Viable-bacterium-delivered copper-irinotecan metal nano-drug with anti-tumor effect and preparation method and application of viable-bacterium-delivered copper-irinotecan metal nano-drug

Abstract

The invention relates to a viable-bacterium-delivered copper-irinotecan metal nano-drug with an anti-tumor effect and a preparation method and application of the viable-bacterium-delivered copper-irinotecan metal nano-drug. The nano drug comprises a copper-irinotecan nano coordination polymer, and lactobacillus rhamnosus coated by dopamine is physically adsorbed or chemically crosslinked on the copper-irinotecan nano coordination polymer. Compared with the prior art, the LGG-PDA-coated Cu-CPT nano-drug provided by the invention has the advantages that by integrating antibacterial treatment, photothermal therapy and STING pathway activation, the defects of the prior art in the aspects of microbiota removal, tumor metastasis inhibition and immune activation are overcome, and multi-mechanism synergy of antibacterial, anti-tumor and immune activation is realized; the multifunctional strategy provides an innovative solution for breast cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a live bacteria-delivered copper-irinotecan metal nanomedicine with anti-tumor effects, a preparation method thereof, and an application thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women globally, with over 2 million new cases and over 600,000 death cases each year, seriously threatening women's health. Despite the continuous progress of treatment methods, the treatment of breast cancer still faces many challenges, including endocrine therapy resistance, recurrence of targeted therapy, and limited chemotherapy efficacy. In recent years, studies have shown that the intratumoral microbiota plays an important role in tumor progression. The presence of bacteria in tumors can promote tumor cell proliferation and survival or accelerate tumor invasion and metastasis. In addition, intratumoral bacteria also inhibit the activity of CD4+ and CD8+ T cells, weaken the anti-tumor immune response, and reduce the efficacy of immunotherapy. The STING (Stimulator of Interferon Genes) pathway is a core regulator of innate immunity, and its activation can induce the production of type I interferons, promote the maturation of dendritic cells (DCs) and the activation of T cells, thereby enhancing the anti-tumor immune response. However, the presence of bacteria in tumors limits the activation of the STING pathway. Photothermal therapy (PTT), as a local treatment method, can destroy the bacterial membrane by generating local high temperatures, accelerate the entry of copper ions into bacteria to exert antibacterial effects, and simultaneously achieve tumor ablation.

[0003] Therefore, combining antibacterial therapy, photothermal therapy, and STING pathway activation is expected to inhibit tumor metastasis by eliminating the microbial barrier and enhancing the function of immune cells.

[0004] CN114617975B discloses the application of copper ion-tannic acid co-assembled antibacterial nanosheets as a carrier for anti-tumor drugs. The antibacterial nanosheets are ultrasonically dispersed in deionized water, and an anti-tumor drug is added. Under dark conditions, the mixture is stirred thoroughly to load the anti-tumor drug into the antibacterial nanosheets. After centrifugation and drying, antibacterial anti-tumor nano-drugs are obtained. The antibacterial nanosheets have good antibacterial properties and biocompatibility, can efficiently load anti-tumor drugs through electrostatic interaction and complexation, and the obtained nano-drugs have a stable structure in the normal physiological environment, and can efficiently release anti-tumor drugs and copper ions in the tumor microacidic and high glutathione environment, exerting a synergistic anti-tumor effect and excellent antibacterial properties, while effectively avoiding the risk of side effects caused by the disordered leakage of metal ions in traditional metal particle antibacterial materials, and are expected to solve the problem that patients are vulnerable to bacterial infections during tumor treatment and improve the therapeutic effect of chemotherapy drugs. This patent emphasizes the dual antibacterial and anti-tumor effects, but does not involve the probiotic delivery system.

[0005] CN112791106B discloses a pharmaceutical composition, which comprises an immune signaling pathway regulator and a certain amount of Lactobacillus rhamnosus, and the amount of Lactobacillus rhamnosus is sufficient to enhance the therapeutic response of a subject to the immune signaling pathway regulator. The pharmaceutical composition may also contain irinotecan, which can improve the response rate of tumor patients receiving PD-1 treatment, inhibit the development of tumors and prolong the survival period; the oral treatment method is simple; the Lactobacillus rhamnosus is acid and alkali tolerant and can pass through the gastrointestinal environment and colonize stably. This scheme mainly enhances the efficacy of immune checkpoint inhibitors (such as PD-1 antibodies) by orally administering Lactobacillus rhamnosus, and does not involve issues such as regulating the tumor acidic environment and enhancing chemotherapy sensitivity. Summary of the Invention

[0006] Based on the problem that the treatment effect of breast cancer remains to be improved in the prior art, the present invention provides a live bacteria-delivered copper-irinotecan metal nano-drug with anti-tumor effect, its preparation method and application.

