A bacterial-mediated drug delivery system, and a preparation method and application thereof

Abstract

The present application relates to a kind of bacterial-mediated drug delivery system and its preparation method and application.A kind of bacterial-mediated drug delivery system, including bacteria, and the chemotherapy drug / photothermal agent complex carried on the bacteria, the bacteria is the composition of one or several of obligate anaerobe and facultative anaerobe, the chemotherapy drug includes cis-aconitate anhydride-prodrug with terminal thiol;A kind of bacterial-mediated drug delivery system is prepared by cis-aconitate anhydride, prodrug, 3-mercapto propionic acid methylamide, photothermal agent and bacteria;A kind of bacterial-mediated drug delivery system is applied in the drug, food or health care product in the prevention and treatment of tumor, intestinal inflammation, obesity.The bacterial-mediated drug delivery system of the present application is combined with the chemotherapy drug / photothermal agent complex by specific binding bacteria, so as to realize the photothermal-chemotherapy combined treatment effect of tumor, and promote the deep delivery of chemotherapy drug, realize better treatment effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a bacterial-mediated drug delivery system, its preparation method, and its application. Background Technology

[0002] Cancer remains a major threat to human health. Most cancer patients have already developed metastases at initial diagnosis. Chemotherapy, a systemic drug treatment, is unaffected by tumor metastasis and is an important adjuvant therapy for patients with metastatic cancer who cannot be treated locally with surgery or radiotherapy. However, chemotherapy drugs lack specificity for tumor tissue and have significant toxic side effects. With the development of nanotechnology, nanomedicine carriers for drug delivery have been widely used because they alter drug distribution in vivo and reduce systemic toxicity. However, due to the high interstitial fluid pressure of tumors and the hypoxia in deep tumor sites caused by the high proliferation of tumor cells, drug-loaded nanocarrier systems have difficulty penetrating deep hypoxic areas of the tumor, resulting in low therapeutic efficiency in deep sites. In recent years, bacterial-mediated microbial therapy has shown great application potential. Some bacteria can target solid tumors through both passive and active mechanisms. For example, obligate anaerobes (such as Clostridium and Bifidobacterium) and facultative anaerobes (such as Salmonella, Lactobacillus, Listeria, Escherichia coli, and Pseudomonas) can establish corresponding microbial communities in the hypoxic areas of tumors and colonize under harsh conditions, multiplying up to 1000 times more than in normal tissues. Furthermore, they also exhibit antitumor and anti-inflammatory activities by secreting exotoxins and enzymes. Therefore, combining bacterial therapy with traditional chemotherapy holds promise for enhancing the targeting of chemotherapy drugs, improving their therapeutic efficiency, and reducing their toxic side effects.

[0003] With advancements in science and technology, new drugs and technologies such as immunotherapy, gene therapy, photodynamic therapy, and photothermal therapy, when combined with chemotherapy, have demonstrated superior therapeutic effects compared to chemotherapy alone. Photothermal therapy involves injecting materials with high photothermal conversion efficiency into the body and using targeted recognition technology to concentrate them near the tumor tissue. Under irradiation by external light sources such as near-infrared rays, the photothermal agent absorbs light of specific wavelengths, converting light energy into heat energy, thus raising the temperature of the lesion tissue and causing cancer cell necrosis. This treatment method has advantages such as minimal side effects, high specificity, and repeatability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first objective of this invention is to provide a bacterial-mediated drug delivery system that achieves combined photothermal-chemotherapy of tumors by specifically binding bacteria and chemotherapeutic drug / photothermal agent complexes, and promotes deeper delivery of chemotherapeutic drugs for better therapeutic effects.

[0005] The second objective of this invention is to provide a method for preparing a bacterial-mediated drug delivery system, which has the advantages of simple processing, high yield, and suitability for industrial application.

