Nano drug composition based on bacterial cell membrane and its preparation method and application

Abstract

The present invention relates to a drug carrier based on bacterial cell membrane and its preparation method and application. The nano drug composition of the present invention includes a drug and a bacterial cell membrane drug-carrying system, wherein the bacterial cell membrane drug-carrying system encapsulates the drug; the bacterial cell membrane drug-carrying system includes a bacterial cell membrane and a liposome; the bacterial cell membrane is wrapped outside the liposome; it can be efficiently enriched in the tumor site, release a drug that can kill Fusobacterium nucleatum, effectively alleviate the immunosuppressive microenvironment caused by Fusobacterium nucleatum, restore the response of colon cancer to anti-cytotoxic T lymphocyte-associated antigen 4 therapy and anti-programmed death receptor therapy, and can also effectively alleviate the resistance of Fusobacterium nucleatum to chemotherapy drugs, and restore the responsiveness of the tumor to the chemotherapy drug 5-fluorouracil. The nano drug composition can also kill a variety of bacteria in the breast cancer tumor site to alleviate tumor metastasis caused by tumor bacteria, and restore the responsiveness of the tumor to the combined chemotherapy regimen.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carriers, in particular to a nano drug composition based on bacterial cell membranes and a preparation method and application thereof. Background Art

[0002] Up to now, tumors are still one of the main factors threatening human health. With the in-depth understanding of tumor biology and microbiology, researchers have found that bacteria in tumor sites play a vital role in tumor growth, metastasis, and treatment, and are also one of the important targets for studying tumor treatment. More and more studies have shown that Fusobacterium nucleatum is enriched in tumor sites through its membrane protein Fap-2 and N-acetylgalactosamine (Gal-GalNAc) highly expressed in colon tumors, breast tumors, and pancreatic tumors, and participates in the physiological process of tumors. Fusobacterium nucleatum recruits immunosuppressive bone marrow-derived suppressor cells (MDSCs), which cause immune cell dysfunction and promote the continuous deterioration of tumors. Fusobacterium nucleatum can also directly inhibit immune cells, such as cytotoxic T lymphocytes and natural killer cells, leading to the generation of an immunosuppressive microenvironment, so that tumor cells escape the monitoring of immune cells and promote tumor proliferation. The immunosuppressive microenvironment caused by Fusobacterium nucleatum further hinders immune checkpoint blockade therapy for tumors, such as anti-programmed death receptor therapy (anti-PD1), anti-programmed death receptor-ligand 1 therapy (anti-PDL1), and anti-cytotoxic T lymphocyte-associated antigen 4 therapy (anti-CTLA4). Fusobacterium nucleatum can also promote the resistance of tumor cells to chemotherapeutic drugs such as oxaliplatin and 5-fluorouracil by regulating the autophagy network of tumor cells through Toll-like receptor-MYD88 and microRNAs. In addition, Fusobacterium nucleatum can also promote host cells to secrete interleukin-8 and chemokine-10 to promote the migration of cancer cells. A large number of recent studies and clinical results have shown that killing bacteria at the tumor site and reshaping the immunosuppressive tumor microenvironment will effectively improve the efficacy of tumor treatment.

[0003] Antibiotics are the most commonly used drugs for treating bacteria. They can inhibit the synthesis of bacterial cell walls, interact with cell membranes, interfere with protein synthesis, and inhibit the replication and transcription of nucleic acids. For example, positively charged polymyxins electrostatically bind to negatively charged lipid A on the bacterial outer membrane, causing the outer membrane to swell; then, they penetrate the outer membrane through the "self-promoted uptake" mechanism, destroying the physical integrity of the cell membrane phospholipid bilayer, leading to osmotic imbalance, causing important components such as nucleotides, amino acids, and phosphates in the cell to leak out, inhibiting bacterial growth or causing bacterial death. Using antibiotics to eliminate bacteria at the tumor site is an effective method.

[0004] Many antibiotics are taken orally and absorbed into the blood in the small intestine. Due to the low efficiency of oral absorption, many unabsorbed antibiotics will reach the colorectum, killing the intestinal flora of the human body, reducing the abundance of the intestinal flora of the human body, destroying the stability of the intestinal flora, and further promoting the occurrence of some diseases, such as causing chronic diseases, allergies or obesity, and even reducing the responsiveness of tumors to immunotherapy. Some antibiotics can also be administered by intravenous injection, but the free drugs are evenly distributed in the body and cannot be well enriched in the tumor site. Large doses of repeated administration are required to achieve therapeutic effects, which will accelerate the emergence of drug-resistant bacteria and pose new and more serious challenges and threats to human health. Researchers have designed various strategies to try to increase the accumulation of antibiotics in the tumor site and improve the effect of antibiotics in killing the tumor site. Liposomes are one of the most widely studied nanoformulations with good biocompatibility and modifiability. At present, antibiotic liposomes have been marketed or entered clinical research. Using liposomes as antibiotic drug carriers, passive targeting can effectively increase the local concentration of antibiotics in the tumor site. However, antibiotic liposome carriers cannot completely kill bacteria at the infection site, which may be due to the fact that there are not many drugs that can reach the infection site through passive targeting. Although there are a large number of ligand-modified nanoparticles that can effectively deliver antibiotics to tumor sites through active targeting, such as hyaluronic acid-modified nanoparticles for targeting CD44 at tumor sites, RGD peptide-modified nanoparticles for targeting αvβ3 integrin, trastuzumab-modified nanoparticles for targeting HER-2, CD14 antibody-modified nanoparticles for targeting CD14, and VEGF antibody-modified nanoparticles for targeting VEGF at tumor sites. However, these ligands are all targeted at tumor-specific receptors. So far, there are no nanocarriers that can actively target bacterially infected tumors. Therefore, it is very necessary to develop a nanocarrier that can effectively deliver antibiotics to bacterially infected tumor sites. Summary of the invention