[0007] The drug provided by the present invention is abbreviated as LGG-PDA@Cu-CPT. This drug is formed by a stable nano-coordination polymer (NCP) through the coordination of copper ions (Cu2+) and the chemotherapy drug irinotecan (CPT), and combines Lactobacillus rhamnosus (LGG) coated with dopamine (PDA) as a delivery carrier to achieve precise targeted delivery of the drug.

[0008] Cu2+ has powerful antibacterial and anticancer effects. It can kill bacteria in tumors by disrupting bacterial membranes, generating reactive oxygen species (ROS), and damaging DNA. At the same time, it activates the STING pathway and enhances the immune response. The polyhydroxy and azacyclic structures of irinotecan can form stable interactions with copper ions, achieving synergistic anti-tumor and antibacterial effects. As an anaerobic bacterium, Lactobacillus rhamnosus GG (LGG) can specifically colonize the hypoxic regions of tumors, realizing precise drug delivery. At the same time, it induces the production of type I interferon-β (IFN-β) through the cGAS / STING / TBK1 / IRF7 signaling pathway, enhancing the anti-tumor immune response.

[0009] The LGG-PDA@Cu-CPT nano-drug provided by the present invention solves the deficiencies of the prior art in the clearance of intratumoral microbiota, tumor metastasis inhibition, and immune activation by integrating antibacterial therapy, photothermal therapy, and STING pathway activation, achieving multi-mechanism synergy of antibacterial, anti-tumor, and immune activation. This multifunctional strategy provides an innovative solution for breast cancer treatment.

[0010] The object of the present invention can be achieved by the following technical solutions:

[0011] The present invention first provides a live bacteria-delivered copper-irinotecan metal nano-drug with anti-tumor effects, abbreviated as LGG-PDA@Cu-CPT, which includes a copper-irinotecan nano-coordination polymer, on which Lactobacillus rhamnosus GG (LGG) coated with dopamine (PDA) is physically adsorbed or chemically cross-linked.

[0012] In the LGG-PDA@Cu-CPT provided by the present invention, the functions of each component are described as follows:

[0013] Copper-irinotecan nano-coordination polymer (Cu-CPT NPs): A stable nano-coordination polymer is formed through the coordination of copper ions (Cu2+) with the chemotherapeutic drug irinotecan (CPT). Cu2+ has broad-spectrum antibacterial and anticancer effects, and the polyhydroxy and azacyclic structures of CPT can form stable interactions with copper ions, achieving synergistic anti-tumor and antibacterial effects.

[0014] Lactobacillus rhamnosus GG (LGG) coated with dopamine (PDA): Utilizing the anaerobic characteristics of LGG, it can specifically colonize the hypoxic regions of tumors, realizing precise drug delivery. The PDA coating not only improves the stability of the nano-drug but also endows it with photothermal therapy function.

[0015] The present invention further provides a preparation method of a live bacteria-delivered copper-irinotecan metal nano-drug with anti-tumor effects, including the following steps:

[0016] Preparation of copper-irinotecan nano-coordination polymer (Cu-CPT NPs): A coordination reaction is carried out between copper ions and irinotecan to form a stable copper-irinotecan nano-coordination polymer;

[0017] Preparation of LGG-PDA@Cu-CPT: Cu-CPT NPs are combined with Lactobacillus rhamnosus coated with dopamine, and the final nano-drug is formed through physical adsorption or chemical cross-linking.

[0018] In one embodiment of the present invention, the preparation method of the copper-irinotecan nano-coordination polymer (Cu-CPT NPs) includes the following steps:

[0019] The first step: By adding a pH regulator and a suspending agent in a solvent in the presence of irinotecan (CPT) and stirring, the lactone bond and carboxyl bond of irinotecan are broken;

[0020] The second step: By adding a CuCl2 compound in the presence of a solvent and stirring together with irinotecan under anaerobic conditions, a Cu-CPT compound is prepared.

[0021] In one embodiment of the present invention, more specifically, in the first step, the pH can be adjusted with triethylamine, the pH of the system is adjusted to 7.4, the suspending agent added is PVP, and stirring is carried out for 1 h.

[0022] In one embodiment of the present invention, more specifically, the solvent for the above reaction can be any solvent that has no adverse effect on the reaction, and methanol and N,N-dimethylformamide are preferably used.