[0006] The third objective of this invention is to provide an application of a bacterial-mediated drug delivery system, which further expands the application of the aforementioned system in multiple fields such as pharmaceuticals, food, and health products.

[0007] To achieve the first objective mentioned above, the present invention provides the following technical solution:

[0008] A bacterial-mediated drug delivery system includes bacteria and a chemotherapeutic drug / photothermal agent complex mounted on the bacteria, wherein the bacteria are one or more of obligate anaerobes and facultative anaerobes, and the chemotherapeutic drug includes a cis-aconitine prodrug having a terminal thiol group.

[0009] Furthermore, the bacteria are one or a combination of several of the following: Helicobacter pylori, Campylobacter, Mycobacterium, Salmonella, Lactobacillus acidophilus, Lactobacillus casei, Clostridium, Listeria, Stylobacterium, Clostridium butyricum, Pseudomonas, Streptococcus, Bifidobacterium, magnetotactic bacteria, and Escherichia coli.

[0010] Furthermore, the *Escherichia coli* is one or a combination of several of *Escherichia coli* Nissle1917, *Escherichia coli* DH5α, and *Escherichia coli* TOP10.

[0011] Furthermore, the prodrugs in the chemotherapeutic agents include one or more of doxorubicin, epirubicin, furazolidone, daunorubicin, amrubicin, their derivatives, and pharmaceutically acceptable salts.

[0012] Furthermore, the photothermal agent includes one or more of the following: organic cyanine dyes, porphyrins, organic nanomaterials, and inorganic nanomaterials.

[0013] Furthermore, the organic cyanine dye includes one or a combination of several of indocyanine green, carbocyanine dyes, neo-indocyanine green IR-820, and near-infrared indocyanine dye IR-808.

[0014] Furthermore, the organic nanomaterials include one or more of polydopamine nanomaterials and semiconductor nanomaterials. The semiconductor nanomaterials are nanomaterials made from semiconductor materials such as silicon and gallium arsenide.

[0015] Furthermore, the inorganic nanomaterials include one or more of the following: metal nanomaterials, metal sulfide nanomaterials, metal oxide nanomaterials, carbon-based nanomaterials, and quantum dots. The metals mentioned above are not limited to, for example, gold and platinum.

[0016] Furthermore, the nanomaterials in the photothermal agent are in the form of at least one of nanospheres, nanoshells, nanorods, nanocages, and nanostars.

[0017] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0018] A method for preparing a bacteria-mediated drug delivery system includes the following steps:

[0019] S1 cis aconitine and the prodrug were reacted in a solvent under the action of triethylamine and nitrogen protection. After the reaction was completed, post-treatment was performed to obtain cis aconitine-prodrug.

[0020] S2 The cis-aconitine-prodrug and 3-mercaptopropionic acid formamide obtained in S1 are reacted in a solvent under the action of glacial acetic acid and under nitrogen protection. After the reaction is completed, post-treatment is performed to obtain cis-aconitine-prodrug-terminal thiol group.

[0021] In S3, the cis-aconitine-prodrug-terminal thiol group and photothermal agent obtained in S2 are subjected to a light-protected reaction in a solvent. After the reaction is completed, post-treatment is performed to obtain a chemotherapy drug / photothermal agent complex.

[0022] The chemotherapy drug / photothermal agent complex obtained in S3 and bacteria are reacted in a solvent under the action of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in the dark. After the reaction is completed, post-processing is performed to obtain a bacterial-mediated drug delivery system.

[0023] Further, in S1, the prodrug is doxorubicin hydrochloride, the solvent is anhydrous N,N'-dimethylformamide; the molar ratio of cis-aconitine anhydride to the prodrug is controlled at (0.05~10.00):1, the molar ratio of cis-aconitine anhydride to triethylamine is 1:(1~2), and the reaction time is 20~30h.

[0024] Further, in S2, the solvent is methanol, and the purity of glacial acetic acid is ≥99.5%; the molar ratio of cis-aconitine-prodrug and 3-mercaptopropionic acid formamide is controlled at 1:(0.1~10.0), and the reaction time is 45~55h.