[0005] In order to solve the problem of low local content of intravenous antibiotics at the tumor site, the present invention provides a nano drug composition based on bacterial cell membranes and its preparation method and application, that is, a nano drug composition that imitates the tumor enrichment behavior of Fusobacterium nucleatum, which can efficiently enrich at the tumor site, release drugs that can kill Fusobacterium nucleatum, effectively alleviate the immunosuppressive microenvironment caused by Fusobacterium nucleatum, restore the response of colon cancer to anti-cytotoxic T lymphocyte-associated antigen 4 therapy and anti-programmed death receptor therapy, and can also effectively alleviate the resistance of tumors caused by Fusobacterium nucleatum to chemotherapy drugs, and restore the responsiveness of tumors to the chemotherapy drug 5-fluorouracil. The nano drug composition can also kill multiple bacteria at the breast cancer tumor site to alleviate tumor metastasis caused by tumor bacteria and restore the responsiveness of tumors to combined chemotherapy regimens.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a nano drug composition based on bacterial cell membrane, comprising a drug and a bacterial cell membrane drug delivery system, wherein the bacterial cell membrane drug delivery system encapsulates the drug; the bacterial cell membrane drug delivery system comprises a bacterial cell membrane and a liposome; and the bacterial cell membrane is wrapped outside the liposome.

[0008] In one embodiment of the present invention, the particle size of the nano drug composition is ≤200 nm.

[0009] In one embodiment of the present invention, the drug is an antibiotic.

[0010] In one embodiment of the present invention, the antibiotics include one or more of β-lactam antibiotics, tetracycline antibiotics, aminoglycoside antibiotics, macrolide antibiotics, lincomycin antibiotics, polypeptide antibiotics, quinolone antibiotics and sulfonamide antibiotics.

[0011] In one embodiment of the present invention, the β-lactam antibiotics include one or more of penicillin G, dicloxacillin, amoxicillin, piperacillin, azlocillin, mezlocillin, ceftazidime, ceftriaxone, cefotaxime, cefoperazone, cefodizime, cefmenoxime, fucam, cefpirome, cefepime, cefazolin, imipenem, panipenem, meropenem, aztreonam, carumonam, cilastatin, aztreonam, clavulanic acid, sulbactam and tazobactam.

[0012] In one embodiment of the present invention, the aminoglycoside antibiotics include one or more of amikacin, tobramycin, gentamicin, netilmicin, sisomicin and streptomycin.

[0013] In one embodiment of the present invention, the macrolide antibiotics include one or more of roxithromycin, clarithromycin and azithromycin.

[0014] In one embodiment of the present invention, the tetracycline antibiotics include one or more of tetracycline, doxycycline and minocycline.

[0015] In one embodiment of the present invention, the lincomycin antibiotics include lincomycin and / or clindamycin.

[0016] In one embodiment of the present invention, the polypeptide antibiotics include one or more of polymyxin B, polymyxin E, vancomycin, norvancomycin and teicoplanin.

[0017] In one embodiment of the present invention, the norone antibiotics include one or more of norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, fleroxacin, elofloxacin, lomefloxacin, sparfloxacin, grefloxacin, rufloxacin, clinafloxacin, balofloxacin and trovafloxacin.

[0018] In one embodiment of the present invention, the sulfonamide antibiotics include cotrimoxazole.

[0019] In one embodiment of the present invention, the antibiotics further include one or more of metronidazole, tinidazole and ornidazole.

[0020] In one embodiment of the present invention, the bacteria in the bacterial cell membrane are selected from Fusobacterium nucleatum.

[0021] In one embodiment of the present invention, the bacterial cell membrane is obtained by bacterial lysis and sucrose density gradient centrifugation extraction, which contains a large number of membrane proteins; the membrane proteins include one or more of Fap-2, FadA, FomA, RadD, OmpF, OmpC and PhoE.

[0022] The second object of the present invention is to provide a method for preparing the bacterial cell membrane-based nanopharmaceutical composition, comprising the following steps:

[0023] The liposomes encapsulated with the drugs are mixed with the bacterial cell membrane solution, and are repeatedly pushed through the polycarbonate membrane to obtain the bacterial cell membrane-based nano drug composition.

[0024] In one embodiment of the present invention, the liposomes are synthesized using phospholipids as raw materials through a reverse microemulsion method, a membrane hydration method, a freeze-thaw cycle method, an organic solvent injection method, an extrusion method, an ultrasonic dispersion method or a high-pressure averaging method.

[0025] In one embodiment of the present invention, the drug-encapsulated liposome is prepared by the following method:

[0026] The organic solution containing phospholipids is mixed with the drug solution, ultrasonicated and stirred to obtain the liposomes encapsulating the drug.

[0027] In one embodiment of the present invention, the phospholipids include one or more of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), phosphatidylcholine (PC), dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylcholine (DSPC).

[0028] In one embodiment of the present invention, the concentration of the drug in the drug solution is 0.5 mg / mL-30 mg / mL, preferably 6 mg / mL.

[0029] In one embodiment of the present invention, the method for preparing the nano drug composition comprises the following steps:

[0030] a) mixing a chloroform solution containing DPPC, CHO and DSPE-PEG with an aqueous solution containing antibiotics, ultrasonicating at a certain power to obtain an oil-in-water emulsion, stirring overnight, removing the chloroform, and obtaining liposomes encapsulating the antibiotics;

[0031] b) preparing Fusobacterium nucleatum membrane;

[0032] c) mixing the liposomes encapsulating the antibiotics with the aqueous solution of the Fusobacterium nucleatum membrane, and repeatedly pushing the obtained mixed solution through a 200 nm polycarbonate membrane to obtain the nano drug composition.