[0023] In one embodiment of the present invention, more specifically, the molar ratio of Cu to irinotecan is 4:1.

[0024] In one embodiment of the present invention, the preparation method of the copper-irinotecan nano-coordination polymer (Cu-CPT NPs) includes the following steps:

[0025] S1. Under anaerobic stirring conditions, the drug irinotecan is added to a methanol solution to form a mixed solution, and the lactone bond, hydroxyl bond, etc. of irinotecan are broken by stirring in a fixed pH system to facilitate coordination and binding with copper ions;

[0026] S2. After stirring for 1 h, the copper chloride solution is slowly dropped into the irinotecan system, stirred under anaerobic conditions for 5 h, and the reaction product is centrifuged to obtain the product Cu-CPT;

[0027] S3. The product Cu-CPT is redispersed, centrifuged and washed several times and then redispersed by ultrasonic treatment with ultrapure water to obtain copper-irinotecan nanoparticles, that is, copper-irinotecan nano-coordination polymer.

[0028] In one embodiment of the present invention, further, the irinotecan solution in step S1 is prepared by dissolving irinotecan hydrochloride trihydrate in a methanol organic solvent, and adding triethylamine dropwise to adjust the pH of the system to 7.4.

[0029] In one embodiment of the present invention, further, in step S1, a suspending agent PVP is also added.

[0030] In one embodiment of the present invention, further, the copper chloride solution in step S2 is prepared by dissolving copper chloride dihydrate in N,N-dimethylformamide, and ultrasonicating to dissolve evenly to obtain a copper chloride solution with a mass concentration of 1.44 mg / ml.

[0031] In one embodiment of the present invention, further, in step S3, the centrifugation speed is 10000 - 12000 g, and the centrifugation time is 10 - 20 minutes.

[0032] In one embodiment of the present invention, the chemical structural formula of irinotecan is as follows:

[0033]

[0034] In one embodiment of the present invention, a method for preparing dopamine-coated Lactobacillus rhamnosus includes the following steps:

[0035] S1. Prepare live Lactobacillus rhamnosus grown to the logarithmic phase, centrifuge and wash for standby;

[0036] S2. Mix the dopamine hydrochloride solution with Lactobacillus rhamnosus, stir and react, and centrifuge the reaction product to separate and obtain dopamine-coated Lactobacillus rhamnosus.

[0037] Further, the solution used for washing the bacterial cells in step S1 is PBS.

[0038] Further, the dopamine hydrochloride solution in step S2 is prepared by dissolving dopamine hydrochloride in Tris-saline (pH = 8.5, 10 mM), and ultrasonicating to dissolve evenly to obtain a dopamine hydrochloride solution with a mass concentration of 2 mg / ml.

[0039] Further, when the dopamine hydrochloride solution is mixed with Lactobacillus rhamnosus in step S2, the dopamine hydrochloride at 2 mg / ml is mixed with the Lactobacillus rhamnosus solution with an OD600 (the absorbance value of the bacterial solution measured at a wavelength of 600 nm) of 1 in a volume ratio of 1:1.

[0040] Further, the conditions for the stirring reaction in step S2 are: stirring at 37 °C and 100 rpm for 10 minutes.

[0041] In one embodiment of the present invention, a method for forming the final nano-drug by combining Cu-CPT NPs and Lactobacillus rhamnosus coated with dopamine through electrostatic adsorption

[0042] In one embodiment of the present invention, when combining Cu-CPT NPs and Lactobacillus rhamnosus coated with dopamine, the mass ratio of Cu-CPT NPs to Lactobacillus rhamnosus coated with dopamine is 4:1.

[0043] The present invention further provides the application of the live bacteria-delivered copper-irinotecan metal nano-drug with anti-tumor effect in the preparation of a drug for treating breast cancer.

[0044] Furthermore, the present invention provides the application of the drug in the preparation of a drug for treating triple-negative breast cancer (TNBC).

[0045] The LGG-PDA@Cu-CPT nano-drug can be used for the treatment of breast cancer, especially for refractory subtypes such as triple-negative breast cancer (TNBC), and overcomes drug resistance and recurrence problems through multi-mechanism synergistic effects.

[0046] Specifically, when realizing the breast cancer treatment function, its mechanism of action includes:

[0047] Intratumoral microbiota clearance: This nano-drug can efficiently clear bacteria in the tumor, eliminate the microbial barrier, and enhance the immunotherapy effect.

[0048] Tumor metastasis inhibition: By inhibiting the pro-metastatic effect of bacteria in the tumor and activating the systemic immune response, tumor metastasis can be effectively inhibited.