[0025] Further, in S3, the solvent is water, the photothermal agent is gold nanorods, and the molar ratio of cis-aconitine-prodrug-terminal thiol group and photothermal agent is controlled to be (0.1~50.0):1, and the reaction time is 20~30h.

[0026] Further, in S4, the solvent is water; the molar ratio of the chemotherapeutic drug / photothermal agent complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is controlled to be 1:(15~25):(15~25), and per 1×10 9 In the CFU-containing bacterial chemotherapeutic drug / photothermal agent complex, the prodrug content is ≥1 μg and the photothermal agent content is ≥0.1 μg. Preferably, per 1×10 9 In the CFU-containing bacterial chemotherapeutic drug / photothermal agent complex, the prodrug content is 1~250μg and the photothermal agent content is 0.1~100μg.

[0027] To achieve the third objective mentioned above, the present invention provides the following technical solution:

[0028] Application of a bacterial-mediated drug delivery system in drugs, foods, or health products for the prevention and treatment of tumors, intestinal inflammation, and obesity.

[0029] In summary, the beneficial technical effects of the present invention are as follows:

[0030] 1. The bacterial-mediated drug delivery system of the present invention can target the tumor microenvironment through the self-driving force and facultative / obligatory anaerobic properties of bacteria, and release drugs in the acidic environment of the tumor. At the same time, by applying near-infrared laser radiation near the tumor area, photothermal ablation of the tumor area can be achieved, thereby realizing the combined photothermal-chemotherapy effect of the tumor and promoting the deep delivery of chemotherapy drugs to achieve better therapeutic effect.

[0031] 2. The bacterial-mediated drug delivery system of the present invention uses a gentle method to bind both the drug and the photothermal agent to the bacteria without affecting the bacterial activity. In addition, the drug-loaded probiotics prepared by the present invention can respond to a weakly acidic environment and release the drug in real time by breaking bonds, thus exhibiting a good photothermal-chemotherapy synergistic therapeutic effect.

[0032] 3. The preparation method of the present invention first connects two acid-sensitive bonds to both ends of the drug. One end can be connected to the bacterial surface, and the other end can be connected to a coordination composite photothermal agent. The resulting drug delivery system can utilize the hypoxia and chemotaxis of bacteria to target and deliver the drug to the tumor or inflammatory site. Under the weakly acidic conditions of the tumor, the bond is broken and the drug is released. In addition, by applying laser stimulation to the tumor site, the loaded photothermal agent can convert the absorbed light energy into heat energy, generate photothermal ablation of the tumor tissue, and increase the permeability of the tumor tissue, promote the drug penetration into deep tissues, and realize the combined therapeutic effect of photothermal-chemotherapy and microbial immunomodulation of tumors, so as to achieve a better anti-tumor effect.

[0033] 4. The bacterial-mediated drug delivery system of the present invention can be used to prepare pharmaceutical compositions, food and health products for functional body regulation, for the diagnosis or treatment of tumors, intestinal inflammation and obesity, and for in vitro detection not for the purpose of disease diagnosis. Attached Figure Description

[0034] Figure 1 The image shows the 1H-NMR spectrum of cis-aconitine-prodrug-terminal thiol group in Example 3 of this invention.

[0035] Figure 2 The image shows the ultraviolet-visible spectrophotometer of the chemotherapy drug / photothermal agent complex in Example 3 of this invention.