[0033] In one embodiment of the present invention, in step a), the mass ratio of DPPC, cholesterol and DSPE-PEG is 13:5:(4-8), preferably 13:5:4.

[0034] In one embodiment of the present invention, in step a), the volume ratio of the chloroform solution containing DPPC, CHO and DSPE-PEG to the antibiotic aqueous solution is 1:3, the ultrasonic time is 20 min, and the ultrasonic power is 20w-200w.

[0035] In one embodiment of the present invention, the mass ratio of the liposome to the membrane protein in the membrane of Fusobacterium nucleatum is 20:(0.5-5), preferably 20:1.

[0036] The third object of the present invention is to provide the use of the nano drug composition in the preparation of drugs for treating tumors.

[0037] The nano drug composition can regulate the tumor microbial microenvironment, enhance tumor immunotherapy, chemotherapy, and reduce tumor lung metastasis caused by bacteria.

[0038] In one embodiment of the present invention, the nano drug composition is administered as an intravenous injection.

[0039] The antibiotics in the nanomedicine composition of the present invention release antibiotics at the tumor site, kill bacteria at the tumor site, reverse the immunosuppressive microenvironment caused by Fusobacterium nucleatum, and increase the efficacy of immune checkpoint blockade therapy. The checkpoint blockade therapy includes, for example, anti-programmed death receptor therapy (anti-PD1), anti-programmed death receptor-ligand 1 therapy (anti-PDL1), and anti-cytotoxic T lymphocyte associated antigen 4 therapy (anti-CTLA4).

[0040] The nano drug composition of the present invention can reverse the chemotherapy resistance caused by Fusobacterium nucleatum.

[0041] The nano drug composition of the present invention can reverse tumor metastasis caused by multiple bacteria in the tumor site.

[0042] The nano drug composition of the present invention can reverse the chemotherapy resistance caused by multiple bacteria in the tumor site.

[0043] Wherein, the multiple bacteria include Fusobacterium nucleatum, Staphylococcus xylosus, Streptococcus sanguinis, and Lactobacillus animalis. The tumor includes colon cancer or breast cancer. The chemotherapy drugs include 5-fluorouracil, methotrexate, oxaliplatin, cyclophosphamide, doxorubicin, paclitaxel, cisplatin, gemcitabine, capecitabine, pemetrexed, S-1, vinorelbine, docetaxel, irinotecan or tamoxifen carboplatin.

[0044] The above technical solution of the present invention has the following advantages compared with the prior art:

[0045] The present invention can effectively kill bacteria in the tumor site by effectively delivering antibiotics to the tumor site and releasing antibiotics, so as to solve the problems of immune resistance, chemotherapy resistance and metastasis caused by bacteria in the tumor site in clinical practice, improve the efficacy of immune checkpoint blockade therapy and chemotherapy, and reduce the problem of tumor metastasis caused by bacteria. The problem of tumor resistance and metastasis caused by bacteria in clinical practice is solved.

[0046] The present invention provides a method for preparing a nano drug composition that has the function of killing bacteria at the tumor site and regulating the tumor immunosuppressive microenvironment. The synthesis method is simple and easy to operate. After intravenous injection, it can efficiently accumulate at the tumor site and increase the antibiotic concentration at the tumor site; it can kill bacteria at the tumor site, effectively regulate the tumor bacterial microenvironment, reverse the immunosuppressive tumor microenvironment, reverse the tumor chemotherapy resistance microenvironment, and reduce tumor metastasis.

[0047] The present invention also discloses the use of nano-drug compositions for reversing the tumor immunosuppressive microenvironment caused by bacteria and increasing the use of tumor immune checkpoint blockade therapy. Intravenous injection of the nano-drug composition, combined with anti-programmed death receptor therapy (anti-PD1) or anti-cytotoxic T lymphocyte-associated antigen 4 therapy (anti-CTLA4), can effectively regulate the tumor immunosuppressive microenvironment, reduce the content of immunosuppressive cells in the tumor, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs); increase the content of immune cells that can kill tumors, CD8+T lymphocytes. Effectively reverse the tumor immunosuppressive microenvironment, activate the body's immune response, and reverse the efficacy of immune checkpoint blockade therapy.

[0048] Simultaneous intravenous injection of the nano-drug composition combined with the chemotherapy drug 5-fluorouracil can effectively regulate the tumor's resistance to chemotherapy drugs and increase the chemotherapy drug's ability to kill tumors.

[0049] Simultaneous intravenous injection of the nano-drug composition can effectively reverse tumor metastasis caused by multiple bacteria at the tumor site.

[0050] Simultaneous intravenous injection of the nanodrug composition can effectively reverse chemotherapy resistance caused by multiple bacteria in the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0052] Figure 1 1 is a relevant characterization diagram of the nano drug composition in Example 1 of the present invention; wherein a is a protein gel electrophoresis diagram of Fusobacterium nucleatum and Fusobacterium nucleatum; b is an energy fluorescence resonance transfer spectrum diagram of the nano drug composition; ce is a particle size distribution diagram, a potential diagram and a transmission electron microscopy diagram of the nano drug composition;

[0053] Figure 2 : are the property test results of the nano drug composition in Example 2 of the present invention; wherein a is a hydrated particle size diagram of the nano drug composition at different times; b is a particle size dispersion index diagram of the nano drug composition at different times; c is the encapsulation rate and loading rate of colistin by the nano drug composition; d is a release curve diagram of colistin in the colistin-nano drug composition; e is an antibacterial diagram of the colistin-nano drug composition;