[0049] The functions and mechanisms of action of the LGG-PDA@Cu-CPT nano-drug provided by the present invention are summarized as follows:

[0050] Antibacterial effect: Cu2+ kills bacteria in the tumor by destroying the bacterial membrane, generating reactive oxygen species (ROS), and damaging DNA. At the same time, the local high temperature generated by photothermal therapy (PTT) further enhances the bacteria clearance effect.

[0051] Anti-tumor effect: The synergistic effect of Cu2+ and CPT realizes efficient killing of tumor cells by inducing apoptosis and immunogenic cell death (ICD) of tumor cells.

[0052] Immune activation effect: The high temperature generated by PTT and the release of Cu2+ synergistically activate the STING pathway, promote the production of type I interferon, enhance the activation of dendritic cells (DCs) and T cells, and enhance the anti-tumor immune response.

[0053] Inhibiting tumor metastasis: By destroying the bacteria in the tumor and the metastasis-promoting factors they secrete, while inducing immunogenic cell death (ICD) of tumor cells, releasing tumor-associated antigens, activating the systemic immune response, and inhibiting tumor metastasis.

[0054] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0055] 1. The present invention has developed a live bacteria-delivered copper-irinotecan metal nanodrug (LGG-PDA@Cu-CPT) with anti-tumor effects. As an anti-tumor drug, irinotecan and copper as an antibacterial drug, using Lactobacillus rhamnosus (LGG) as a carrier, giving full play to the anaerobic characteristics and tumor targeting of LGG, which can specifically colonize in the hypoxic area of the tumor, achieve precise drug delivery, effectively improve the enrichment degree of the nanodrug at the tumor site, and prolong the action time of the drug in the body. At the same time, LGG induces the production of type I interferon β (IFN-β) through the cGAS / STING / TBK1 signaling pathway, further enhancing the anti-tumor immune response and realizing the dual characteristics of antibacterial and anti-tumor.

[0056] 2. The present invention solves the problem of the high toxicity of copper ions (Cu2+) through nanotechnology. Although traditional copper ions have broad-spectrum antibacterial and anti-cancer effects, their high toxicity limits their direct application. The present invention forms a stable nano-coordination polymer (NCP) by coordinating copper ions with irinotecan (CPT), significantly reducing the toxicity of copper ions, while improving its stability and biocompatibility. The nano-sized copper ions not only retain their antibacterial and anti-cancer activities, but also further enhance the therapeutic effect by binding to CPT, achieving efficient and low-toxic tumor treatment.

[0057] 3. The present invention overcomes the inhibition of immunotherapy by the tumor microbiota through multi-mechanism synergistic action. The presence of bacteria in the tumor weakens the anti-tumor immune response by inhibiting the activities of CD4+ and CD8+ T cells, while the broad-spectrum antibacterial effect of Cu2+ and the local hyperthermia effect of photothermal therapy (PTT) efficiently eliminate the bacteria in the tumor, eliminate the microbial barrier, further enhance the bacteria clearance effect, and induce immunogenic cell death of tumor cells. At the same time, the release of CPT and LGG synergistically activates the STING pathway, promotes the production of type I interferon, enhances the activation of dendritic cells (DCs) and T cells, and significantly improves the anti-tumor immune response.

[0058] 4. Through the synergistic effect of copper ions and irinotecan, the present invention overcomes the problems of drug resistance and recurrence in breast cancer treatment. The synergistic anti-tumor effect of Cu2+ and CPT realizes the precise release of drugs through nano-coordination polymers (NCPs), enhancing the killing effect on tumor cells. In addition, immunogenic cell death (ICD) induced by photothermal therapy (PTT) further releases tumor-associated antigens, activates the systemic immune response, and effectively inhibits tumor recurrence and metastasis.

[0059] 5. By integrating antibacterial therapy, photothermal therapy, and STING pathway activation, the present invention achieves multi-mechanism synergistic therapy. The LGG-PDA@Cu-CPT nanodrug can not only efficiently eliminate the tumor microbiota but also enhance the anti-tumor immune response through STING pathway activation, while inhibiting tumor metastasis and minimizing systemic toxicity at the same time. This multi-functional integration strategy provides an innovative multi-mechanism synergistic therapy plan for breast cancer treatment, with significant clinical application prospects. Brief Description of the Drawings

[0060] Figure 1 It is the TEM morphological characterization image of Cu-CPT nanoparticles.

[0061] Figure 2 It is the standard curve range of irinotecan concentration.