[0036] Figure 3 Different concentrations of chemotherapy drug / photothermal agent complex and 2×10 in Example 3 of this invention 9 The amount of drug adsorption by CFU-E. coli at different times;

[0037] Figure 4 This is a laser confocal microscope image of the bacterial-mediated drug delivery system of Embodiment 3 of the present invention;

[0038] Figure 5 This is a diagram showing the cumulative in vitro drug release of the bacterial-mediated drug delivery system of Example 4 of the present invention;

[0039] Figure 6 A graph showing the survival rate of HUVEC cells using the bacterial-mediated drug delivery system of Embodiment 4 of the present invention;

[0040] Figure 7 The image shows the photothermal performance of EcN in Embodiment 4 of the present invention;

[0041] Figure 8 The graph shows the changes in tumor-bearing mice treated with photothermal-chemotherapy using the bacterial-mediated drug delivery system of Example 4 of the present invention. Implementation

[0042] To make the technical means, creative features, objectives and effects of this invention clearer and easier to understand, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0043] Example 1: A bacterial-mediated drug delivery system disclosed in this invention includes bacteria and a chemotherapeutic drug / photothermal agent complex mounted on the bacteria. The bacteria are one or more obligate anaerobes and facultative anaerobes, and the chemotherapeutic drug includes acitretin prodrugs with terminal thiol groups.

[0044] Specifically, the bacteria are one or a combination of several selected from Helicobacter pylori, Campylobacter, Mycobacterium, Salmonella, Lactobacillus acidophilus, Lactobacillus casei, Clostridium, Listeria, Stylobacterium, Clostridium butyricum, Pseudomonas, Streptococcus, Bifidobacterium, magnetotactic bacteria, and Escherichia coli. Among them, the Escherichia coli is one or a combination of several selected from Escherichia coli Nissle 1917, Escherichia coli DH5α, and Escherichia coli TOP10. In this embodiment, Escherichia coli Nissle 1917 is preferred.

[0045] The prodrugs in chemotherapy drugs include one or more of doxorubicin, epirubicin, furazolidone, daunorubicin, amrubicin, their derivatives, and pharmaceutically acceptable salts. In this embodiment, doxorubicin or doxorubicin hydrochloride is preferred.

[0046] The photothermal agent comprises one or more of the following: organic cyanine dyes, porphyrins, organic nanomaterials, and inorganic nanomaterials. The organic cyanine dyes include one or more of the following: indocyanine green, carbocyanine dyes, neo-indocyanine green IR-820, and near-infrared indocyanine dye IR-808. The organic nanomaterials include one or more of the following: polydopamine nanomaterials and semiconductor nanomaterials. The semiconductor nanomaterials are nanomaterials made from semiconductor materials such as silicon and gallium arsenide. The organic nanomaterials include one or more of the following: metal nanomaterials, metal sulfide nanomaterials, metal oxide nanomaterials, carbon-based nanomaterials, and quantum dots. The metals mentioned above are not limited to, for example, gold and platinum. Furthermore, the nanomaterials in the photothermal agent are in the form of at least one of the following: nanospheres, nanoshells, nanorods, nanocages, and nanostars. In this embodiment, gold nanorods are preferred.

[0047] Example 2: A method for preparing a bacterial-mediated drug delivery system disclosed in this invention, which differs from Example 1 in that it includes the following steps:

[0048] S1 cis aconitine and the prodrug were reacted in a solvent under the action of triethylamine and nitrogen protection. After the reaction was completed, post-treatment was performed to obtain cis aconitine-prodrug.

[0049] The prodrug is doxorubicin hydrochloride, and the solvent is anhydrous N,N'-dimethylformamide. The molar ratio of cis-aconitine anhydride to the prodrug is controlled at (0.05~10.00):1, preferably (0.05, 0.01, 0.50, 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00 or 10.00):1. The molar ratio of cis-aconitine anhydride to triethylamine is 1:(1~2). The reaction time is 20~30h, preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30h.

[0050] S2 The cis-aconitine-prodrug and 3-mercaptopropionic acid formamide obtained in S1 are reacted in a solvent under the action of glacial acetic acid and under nitrogen protection. After the reaction is completed, post-treatment is performed to obtain cis-aconitine-prodrug-terminal thiol group.