[0054] Figure 3 is the absorption capacity of different colon cancer cells to the nano drug composition in Example 3 of the present invention; wherein a is a flow cytometric representation of the fluorescence intensity of HT-29 colon cancer cells absorbing the nano drug composition; b is the fluorescence intensity of different colon cancer cells absorbing the nano drug composition;

[0055] Figure 4 is the behavior of the nano drug composition in Example 4 of the present invention in mice; wherein a is the pharmacokinetic diagram of the nano drug composition; b is the fluorescence intensity of the nano drug composition at the tumor site at different time points; c is the fluorescence intensity of the nano drug composition and liposomes in various organs and tumors after the mice were killed; d is the drug amount of the nano drug composition and liposomes in various organs and tumors after the mice were killed;

[0056] Figure 5 This is the in vivo antibacterial experiment of the nanomaterial in Example 5 of the present invention;

[0057] Figure 6The nano drug composition in Example 6 of the present invention increases the efficacy of immune checkpoint blockade therapy on subcutaneous colon cancer; wherein a is a tumor volume diagram of six groups of mice at different times; b is a tumor photo and tumor mass diagram of four groups of mice at the same time; c is a diagram of changes in immune-promoting cells in tumors of four groups of mice;

[0058] Figure 7 is a graph showing the efficacy of the nanomedicine composition in Example 7 of the present invention in increasing immune checkpoint blockade therapy on spontaneous colon cancer;

[0059] Figure 8 This is a graph showing the efficacy of the nano drug composition in Example 8 of the present invention in increasing chemotherapeutic drugs on subcutaneous breast cancer tumors in mice;

[0060] Fig. 9 The nanomedicine composition in Example 9 of the present invention alleviates breast cancer lung metastasis caused by breast cancer tumor bacteria; wherein a is a graph showing that the nanomedicine composition effectively alleviates breast cancer metastasis caused by bacteria; b is a graph showing that the nanomedicine composition alleviates breast cancer lung metastasis caused by bacteria; c is a graph showing the bacterial content in the tumor after treatment with the nanomedicine composition; d is a graph showing the bacterial content in the lung after treatment with the nanomedicine composition;

[0061] Fig.10 The nanomedicine composition alleviates chemotherapy resistance caused by breast cancer tumor bacteria, where a is a graph of tumor volume of five groups of mice at different times; b is a graph of tumor mass of five groups of mice at different times. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0063] Example 1: Preparation method of nano drug composition

[0064] S1: Weigh 13mg DPPC, 5mg CHO, 4mg DSPE-PEG, add to 6mL chloroform, then add 2mL 6mg / mL colistin aqueous solution to 6mL chloroform solution. Then transfer the mixture of chloroform and water (layering) to ice, insert the ultrasonic probe into the solution, set the ultrasonic power to 100w, ultrasonicate for 3 seconds, stop for 5 seconds, and ultrasonicate for a total of 20 minutes; obtain an oil-in-water emulsion. Transfer the obtained emulsion to an open glass bottle, add a magnetic stirrer, stir at 200rpm, stir at 25 degrees Celsius for 12 hours to volatilize chloroform, and obtain liposomes encapsulating colistin.

[0065] S2: The bacterial strain is drawn into a liquid thioglycollate medium and cultured at 37°C under anaerobic conditions until the absorbance at 600nm is about 0.8-1.2. Subsequently, Fusobacterium nucleatum is collected by centrifugation at 10,000 rpm, and Fusobacterium nucleatum is resuspended in buffer A, which includes 0.2 mol / L tris(hydroxymethyl)aminomethane hydrochloride, 1 mol / L sucrose, and 20 mg / L lysozyme. Then shake at 37°C and 120 rpm for 1 hour. Then 90 mL of an aqueous solution containing 10 mg / L deoxyribonuclease I is added to the above solution containing Fusobacterium nucleatum, shaken 20 times, and the cells are collected by centrifugation at 10,000 rpm, and the supernatant is discarded. Then resuspend in buffer B, which contains 20 mmol / L tris(hydroxymethyl)aminomethane hydrochloride, 50 mmol / L sodium chloride, 5 mmol / L disodium ethylenediaminetetraacetic acid, and 200 g / L sucrose. The buffer solution B containing Fusobacterium nucleatum was ultrasonically broken in an ice bath for 20 minutes at 400w power, 3 seconds on, and 5 seconds off. The liquid after ultrasonication was centrifuged at 10000rpm, the precipitate was discarded, and the supernatant was retained. Subsequently, 3mL of the above lysate was placed on the top of a discontinuous sucrose gradient (from bottom to top, 500mg / mL 2.5mL, 460mg / mL 1.25mL, 420mg / mL 2.5mL, 360mg / mL 2.5mL, 320mg / mL 2.5mL and 270mg / mL 2.5ml). The above sucrose solution was prepared in a solution of 20mmol / L tris(hydroxymethyl)aminomethane hydrochloride, 50mmol / L sodium chloride, and 5mmol / L disodium ethylenediaminetetraacetic acid. The above centrifuge tube was placed on an ultracentrifuge and centrifuged at 50000g and 4℃ vacuum conditions for 1 hour. After the centrifugation was completed, the turbid liquid in the middle layer of the centrifuge tube was collected, which was the cell membrane of Fusobacterium nucleatum. Collect this part of the Fusobacterium nucleatum membrane into an ultracentrifuge tube, centrifuge at 100,000 g under 4°C vacuum conditions for 1 hour, discard the supernatant, add PBS and pipette evenly, and place the Fusobacterium nucleatum membrane at -80°C for use.

[0066] S3: 1 mg of Fusobacterium nucleatum membrane is added to the liposomes encapsulated with antibiotics obtained in step S1 to obtain a mixed solution. The mixed solution is repeatedly pushed through a 200 nm polycarbonate membrane to obtain a nanomedicine composition simulating Fusobacterium nucleatum, that is, a nanomedicine composition based on bacterial cell membrane. The nanomedicine composition is then placed in a dialysis bag with a molecular weight of 8000 Daltons and dialyzed in a PBS solution to remove the unencapsulated colistin.