[0062] Figure 3 It is the ultraviolet absorption spectrogram for verifying the ROS generation effect of different formulation groups.

[0063] Figure 4 It is the comparison chart of the antibacterial ability of Cu-CPT nanoparticles with different concentration gradients.

[0064] Figure 5 It is the comparison chart of the cytotoxicity of Cu-CPT nanoparticles with different concentration gradients.

[0065] Figure 6 It is the STING Western blot of in vitro experiments after stimulation with different formulation groups. Detailed Embodiments

[0066] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0067] This implementation is carried out on the premise of the technical solution of the present invention, giving detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0068] In the following embodiments, the specific sources of each reagent are as follows:

[0069]

[0070]

[0071] For the remaining raw materials, reagents or treatment techniques without specific description, they are all indicated as conventional commercially available raw materials or conventional treatment techniques in the art.

[0072] Example 1

[0073] This example provides a preparation method of a live bacteria-delivered copper-irinotecan metal nanodrug with anti-tumor effect.

[0074] (1) Preparation of copper-irinotecan complex nanoparticles, including the following steps:

[0075] Preparation of CPT solution: Weigh polyvinylpyrrolidone K30 (PVP-K30, 66 mg) into 5 ml of methanol, add triethylamine (TEA, 0.2 ml), and gently shake until completely dissolved. Subsequently, weigh irinotecan hydrochloride hydrate (CPT, 6.6 mg) and add it to the above solution, and dissolve it in a digital control ultrasonic bath to obtain a CPT solution. Transfer the CPT solution to a three-necked flask protected by nitrogen and stir for 1 h.

[0076] Preparation of copper chloride solution: Weigh copper chloride dihydrate (7.2 mg) into 5 ml of N,N-dimethylformamide (DMF), and ultrasonically treat it until completely dissolved to obtain a copper chloride solution.

[0077] Preparation of Cu-CPT nanoparticles (Cu-CPT NPs): Dropwise add the copper chloride solution to the CPT solution, and stir at a speed of 400 rpm for 5 hours. After the reaction is completed, centrifuge (12,000 rpm, 10 minutes), collect the precipitate, discard the supernatant, and resuspend the washed precipitate with ultrapure water to obtain a solution containing Cu-CPT NPs, which is also the copper-irinotecan nano-coordination polymer (Cu-CPT NPs) in the inventive content of this application.

[0078] (2) Preparation of a live bacteria-loaded copper-irinotecan nanodrug system

[0079] Prepare live Lactobacillus rhamnosus grown to the logarithmic phase, centrifuge, wash with PBS and resuspend in PBS for standby. Then mix the 2 mg / mL dopamine hydrochloride solution with Lactobacillus rhamnosus. When mixing the dopamine hydrochloride solution with Lactobacillus rhamnosus, mix the dopamine hydrochloride at 2 mg / ml and the Lactobacillus rhamnosus solution with an OD600 of 1 in a volume ratio of 1:1, stir at 37 °C and 100 rpm for 10 minutes, centrifuge the reaction product, and separate to obtain dopamine-coated Lactobacillus rhamnosus (LGG-PDA).

[0080] The copper-irinotecan nano-coordination polymer and dopamine-coated Lactobacillus rhamnosus were stirred at 37 °C for 20 min. The copper-irinotecan nano-coordination polymer and dopamine-coated Lactobacillus rhamnosus were combined by electrostatic adsorption to form the final nano-drug LGG-PDA@Cu-CPT.

[0081] (III) Particle size determination of Cu-CPT NPs

[0082] The hydrodynamic diameter and polydispersity index (PDI) of the nanoparticles in the nanoparticle solution were measured using the dynamic light scattering (DLS) mode of a nanoparticle size analyzer (Nanobrook omni, Brookhaven). The sample preparation method was as follows: The prepared Cu-CPT NPs were diluted 10 times and then added to a quartz cuvette with four-sided transparency for detection. Each sample was detected for 20 cycles and measured three times. The nanoparticles were prepared three times repeatedly, and the above particle size detection was performed to obtain the average particle size of the nanoparticles.

[0083] Experimental group Particle size(nm) PDI 1 37.8±1.5 0.120±0.03 2 38.5±1.2 0.115±0.025 3 38.2±1.0 0.13±0.028 Average value 38.0±1.2 0.122±0。028

[0084] The particle size of the nanoparticles was detected by a particle size analyzer. The same method was repeated three times and the average value was taken. It was monitored that the particle size of Cu-CPT NPs was 38.0 ± 1.2 nm, and the polydispersity coefficient PDI was 0.122 ± 0.028. This indicates that the particle size is in the nanometer range and the particle size distribution is uniform. The experimental results prove that the prepared nanoparticles lay the foundation for efficient drug delivery and anti-tumor effects.