[0051] The solvent is methanol, and the purity of glacial acetic acid is ≥99.5%. The molar ratio of cis-aconitine anhydride-prodrug and 3-mercaptopropionic acid formamide is controlled at 1:(0.1~10.0), preferably 1:(0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0), and the reaction time is 45~55h, preferably 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55h.

[0052] In S3, the cis-aconitine-prodrug-terminal thiol group and photothermal agent obtained in S2 are subjected to a light-protected reaction in a solvent. After the reaction is completed, post-treatment is performed to obtain a chemotherapy drug / photothermal agent complex.

[0053] The solvent is water, and the photothermal agent is gold nanorods. The molar ratio of cis-aconitine anhydride-prodrug-terminal thiol group to photothermal agent is controlled to be (0.1~50.0):1, preferably (0.1, 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0 or 50.0):1, and the reaction time is 20~30h, preferably 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30h.

[0054] S4 The chemotherapy drug / photothermal agent complex obtained in S3 and bacteria are reacted in a solvent in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in the dark. After the reaction is completed, post-processing is performed to obtain a bacterial-mediated drug delivery system.

[0055] The solvent is water; the molar ratio of the chemotherapy drug / photothermal agent complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is controlled at 1:(15~25):(15~25), and per 1×10 9 In the CFU-containing bacterial chemotherapeutic drug / photothermal agent complex, the prodrug content is 1~250μg and the photothermal agent content is 0.1~100μg.

[0056] Example 3: A method for preparing a bacterial-mediated drug delivery system disclosed in this invention, which differs from Example 2 in that it specifically includes the following steps:

[0057] Preparation of S1 cis-aconitine prodrug

[0058] Doxorubicin hydrochloride was ultrasonically dissolved in an appropriate amount of anhydrous N,N'-dimethylformamide (DMF), and then cis-aconitine anhydride (CA) and triethylamine (TEA) were added. The reaction was carried out under nitrogen protection for 24 h. After the reaction was completed, the solution was first added to an appropriate amount of ethyl acetate and then continuously extracted and washed with saturated sodium chloride solutions at pH=3.0 and pH=7.4, respectively. The organic phase was collected, and then the water in the organic phase was removed with anhydrous sodium sulfate. The dehydrated organic phase was then concentrated by rotary evaporation and vacuum dried to constant weight at room temperature to obtain a red powder sample (CA-Dox).

[0059]

[0060] Preparation of S2 cis-aconitine-prodrug-terminal thiol group

[0061] CA-Dox was dissolved in methanol, and an appropriate amount of 99.5% glacial acetic acid was added. A methanol solution of 3-mercaptopropionic acid formamide was then added dropwise to the solution. The reaction was carried out at room temperature for 48 hours under nitrogen protection. After the reaction, the mixture was precipitated with glacial ether and washed three times by centrifugation. It was then vacuum dried for 24 hours to constant weight to obtain a red solid, cis-aconitine anhydride-prodrug-terminal thiol group (CA-Dox-Hyd-SH). The spectral diagram is shown below. Figure 1 ;

[0062]

[0063] Preparation of S3 chemotherapy drug / photothermal agent complex

[0064] S31. Accurately weigh an appropriate amount of hexadecyltrimethylammonium bromide (CTAB), heat and dissolve it to prepare an aqueous solution with a concentration of 0.2 mol / L. Take 5 mL of CTAB aqueous solution in a vial, add 5 mL of 0.0005 mol / L chloroauric acid aqueous solution, stir evenly, then add 0.6 mL of 0.01 mol / L freshly prepared sodium borohydride ice solution, stir vigorously for 3 min to generate a brownish-yellow gold seed solution. Store the gold seed solution at 28℃ for 2 h before use.