[0067] The relevant characteristics of the nano drug composition obtained in this example are as follows Figure 1As shown, a is the sodium dodecyl sulfate polyacrylamide gel electrophoresis diagram of Fusobacterium nucleatum and Fusobacterium nucleatum membrane, b is its fluorescence resonance energy transfer curve, c is the hydrated particle size of the nano drug composition, d is the potential diagram of the surface charge of the nano drug composition, and e is its transmission electron microscope diagram. In the sodium dodecyl sulfate polyacrylamide gel electrophoresis diagram, 1 is a protein marker used to indicate the molecular weight of the protein, 2 is the whole bacterial lysate of Fusobacterium nucleatum, and 3 is the Fusobacterium nucleatum membrane. It can be seen that the Fusobacterium nucleatum membrane retains the adhesion protein Fap-2 of Fusobacterium nucleatum. The applicant marked FITC on the liposome and TRITC on the Fusobacterium nucleatum membrane. The two form a fluorescence resonance energy transfer fluorescent group pair. When the distance between the two fluorescent groups is appropriate, the phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed, that is, when excited by the excitation wavelength of the former group, the fluorescence emitted by the latter group can be observed. From the energy fluorescence resonance transfer curve, it can be seen that as the number of extrusions increases, the FITC fluorescence of the liposome membrane decreases and the TRITC fluorescence of the Fusobacterium nucleatum membrane increases, indicating that the Fusobacterium nucleatum membrane is wrapped on the liposome. From the hydrated particle size diagram, it can be seen that the hydrated particle size of the nano drug composition is about 190 nanometers. From the potential diagram, it can be seen that the potential of the nano drug composition is about -10 mA, which is between the liposome and the Fusobacterium nucleatum membrane, further proving that the nano drug composition and the liposome have successfully fused. From the transmission electron microscope diagram, it can be seen that the shape of the nano drug composition is round and the particle size distribution is uniform.

[0068] Example 2: Property detection of nano drug composition

[0069] The nano drug composition prepared in Example 1 was subjected to stability testing, loading capacity testing, release testing, and in vitro antibacterial testing.

[0070] The modified nano drug composition obtained in Example 1 was subjected to stability testing. The nano drug composition was dispersed in RPMI 1640 medium containing 10% serum, and its stability was determined by detecting the change in particle size and particle size dispersion index of the nano drug composition at different time points. Figure 2 As shown in a and b in the figure; it can be seen that the nano drug composition prepared in Example 1 maintains stability in the RPMI 1640 medium containing 10% serum. Compared with the simple Fusobacterium nucleatum membrane, the nano drug composition of the present application has no significant change in particle size within 72 hours, proving that the modified nano drug composition prepared in Example 1 has good stability.

[0071] The loading capacity of the nano drug composition prepared in Example 1 was tested. The nano drug composition obtained in Example 1 was added with an equal volume of methanol to release the colistin in the nano drug composition. High performance liquid chromatography was used, and the mobile phase was a mixture of sodium sulfate aqueous solution [30mM (pH=2.5)] and acetonitrile, the volume ratio was 77:23, and the flow rate was 1mL / min. Colistin was detected at a wavelength of 205nm, the separation column was C18, and the peak time was 8 minutes. Figure 2 As shown in c; the results show that the nano drug composition prepared in Example 1 can load colistin very well, with an encapsulation rate of about 13.2% and a loading rate of about 6.6%.

[0072] The drug release capacity of the nano drug composition prepared in Example 1 was tested. The nano drug composition was transferred to a dialysis bag with a molecular weight cutoff of 8000 Daltons and immersed in 1640RPMI culture medium and PBS buffer containing 10% serum at room temperature. At a predetermined time point, the external solution was collected and HPLC was used. The mobile phase was a mixture of sodium sulfate aqueous solution [30mM (pH=2.5)] and acetonitrile, with a volume ratio of 77:23 and a flow rate of 1mL / min. Colistin was detected at a wavelength of 205nm, the separation column was C18, and the peak time was 8 minutes. The test results are as follows: Figure 2 As shown in d, the results show that the nano drug composition prepared in Example 1 can release antibiotics in a sustained manner.

[0073] The nano drug composition prepared in Example 1 was tested for antibacterial properties. 100 μl of nano drug composition of different concentrations (colistin concentration: from 0.0171 μg / ml to 2.1875 μg / ml) was mixed with 100 μl of 10,000,000 / ml Fusobacterium nucleatum, and cultured anaerobically at 37°C for 72 hours. Then 30 μl of bacterial solution was added with 10 μl of bacterial activity detection solution. After 10 minutes, bioluminescence was detected with an ELISA instrument to detect bacterial activity. Bacterial relative survival rate (%) = bacterial luminescence intensity of experimental group / bacterial luminescence intensity of control group × 100%. The results are shown in the figure. Figure 2 As shown in e; the results show that the killing ability of the nano drug composition prepared in Example 1 on Fusobacterium nucleatum is lower than that of free colistin, which is due to the sustained release of antibiotics by the nano drug composition.