[0085] (IV) Morphological detection of Cu-CPT NPs under transmission electron microscopy

[0086] The morphology and size of the nanoparticles were further characterized by transmission electron microscopy (TEM, LIBRA200CS, Carl Zeiss). First, a copper grid (200 mesh) with a carbon film was placed on a glass slide (matte side up). Then, 4 μL of the above-prepared Cu-CPT nanoparticles was dropped onto the matte side of the copper grid and left to dry overnight in a fume hood before being sent for detection. The TEM detection results of the silk fibroin-copper nanoparticles were consistent with the results of the nanoparticle size analyzer. As Figure 1 shown, the morphology of Cu-CPT NPs was uniform, with an average particle size of 38.0 ± 1.2 nm and a relatively uniform particle size distribution.

[0087] (V) Investigation of the encapsulation capacity of irinotecan in Cu-CPT NPs

[0088] The maximum absorption wavelength was determined after a full-wavelength ultraviolet scan of the irinotecan solution.

[0089] The maximum absorption wavelength of copper ions was measured to be 370 nm. A standard curve of irinotecan concentration was prepared asFigure 2 As shown, the correlation coefficient r of the standard curve is 0.9991, meeting the relevant regulatory requirements.

[0090] According to the preparation method, 6.6 mg of irinotecan hydrochloride hydrate (CPT) was used in the preparation of Cu-CPT nanoparticles. Assuming that CPT was completely encapsulated in the Cu-CPT nanoparticles, 1 mL of the Cu-CPT nanoparticle solution theoretically contained 6.6 mg of CPT. 1 mL of ethylenediaminetetraacetic acid (EDTA) was added, and the OD value at 370 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader at the maximum absorption wavelength of CPT determined in the above experiment. Substituting into the standard curve equation and performing subsequent calculations, the content of CPT encapsulated in the copper-irinotecan nanoparticle solution was 1.12 mg.

[0091] (VI) Investigation of the generation of hydroxyl radicals by LGG-PDA@Cu-CPT

[0092] Methylene blue (MB) reacts with hydroxyl radicals to reduce MB to colorless MB. The decrease in the absorbance of methylene blue is linearly related to the amount of Fenton reagent added. Therefore, the fading degree (ΔA) of methylene blue can be measured at 660 / 664 nm for indirect photometric determination of hydroxyl radicals.

[0093] The specific experimental procedure was to divide the solution systems into four groups and number them, namely the control group, which only contained the MB group, the MB + H2O2 group, the MB + Cu-CPT NPS + H2O2 group (the concentration of Cu-CPT NPS was 100 μg / ml), and the MB + LGG-PDA@Cu-CPT + H2O2 group (the concentration of LGG-PDA@Cu-CPT was 100 μg / ml). The concentration of MB was 32 μg / mL, and the concentration of hydrogen peroxide was 100 mM. MB absorbs at the ultraviolet light wavelength position. It can react with OH to form a colorless substance, resulting in a decrease in the absorbance of the system. The ultraviolet light wavelength absorption value in the MB solution system is inversely proportional to the hydroxyl radical level. The lower the ultraviolet light wavelength absorption value, the higher the hydroxyl radical level in the system. As Figure 3 shown, the maximum absorption wavelength of MB is 660 nm. The control group had the highest ultraviolet absorption wavelength. The peroxy bond in hydrogen peroxide is unstable, and light and heat can promote its homolytic cleavage to form hydroxyl radicals, which can be manifested as the generation of a certain amount of hydroxyl radicals in the two groups with the addition of hydrogen peroxide, resulting in a lower ultraviolet absorbance than that of the control group. The addition of Cu-CPT NPS and LGG-PDA@Cu-CPT to the system had similar ultraviolet absorption wavelengths, and the ultraviolet absorbances were both lower than that of the control group, indicating that the prepared LGG-PDA@Cu-CPT nanoparticles could also autonomously generate a certain amount of hydroxyl radicals.