[0065] In a 28℃ water bath, 5 mL of 0.02 mol / L CTAB aqueous solution was added to a 20 mL vial, along with 0.25 mL of 0.004 mol / L nitrate aqueous solution and 5 mL of 0.001 mol / L chloroauric acid aqueous solution. After mixing thoroughly, 70 μL of freshly prepared 0.0788 mol / L ascorbic acid ice solution was added and stirred until the solution changed from yellow to colorless, thus preparing the gold nanorod growth solution.

[0066] S33 was incubated in a 28℃ water bath with 12μL of gold seed solution added to 10mL of the prepared gold nanorod growth solution for 12h to obtain a purple-red gold nanorod solution. The solution was centrifuged three times with pure water at room temperature (25℃, 9000rpm, 10min) to remove the template molecule CTAB, thus obtaining the gold nanorod solution (AuNRs).

[0067] S34 was prepared by adding an appropriate amount of deionized water to the CA-Dox-Hyd-SH obtained in S2 and dispersing it into a suspension using a cell disruptor. AuNRs were then added, and the mixture was stirred in the dark for 24 hours to obtain the chemotherapeutic drug / photothermal agent complex (CA-Dox-Hyd-SH / AuNRs complex).

[0068] The UV-Vis spectra of CA-Dox-Hyd-SH prepared in this embodiment before and after the reaction with gold nanorods are as follows: Figure 2 As shown, compared with gold nanorods (829 nm), the local surface plasmon resonance peak of the CA-Dox-Hyd-SH / AuNRs complex showed a red shift, while the maximum absorption wavelength of the solution reacting doxorubicin without thiol modification with gold nanorods did not show a red shift. This is because the outer medium of the gold nanorods changed, and the change in dielectric constant led to the red shift of the absorption peak, proving the successful preparation of the CA-Dox-Hyd-SH / AuNRs complex.

[0069] Preparation of S4 bacteria-mediated drug delivery system

[0070] S41. An appropriate amount of Escherichia coli Nissle1917 bacterial suspension (EcN) was inoculated into lysate broth liquid medium and activated by constant temperature shaking at 37℃ and 200rpm overnight.

[0071] S42 collected 1 mL of viable E. coli and used an amide condensation reaction activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to bind CA-Dox-Hyd-SH / AuNPs to EcN, as detailed below:

[0072] Weigh out the CA-Dox-Hyd-SH / AuNPs complex, EDC, and NHS in a molar ratio of 1:20:20, dissolve them in sterile water to a final volume of 2 mL, then add 1 mL of EcN suspension. Incubate in a 37°C, 200 rpm shaking incubator in the dark for the reaction. After the reaction is complete, centrifuge at 4500 rpm for 5 min to remove the unreacted supernatant. Wash the supernatant 2-3 times with sterile water and resuspend to a final volume of 1 mL to obtain the bacterial-mediated drug delivery system (EcN-Dox-Au).

[0073] like Figure 3As shown, the adsorption rate decreases with increasing drug complex concentration. When reacted with 2 mL of 250 μg / mL CA-Dox-Hyd-SH / AuNPs for 4 h, the adsorption rate decreased by 2 × 10⁻⁶ μg / mL. 9 CFU's EcN can load 135.4 μg of chemotherapy drug / photothermal agent complex (27.09% of the total drug dosage). Bright red fluorescence was also observed in the EcN loaded with the chemotherapy drug / photothermal agent complex under laser confocal microscopy. Figure 4 This indicates that CA-Dox-Hyd-SH / AuNPs have been successfully loaded onto the EcN surface.

[0074] Example 4: This example demonstrates the application of a bacterial-mediated drug delivery system disclosed in this invention in drugs, foods, or health products for the prevention and treatment of tumors, enteritis, and obesity. The photothermal properties, drug release behavior, in vitro cytotoxicity, and photothermal-chemotherapy synergistic effect in tumor-bearing mice were further investigated using the bacterial-mediated drug delivery system (EcN-Dox-Au) prepared in Example 3.