[0074] Example 3: Detection of the ability of nanomedicine compositions to target colon cancer cells

[0075] This example detects the targeting ability of the nanomedicine composition to colon cancer cells that highly express Gal-GalNAc. The nanomedicine composition prepared in Example 1 was labeled with fluorescent FITC, and then incubated with mouse colon cancer cell lines CT-26, MC-38, human colon cancer cell lines HT-29, and CaCO-2 in culture medium for 2 hours. The control group was the liposome (Liposome) prepared in Example 1, the nanomedicine composition of Escherichia coli membrane liposomes (LipoEM), and the nanomedicine composition pretreated with Gal-GalNAc (Liposome+Gal-GalNAc): Gal-GalNAc was used to pre-bind the Fap-2 protein on the nanomedicine composition. Among them, N-acetylgalactosamine can prevent the nanomedicine composition from binding to colon cancer cells that highly express N-acetylgalactosamine. After 2 hours, the cells were collected and the materials were washed away with PBS.

[0076] Then flow cytometry was used to detect the fluorescence intensity of cancer cells. Figure 3 As shown in a and b, the Fusobacterium nucleatum liposome nanomedicine composition prepared in Example 1 can better target colon cancer cells compared with liposomes, Escherichia coli membrane liposome nanomedicine compositions, and Gal-GalNAc pretreated nanomedicine compositions.

[0077] Example 4: In vivo behavior of nanomedicine composition in mice after tail vein injection

[0078] Through Example 3, the applicant found that the nano drug composition prepared in Example 1 has a good targeting ability to colon cancer cells in vitro.

[0079] In order to further understand the behavior of the modified nano drug composition prepared in Example 1 in mice after intravenous injection, the nano drug composition was labeled with a fluorescent small molecule, and its behavior in mice was understood by in vivo fluorescence imaging. In order to understand the blood circulation behavior of the nano drug composition in mice, the nano drug composition labeled with the DiR fluorescent molecule was intravenously injected into mice, and the liposomes prepared in Example 1 were labeled with the fluorescent small molecule DiR as a control group. Blood was collected at a specific time point, and the fluorescent signal value of the drug in the blood was measured by lysis, and then the blood circulation half-life of the nano drug composition was calculated. The test results are shown in FIG. Figure 4 As shown in a in the figure; the results show that both the nanomedicine composition and the liposome have a long blood circulation time. In addition, the nanomedicine composition labeled with the fluorescent small molecule DiR was intravenously injected into mice with colon cancer tumors. At different time points, the small animal in vivo imaging system was used to collect real-time images of the mice and calculate the fluorescence intensity to observe the enrichment of the nanomedicine composition in the tumor site. The test results are shown in Figure 4As shown in b; the results show that as time passes, the fluorescence signal of the nanomedicine composition at the tumor site gradually becomes stronger than that of the liposome group, and still maintains a high fluorescence signal at the 72h time point, indicating that the nanomedicine composition has good tumor enrichment behavior. Subsequently, the mice were killed, the main organs and tumors of the mice were removed, and the small animal in vivo imaging system was used to collect real-time images of the mice and count the fluorescence intensity of each organ and tumor to observe the distribution behavior of the nanomedicine composition and liposomes in the body. Figure 4 As can be seen from c, compared with liposomes, the Fusobacterium nucleatum liposome nanomedicine composition prepared in Example 1 can be better enriched in the tumor site. Further, the applicant weighed and crushed the various organs of the mouse, and then added an extract (water: trichloroacetic acid: Triton X-100: acetonitrile 68:4:20), extracted at 4°C for 48 hours, collected the extract, and used high performance liquid chromatography, the mobile phase was a mixture of sodium chlorate aqueous solution [30mM (pH = 2.5)] and acetonitrile, the volume ratio was 77:23, the flow rate was 1 ml / min, colistin was detected at a wavelength of 205nm, the separation column was C18, and the test results were as follows: Figure 4 As shown in d, the results show that the nano drug composition prepared in Example 1 can better deliver colistin to the tumor site than the liposome.

[0080] Example 5: In vivo antibacterial effect of colistin-nano drug composition after tail vein injection

[0081] In order to detect the in vivo antibacterial ability of the colistin-nano drug composition prepared in Example 1, 10,000,000 Fusobacterium nucleatum were first injected into the tumor of mice with colon cancer tumors, and then the colistin-nano drug composition was injected into the mice intravenously. After the injection of the drug, the mice were killed, the tumors were removed from the mice under sterile conditions, and then the tumors were homogenized and applied to the BHI medium liquid brain heart infusion broth with antibiotics (containing 5g / L yeast extract, 0.5g / L cysteine ​​hydrochloride, 5mg / L hemin, 1uL / L vitamin K1, 4mg / L vancomycin hydrochloride, 4mg / L erythromycin, 1mg / L crystal violet), and then cultured at 37°C for 72h under anaerobic conditions, and then counted using a colony counter to detect the bacterial content in the tumor. The experimental results are shown in the figure. Figure 5 As shown, it can be seen that the colistin nano drug composition synthesized in Example 1 can kill Fusobacterium nucleatum in the tumor site better than liposomes and free antibiotics, indicating that the colistin-nano drug composition can be used as an effective tumor bacterial antibacterial drug.

[0082] Example 6: Colistin-nanomedicine combination increases the efficacy of immune checkpoint blockade therapy in subcutaneous colon cancer

