta potential of EcN / COS.

[0111] The three functionalized PDA EcN / COS was tested for anti-tumor activity in vitro. Figure 15 As shown. In Comparative Example 1-(1) without adding CTAB and Poloxamer 188, PDA EcN and COS nanoparticles are naturally composited, and the composite efficiency is poor. The prepared composite preparation has poor in vitro anti-tumor effect. In Comparative Example 1-(2), Poloxamer 188 is added. Since Poloxamer 188 reduces the interfacial tension of the aqueous phase, it promotes the collision probability between EcN / PDA and COS nanoparticles. PDA The interfacial bridging effect between EcN and COS nanoparticles improves PDA The efficiency of the composite preparation between EcN and COS nanoparticles was significantly improved compared with the composite preparation prepared in comparative example 1-(1). In contrast, the addition of CTAB in comparative example 1-(3) improved the anti-tumor effect in vitro. PDA The composite efficiency between EcN and COS nanoparticles is low, but because there is no Poloxamer 188 on the PDA surface to adjust the interfacial tension, the distribution uniformity of COS nanoparticles attached to the PDA surface is extremely poor, and the nanoparticles maintain their original round morphology, such as Figure 16 In addition, in comparative examples 1-(3), without the protection of the hydration layer, CTAB had a certain destructive effect on E. coli (the activity of EcN was 83.4% of that before functionalization), resulting in a decrease in the anti-tumor effect of the final preparation, which was equivalent to that of the natural compound preparation group.

[0112] Comparative Example 2

[0113] Compared with Example 3, the difference is that the functionalization step (iii) is as follows:

[0114] Take 1 mL of the prepared PDAThe EcN resuspension was added with CTAB, and then mixed with 1 mL of COS nanoparticle emulsion, and then Poloxamer 188 was added to obtain a mixture, vortexed for 2 min, centrifuged at 5000 rpm for 7 min, washed once with physiological saline, and resuspended to 1 mL to obtain PDA EcN / COS, the concentration of Poloxamer 188 in the mixed solution is 0.06% w / v, and the concentration of CTAB is 0.03 mmol / L.

[0115] Prepared PDA The inhibitory effect of EcN / COS on tumor cells was significantly reduced. PDA The activity of EcN in EcN / COS was 78.9% of that before functionalization. PDA Insertion of EcN into a resuspended solution onto a PDA surface significantly impairs EcN activity. In Example 3, however, the presence of Poloxamer 188 forms a hydration layer, effectively shielding the EcN from CTAB damage. Adding only CTAB during the functionalization process, regardless of the timing, will cause varying degrees of damage to the EcN.

Claims

1. A method for preparing an Escherichia coli preparation, characterized in that: Preparation of chitosan oligosaccharide nanoparticle emulsion and dopamine-loaded Escherichia coli PDA EcN, and then chitosan oligosaccharide nanoparticles were used to treat dopamine-loaded Escherichia coli PDA EcN is functionalized, and the functionalization is performed by adding poloxamer 188 to the dopamine-encapsulated Escherichia coli resuspension, then mixing it with the chitosan oligosaccharide nanoparticle emulsion, adding cetyltrimethylammonium bromide CTAB to form a mixed solution, vortexing for 2 to 3 minutes, and then centrifuging and washing to obtain the Escherichia coli preparation, wherein the amount of poloxamer 188 added to the mixed solution is 0.05 to 0.08% w / v, and the amount of CTAB added is 0.02 to 0.05 mmol / L, and the preparation of the chitosan oligosaccharide nanoparticle emulsion is performed by taking chitosan oligosaccharide COS and adding Distilled water is used to prepare a COS solution, and then polyacrylic acid PAA is added to distilled water to prepare a PAA solution, and the PAA solution is added dropwise to the COS solution, and ultrasonic treatment is performed to obtain a COS nanoparticle emulsion. The concentration of the COS solution is 1.5-2.5 mg / mL, the concentration of the PAA solution is 0.8-1.2 mg / mL, and the volume ratio of the COS solution to the PAA solution is 4-6:

1. The preparation of dopamine-encapsulated Escherichia coli is to take dopamine hydrochloride and add it to Tris-HCl buffer to form PDA Tris-HCl buffer, resuspend the Escherichia coli EcN, and centrifuge to obtain the dopamine-encapsulated Escherichia coli. PDA EcN.

2. The method for preparing an Escherichia coli preparation according to claim 1, wherein: The ultrasonic temperature is room temperature, the ultrasonic power is 35-45 kHz, and the ultrasonic time is 3-5 min.

3. The method for preparing an Escherichia coli preparation according to claim 2, wherein: The PDA Tris-HCl buffer has a concentration of 550-650 μL / mL, a concentration of 8-12 mmol / L, and a pH of 8.

5.

4. The method for preparing an Escherichia coli preparation according to claim 3, wherein: The resuspension is to add 8~10 mL OD 600 The E. coli bacterial solution with a pH value of 0.8 was centrifuged and resuspended into 10 mL using PDA Tris-HCl buffer.

5. Use of the Escherichia coli preparation prepared by the preparation method according to any one of claims 1 to 4 in the preparation of anti-breast cancer drugs.

6. The use according to claim 5, characterized in that: The Escherichia coli preparation is used in the preparation of an anti-triple-negative breast cancer tumor drug.

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