[0075] (1) Investigate the photothermal properties of EcN-Dox-Au

[0076] 1 mL of drug-loaded E. coli suspensions with different gold rod concentrations were placed in centrifuge tubes and analyzed using NIR (808 nm, 2 w / cm²). 2 The drug-loaded E. coli suspension was irradiated for 10 min. The temperature of the solution was measured every 2 min using a thermal imager. Under a certain laser power irradiation, the heating rate of EcN-Dox-Au was positively correlated with the increase of the concentration of gold nanorods loaded with it. Figure 5 This indicates that EcN-Dox-Au has good photothermal conversion ability under near-infrared light irradiation.

[0077] (2) Investigate the drug release behavior of EcN-Dox-Au

[0078] To investigate the in vitro release of drug-loaded EcN, Dox release experiments were conducted under different release conditions at 37°C or 45°C using PBS buffer at pH 7.4, pH 6.5, and pH 5.0. The release solution was collected at specified time intervals, and then an equal volume of release medium was added for further incubation. The cumulative in vitro drug release curves of EcN-Dox-Au are shown below. Figure 6 As shown, the release of Dox from EcN-Dox-Au is pH-dependent and temperature-sensitive. The drug release rate is slightly higher in the slightly acidic tumor microenvironment (pH 6.8) than in the physiological environment, while the release is even faster in the tumor lysosomal microenvironment (pH 5.0). Furthermore, the release of Dox from EcN-Dox-Au is temperature-dependent, with a significantly higher release rate at 45°C than at 37°C.

[0079] (3) Investigate the in vitro cytotoxicity of EcN

[0080] The cytotoxicity of EcN to HUVEC cells was evaluated using the MTT assay. HUVECs were cultured to the logarithmic growth phase and then seeded into 96-well plates, incubated for 24 h in a CO2 incubator. After good cell growth, different concentrations of EcN in culture medium suspension were added and co-cultured for 24 h. Then, 10 μL of MTT solution (5 μg / mL) was added to each well, and the cells were incubated for 4 h. The old culture medium was then removed, and 150 μL of LDMSO was added to dissolve formazan. The absorbance at 570 nm was measured using a microplate reader, and cell viability was calculated. The viability results of different concentrations of EcN on HUVEC cells are shown below. Figure 7 Blank bacterial vectors at 2×10 7 At CFU levels below a certain threshold, EcN exhibits low toxicity to HUVECs, with cell survival rates exceeding 80%, indicating that EcN is a drug carrier with good biocompatibility.

[0081] (4) To investigate the photothermal-chemotherapy synergistic effect of EcN-Dox-Au in tumor-bearing mice.

[0082] MCF-7 breast cancer cells were subcutaneously injected into Balb / cnude mice, and tumors were allowed to grow to approximately 100 mm. 3 The following experiment was conducted. Mice were randomly divided into two groups: a saline group and an EcN-Dox-Au + laser treatment group. The saline group received 200 μL of sterile saline per injection. The laser treatment group received 0.2 mL of EcN-Dox-Au via tail vein injection 24 hours later, followed by NIR laser treatment (808 nm, 2 W / cm²). 2 Irradiate for 2 minutes. Administer the medication every 3 days for a 21-day cycle, while simultaneously measuring changes in tumor size using calipers. Figure 8 The study found that the tumors in mice treated with the drug-loaded probiotics and laser therapy were completely eliminated after treatment, and there was no recurrence after 53 days.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a bacterial-mediated drug delivery system, characterized by: The drug delivery system comprises bacteria and a chemotherapeutic drug / photothermal agent complex carried on the bacteria, the bacteria being one or a combination of several of obligate anaerobes and facultative anaerobes, and the chemotherapeutic drug comprising cis-aconitate anhydride- prodrug with a terminal thiol group. The preparation method comprises the following steps, S1. Cis-aconitate anhydride and prodrug are reacted in a solvent under the action of triethylamine and protection of nitrogen, after reaction, post-treatment is performed to obtain cis-aconitate anhydride-prodrug; S2. Cis-aconitate anhydride-prodrug obtained in S1 and 3-mercapto propionic acid methylamide are reacted in a solvent under the action of glacial acetic acid and protection of nitrogen, after reaction, post-treatment is performed to obtain cis-aconitate anhydride-prodrug-terminal thiol group; S3. Cis-aconitate anhydride-prodrug-terminal thiol group obtained in S2 and a photothermal agent are reacted in a solvent to perform a light-avoiding reaction, after reaction, post-treatment is performed to obtain a chemotherapeutic drug / photothermal agent complex; S4. The chemotherapeutic drug / photothermal agent complex obtained in S3 and bacteria are reacted in a solvent under the action of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide to perform a light-avoiding reaction, after reaction, post-treatment is performed to obtain a bacteria-mediated drug delivery system.