[0083] In order to detect the efficacy of the nano drug composition prepared in Example 1 in increasing immune checkpoint blockade therapy for subcutaneous colon cancer, mice with subcutaneous colon cancer tumor models were divided into six groups: 1: control group (no treatment); 2: anti-CTLA4 (intravenous injection); 3: anti-CTLA4 (intravenous injection) + Fusobacterium nucleatum (intratumoral injection); 4: anti-CTLA4 (intravenous injection) + Fusobacterium nucleatum (intratumoral injection) + colistin (intravenous injection); 5: anti-CTLA4 (intravenous injection) + Fusobacterium nucleatum (intratumoral injection) + colistin-liposome (intravenous injection); 6: anti-CTLA4 (intravenous injection) + Fusobacterium nucleatum (intratumoral injection) + colistin-nano drug composition (intravenous injection). Fusobacterium nucleatum was injected intratumorally at a dose of 1,000,000 bacteria per tumor on day 0, and anti-CTLA4 was injected intravenously at a dose of 1 mg / kg on days 1, 3, and 5. Colistin, colistin-liposome, Example 1 The colistin-nano drug composition was intravenously injected at a dose of 15 mg / kg of colistin sulfate on days 1, 3, and 5. The tumor size was recorded with a vernier caliper every two days, and the tumor volume was calculated as follows: tumor volume = length × width × width / 2. The experimental results are shown in Figure 6 As shown in a in Figure 6 Compared with the first, third, fourth and fifth groups, the tumor growth of the sixth group was effectively inhibited, indicating that the nanomedicine composition can effectively reverse the resistance of subcutaneous colon cancer tumors caused by Fusobacterium nucleatum to anti-CTLA4. In another parallel experiment, after the above treatment, the applicant removed the mouse tumors such as Figure 6 As shown in b, the tumor volume and tumor weight further illustrate that the colistin-nano drug combination treatment can effectively reverse the resistance of subcutaneous colon cancer tumors caused by Fusobacterium nucleatum to anti-CTLA4. The tumor was then dispersed into a single cell suspension, and after antibody staining, the changes in immune-promoting cells and immune-suppressive cells in the tumor were analyzed by flow cytometry. Figure 6 As can be seen in c, after treatment with the colistin-nano drug composition, the proportion of immune-promoting cells CD8+IFNγ+T cells in the tumor returned to the level of the anti-CTLA4 (intravenous injection) group, proving that the colistin-nano drug composition synthesized in Example 1 can effectively alleviate the failure of immune-promoting cells caused by Fusobacterium nucleatum.

[0084] Example 7: Colistin-nanomedicine combination increases the efficacy of immune checkpoint blockade therapy against spontaneous colon cancer

[0085] In order to detect the efficacy of the colistin-nano drug composition prepared in Example 1 to increase the immune checkpoint blockade therapy for spontaneous colon cancer, azomethane and dextran sulfate were first used to induce C57 mice to spontaneously generate colon cancer. The specific steps are as follows: the AOM dose was 10 mg / kg, and it was administered by intraperitoneal injection on days 0 and 20, respectively. At the same time, drinking water containing 3% dextran sulfate was consumed for 5 consecutive days, and then replaced with ordinary water for 14 consecutive days, for a total of 3 times. After 52 days, streptomycin and penicillin were gavaged to remove intestinal bacteria, and then Fn was gavaged every day to establish a spontaneous colorectal tumor model infected with Fn. Mice with spontaneous tumor models were divided into six groups: 1: control group (no treatment); 2: Fusobacterium nucleatum (oral); 3: anti-CTLA4 (intravenous injection); 4: anti-CTLA4 (intravenous injection) + Fusobacterium nucleatum (oral); 5: anti-CTLA4 (intravenous injection) + Fusobacterium nucleatum (oral) + colistin-nano drug composition (intravenous injection). 2000000000 Fusobacterium nucleatum was administered orally every day until the end of the experiment. Anti-CTLA4 was intravenously injected at a dose of 1 mg / kg on days 60, 67, 74, 81, 88, and 95, and colistin-nano drug composition was intravenously injected on days 60, 67, 74, 81, 88, and 95. Mice were killed after 8 weeks and their intestines were collected. Tumor counts were performed. The experimental structure is as follows Figure 7 As shown, the number of spontaneous colon cancers in mice treated with the colistin-nano drug composition was greatly reduced, indicating that in the spontaneous colon cancer model, the colistin-nano drug composition can be combined with anti-CTLA4 treatment to achieve efficient treatment.

[0086] Example 8: Colistin-nano drug composition increases the efficacy of chemotherapy drugs against subcutaneous breast cancer tumors in mice.

[0087] In order to detect the effect of the colistin-nano drug composition prepared in Example 1 on increasing chemotherapy for subcutaneous colon cancer, mice with subcutaneous colon cancer tumor models were divided into six groups: 1: control group (no treatment); 2: 5Fu (intraperitoneal injection); 3: 5Fu (intraperitoneal injection) + Fusobacterium nucleatum (intratumoral injection); 4: 5Fu (intraperitoneal injection) + Fusobacterium nucleatum (intratumoral injection) + colistin (intravenous injection); 5: 5Fu (intraperitoneal injection) + Fusobacterium nucleatum (intratumoral injection) + colistin-liposome (intravenous injection); 6: 5Fu (intraperitoneal injection) + Fusobacterium nucleatum (intratumoral injection) + colistin-nano drug composition (intravenous injection). Fusobacterium nucleatum was injected intratumorally at a dose of 1,000,000 bacteria per tumor on day 0, and 5Fu was injected intraperitoneally at a dose of 25 mg / kg on days 1, 3, 5, 7, 9, 11, and 13. Colistin, colistin-liposome, Example 1 The colistin-nano drug composition was intravenously injected at a dose of 15 mg / kg of colistin sulfate on days 1, 3, and 5. The tumor size was recorded with a vernier caliper every two days, and the tumor volume was calculated as follows: tumor volume = length × width × width / 2. The experimental results are shown in Figure 8 As shown, after treatment with the colistin-nano drug composition, the resistance of breast cancer tumors caused by Fusobacterium nucleatum to 5Fu was completely alleviated, indicating that the colistin-nano drug composition combined with the chemotherapy drug 5Fu can achieve efficient treatment of breast cancer tumors.

[0088] Example 9: Doxycycline-nanomedicine composition alleviates breast cancer lung metastasis caused by breast cancer tumor bacteria.