[0094] (VII) Investigation of the antibacterial ability of different concentrations of LGG-PDA@Cu-CPT

[0095] Fusobacterium nucleatum was exposed to different concentrations of Cu-CPT NPS, LGG-PDA@Cu-CPT (-L, indicating no laser irradiation), and LGG-PDA@Cu-CPT (+L, irradiated with 808 nm laser) (40 μg / mL, 32 μg / mL, 24 μg / mL, 16 μg / mL, 12 μg / mL) for 18 hours. Then, the bacterial colonies were diluted by spotting on a plate, and the diluted bacterial suspension was spotted onto a blood agar plate. 12 spots were made for each dilution to ensure uniform distribution of the colonies. The blood agar plate was placed in an incubator at 37 °C for 24 - 48 hours to observe the colony formation. According to the colony counting results, the inhibition rate of different concentrations on bacteria was calculated. By Figure 4 It can be seen that the antibacterial ability of LGG-PDA@Cu-CPT (-L) and LGG-PDA@Cu-CPT (+L) against Fusobacterium nucleatum shows concentration dependence, and the antibacterial ability increases with the increase in concentration. And the antibacterial ability is the strongest under photothermal conditions.

[0096] (VIII) Toxicity evaluation of LGG-PDA@Cu-CPT on tumor cells

[0097] For the prepared nanocomposite LGG-PDA@Cu-CPT, its toxicity against tumor cells was verified. The experimental method is as follows: The CCK-8 method was used to detect the toxicity of the nanoparticles to cells, and by analyzing the changes in cell viability among different groups after administration, the toxicity of different formulation groups to cells was further explored.

[0098] Mouse breast cancer cell 4T1 was selected as the tumor cell model. The specific method is as follows: Digest the 4T1 cells in the logarithmic growth phase, at 10 4The density of each well was inoculated into a 96-well plate and placed in a cell incubator at 37 °C and 5% CO2 for 12 h until the cells adhered to the wall. During this period, the growth state of the cells was observed in a timely manner to ensure that their growth state was good and the growth density in the wells was appropriate. After the cells adhered to the wall, the culture medium was aspirated and discarded, and the cells were washed 2-3 times with sterile PBS. The sterile PBS was warmed in a 37 °C water bath for 5 min. After the cells adhered to the wall, the culture medium was aspirated and discarded, and the cells were washed 2-3 times with sterile PBS. 200 μL of CPT, Cu-CPT NPS, LGG-PDA@Cu-CPT(-L), and LGG-PDA@Cu-CPT(+L) prepared in 1640 medium at concentrations of (5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 80 μg / ml, recorded as the concentration of CPT) were added to each well, and five replicate wells were set for each group. Incubation continued for 24 hours. After the incubation was completed, the cells were washed 2-3 times with sterile PBS, and 100 μL of CCK-8 solution diluted with the medium was added to each well, so that the actual added amount of the CCK-8 solution in each well was 10 μL. After continued incubation for 1 hour at 37 °C and 5% CO2 in the cell incubator, the absorbance was measured at 450 nm. As Figure 5 shown, in all experimental groups, CCK-8 had concentration-dependent cytotoxicity. As the drug concentration increased, the cell viability of each group was significantly differentiated, showing the following trend: CPT > Cu-CPT ≈ LGG-PDA@Cu-CPT(-L) > LGG-PDA@Cu-CPT(+L). Compared with the CPT group (IC50 = 25.06 ± 1.24 μg / mL), the Cu-CPT group (IC50 = 10.83 ± 0.25 μg / mL) and the LGG-PDA@Cu-CPT(-L) group (IC50 = 9.47 ± 0.35 μg / mL) showed enhanced cytotoxicity, indicating that the nanosystem effectively promoted the delivery of the drug to tumor cells. The LGG-PDA@Cu-CPT(+L) group had the highest cytotoxicity (IC50 = 5.68 ± 0.26 μg / mL) and had a good killing effect.

[0099] (IX) STING Western blot analysis

[0100] 4T1 cells were co-incubated with CPT, Cu-CPT NPS, LGG-PDA@Cu-CPT(-L), and LGG-PDA@Cu-CPT(+L) in a 37 °C cell incubator for 24 hours. Then, the cells were collected and lysed in ice-cold RIPA buffer containing protease inhibitor (PMSF). All samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a 0.2 m nitrocellulose membrane. The membrane was blocked with 5% skim milk at 25 °C for 1 hour and then incubated with p-STING, p-TBK1, and p-IRF3 antibodies overnight at 4 °C. Subsequently, the membrane was washed with TBET and incubated with secondary antibodies (antibodies that can specifically recognize the primary antibody and do not directly react with the target antigen but bind to the Fc segment of the primary antibody. In this example, Goat anti-Mouse IgG(H+L) Secondary Antibody, HRP and Goat anti-Rabbit IgG(H+L) Secondary Antibody, HRP (Thermo Fisher) were used respectively) at room temperature for 45 minutes. Specific proteins were visualized by enhanced chemiluminescence. As Figure 6 shown in the protein blot figure, the labels 1-5 in the figure are the control group (Control), free drug group (CPT), formulation group (Cu-CPT NPS), final formulation group LGG-PDA@Cu-CPT(-L), and LGG-PDA@Cu-CPT(+L) (LGG-PDA@Cu-CPT), respectively. From Figure 6 it can be seen that the expression levels of related proteins in the Cu-CPT NPs and LGG-PDA@Cu-CPT groups are higher than those in other groups, which is attributed to the activation ability of CPT and LGG on the STING pathway. Among them, the expression in the laser treatment group is the highest, which may be due to the improvement of the immune effect by photothermal therapy and the promotion of tumor ablation.