2. The method of claim 1, wherein the bacterial-mediated drug delivery system is prepared by the steps of: a) providing a bacterial cell; b) introducing a nucleic acid construct into the bacterial cell; c) culturing the bacterial cell; and d) isolating the bacterial cell. The bacteria are one or a combination of several of Helicobacter, Campylobacter, Mycobacterium, Salmonella, Lactobacillus acidophilus, Lactobacillus casei, Clostridium, Listeria, Bacillus, Clostridium butyricum, Pseudomonas, Streptococcus, Bifidobacterium, Magnetotactic bacteria and Escherichia coli.

3. The method for preparing a bacterial-mediated drug delivery system according to claim 1, characterized in that: The prodrug in the chemotherapeutic drug comprises one or a combination of several of doxorubicin, epirubicin, furan doxorubicin, daunorubicin, amonofide, derivatives and pharmaceutically acceptable salts thereof.

4. The method for preparing a bacterial-mediated drug delivery system according to claim 1, characterized in that: The photothermal agent comprises one or a combination of several of organic cyanine dye, porphyrin, organic nanomaterial and inorganic nanomaterial.

5. The method for preparing a bacterial-mediated drug delivery system according to claim 1, characterized in that: In S1, the prodrug is doxorubicin hydrochloride, the solvent is anhydrous N, N'-dimethylformamide, the molar ratio of cis-aconitate anhydride to prodrug is controlled to be (0.05-10.00):1, the molar ratio of cis-aconitate anhydride to triethylamine is controlled to be 1:(1-2), and the reaction time is 20-30 h.

6. The method for preparing a bacterial-mediated drug delivery system according to claim 1, characterized in that: In S2, the solvent is methanol, the purity of glacial acetic acid is ≥99.5%, and the molar ratio of cis-aconitate anhydride-prodrug to 3-mercapto propionic acid methylamide is controlled to be 1:(0.1-10.0), and the reaction time is 45-55 h.

7. The method for preparing a bacterial-mediated drug delivery system according to claim 1, characterized in that: In S3, the solvent is water, the photothermal agent is gold nanorod, the molar ratio of cis-aconitate anhydride-prodrug-terminal thiol group to photothermal agent is controlled to be (0.1-50.0):1, and the reaction time is 20-30 h.

8. The method for preparing a bacterial-mediated drug delivery system according to claim 1, characterized in that: In the S4, the solvent is water; the molar ratio of the chemotherapeutic drug / photothermal agent complex, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is 1: (15-25): (15-25), and the amount of the chemotherapeutic drug / photothermal agent complex is 1×10 9 The content of the prodrug in the chemotherapeutic drug / photothermal agent complex corresponding to the CFU of bacteria is ≥1 μg, and the content of the photothermal agent is ≥0.1 μg.

9. Use of a bacterial-mediated drug delivery system, characterized in that: The drug delivery system prepared by the preparation method according to any one of claims 1-8 is used in the preparation of a drug for preventing and treating tumors, intestinal inflammation and obesity.

Images