[0089] In order to detect the effect of the doxycycline-nano drug composition prepared in Example 1 on relieving breast cancer lung metastasis caused by breast cancer tumor bacteria. Mice with in situ breast cancer were divided into three groups: 1: control group; 2: bacteria (intratumor); 3: bacteria (intratumor) + doxycycline-nano drug composition. On days 1, 8, 15, and 22, 2,500,000 Fusobacterium nucleatum, 2,500,000 Staphylococcus xylosus, 2,500,000 Streptococcus sanguinis, and 2,500,000 Lactobacillus animalis were injected into the tumors of the second and third groups of mice with in situ breast cancer. Then, on days 2, 9, 16, and 23, the third group of mice were injected with the doxycycline-nano drug composition prepared in the same steps as in Example 1, except that the drug colistin was replaced with doxycycline. The tumor growth and metastasis of mice were observed by a small animal in vivo imaging device. Fig. 9 As shown in a, the use of doxycycline nanomedicine composition can effectively slow down breast cancer metastasis caused by bacteria. The lungs were then removed for small animal imaging, such as Fig. 9As shown in Figure b, the use of doxycycline-nano drug combination can indeed alleviate breast cancer lung metastasis caused by bacteria. Next, the in situ breast cancer and lung homogenate were spread on blood plate culture medium, and then cultured at 37°C for 72 hours under anaerobic conditions. Then, the bacterial content in the tumor and lung was detected using a colony counter. The experimental results are shown in Figure 2. Fig. 9 As shown in c and d, it can be seen that the synthesized doxycycline-nano drug composition can effectively kill various bacteria in breast cancer tumor sites and lung metastases, indicating that the doxycycline-nano drug composition can be used as an effective tumor bacterial antibacterial drug and reduce lung metastasis caused by tumor bacteria.

[0090] Example 10: Doxycycline-nanomedicine combination alleviates chemotherapy resistance caused by breast cancer tumor bacteria

[0091] In order to detect the effect of the doxycycline-nano drug composition prepared in Example 1 on relieving the chemotherapy resistance caused by breast cancer tumor bacteria. Mice with in situ breast cancer were divided into five groups: 1: control group (no treatment); 2: bacteria (intratumoral injection); 3: combined chemotherapy (intraperitoneal injection); 4: combined chemotherapy (intraperitoneal injection) + bacteria (intratumoral injection); 5: combined chemotherapy (intraperitoneal injection) + bacteria (intratumoral injection) + doxycycline-nano drug composition (intravenous injection). On day 0, 2,500,000 Fusobacterium nucleatum, 2,500,000 Staphylococcus xylosus, 2,500,000 Streptococcus sanguinis, and 2,500,000 Lactobacillus animalis were injected into the tumors of the second, fourth, and fifth groups of mice with in situ breast cancer, and then on days 1, 3, and 5, the fifth group of mice were injected with the doxycycline-nano drug composition prepared in the same steps as in Example 1, with the drug colistin replaced by doxycycline. The third, fourth and fifth groups were given combined chemotherapy, with 5Fu injected intraperitoneally at a dose of 25 mg / kg on days 1, 3, 5, 7, 9, 11 and 13, methotrexate injected intraperitoneally at a dose of 20 mg / kg on days 1 and 5, and cyclophosphamide injected intraperitoneally at a dose of 100 mg / kg on days 1 and 5. Tumor size was recorded with a vernier caliper every two days. Tumor volume was calculated as follows: tumor volume = length × width × width / 2. The experimental results are shown in Table 1. Fig.10 As shown in a, after treatment with the doxycycline-nano drug composition, the resistance of breast cancer tumors caused by various bacteria to chemotherapy drugs was completely alleviated, indicating that the nano drug composition combined with chemotherapy drugs can achieve efficient treatment of breast cancer tumors. On the 18th day, the tumor was removed, photographed and weighed. The results are as follows Fig.10 As shown in b, the tumor volume and tumor weight further illustrate that doxycycline-nano drug composition treatment can effectively reverse the chemotherapy resistance of breast cancer tumors caused by tumor bacteria.

[0092] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A nano drug composition based on bacterial cell membrane, characterized in that: It includes a drug and a bacterial cell membrane drug-carrying system, wherein the bacterial cell membrane drug-carrying system encapsulates the drug; the bacterial cell membrane drug-carrying system includes a bacterial cell membrane and a liposome; the bacterial cell membrane is wrapped outside the liposome; The drug is an antibiotic; The bacteria in the bacterial cell membrane are selected from Fusobacterium nucleatum.

2. The nano drug composition according to claim 1, characterized in that: The particle size of the nano drug composition is ≤200 nm.

3. The nano drug composition according to claim 1, characterized in that: The antibiotics include one or more of β-lactam antibiotics, tetracycline antibiotics, aminoglycoside antibiotics, macrolide antibiotics, lincomycin antibiotics, polypeptide antibiotics, quinolone antibiotics and sulfonamide antibiotics.

4. A method for preparing the bacterial cell membrane-based nanomedicine composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: The liposomes encapsulated with the drugs are mixed with the bacterial cell membrane solution, and are repeatedly pushed through the polycarbonate membrane to obtain the bacterial cell membrane-based nano drug composition.

5. The preparation method according to claim 4, characterized in that: The drug-encapsulated liposome is prepared by the following method: The organic solution containing phospholipids is mixed with the drug solution, ultrasonicated and stirred to obtain the liposomes encapsulating the drug.

6. The preparation method according to claim 5, characterized in that: The phospholipid is selected from one or more of dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, phosphatidylcholine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylcholine.

7. The preparation method according to claim 5, characterized in that: The concentration of the drug in the drug solution is 0.5 mg / mL-30 mg / mL.

8. Use of the nano drug composition according to any one of claims 1 to 3 in the preparation of drugs for treating tumors; the tumors are colon cancer and breast cancer.

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