[0101] Screening of experimental conditions in Example 2

[0102] To ensure that the prepared nanoparticles have a smaller particle size and better dispersion, the factors that may affect each experimental link were investigated. As shown in Table 1 and Table 2, repeated experiments were carried out multiple times to screen the optimal experimental scheme. Under the condition of determining the volume of the reaction system, the effect of the ratio of copper to irinotecan on the final particle size of the nanoparticles was explored, and it was found that when the ratio of copper to irinotecan was 4:1 under this condition, the particle size value was the smallest. When exploring the stirring speed under the condition of determining the ratio of copper ions to irinotecan, it was found that a smaller reaction system was more conducive to the particle size of the nanoparticles finally obtained in the experiment, and the reaction system with a stirring speed of 400 rpm showed the best performance. Considering the selection of comprehensive experimental conditions, the ratio of copper to irinotecan of 4:1 and a reaction of 400 rpm were the best. And the preparation scheme designed in this project has repeatability.

[0103] Table 1 Screening Table of the Optimal Ratio of Copper and Irinotecan in a 10 mL Reaction System

[0104]

[0105] Table 2 Screening Table of the Optimal Stirring Rate in a 10 mL Reaction System

[0106] 200rpm 400rpm 600rpm 800rpm 1 850.3±5.2 37.8±1.5 250.7±3.1 550.4±4.8 2 830.5±4.8 38.5±1.2 240.9±2.9 530.2±5.1a 3 820.1±5.0 38.2±1.0 230.8±3.0 540.6±4.7 Average value 833.6±5.0 38.0±1.2 240.8±3.0 540.4±4.9a

[0107] Tables 1 and 2 demonstrate that nanoparticles with the best particle size can be synthesized when Cu:CPT = 4:1 and the stirring speed is 400 rpm.

[0108] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect, characterized in that: The invention comprises a copper-irinotecan nano coordination polymer, on which dopamine-coated lactobacillus rhamnosus is physically adsorbed or chemically cross-linked.

2. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 1, characterized in that: The following steps are involved: Preparation of copper-irinotecan nano-coordination polymer: Copper ions are coordinated with irinotecan to form a stable copper-irinotecan nano-coordination polymer; Preparation of LGG-PDA@Cu-CPT: The copper-irinotecan nano-coordination polymer is combined with dopamine-coated Lactobacillus rhamnosus to form the final nanodrug through physical adsorption or chemical cross-linking, that is, the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect.

3. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 2, characterized in that: The preparation method of the copper-irinotecan nano coordination polymer comprises the following steps: S1. Under anaerobic stirring conditions, the drug irinotecan is added to a methanol solution to form a mixed solution, and the lactone bond and hydroxyl bond of irinotecan are disconnected by stirring in a fixed pH system to facilitate coordination and binding with copper ions; S2, after stirring for 1 hour, dripping the copper chloride solution into the irinotecan system, stirring for 5 hours under anaerobic conditions, and centrifuging the reaction product to obtain the product Cu-CPT; S3, re-dispersing the product Cu-CPT, centrifuging, washing and separating for multiple times, and then adding ultrapure water for ultrasonic re-dispersion to obtain copper-irinotecan nanoparticles, namely, copper-irinotecan nano-coordination polymers.

4. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 3, characterized in that: The irinotecan solution in step S1 is prepared by dissolving irinotecan hydrochloride trihydrate in methanol organic solvent, and adding triethylamine dropwise to adjust the pH of the system to 7.

4.

5. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 3, characterized in that: In step S1, a suspending agent, PVP, is also added.

6. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 2, characterized in that: The preparation method of dopamine-coated Lactobacillus rhamnosus comprises the following steps: S1. Prepare live Lactobacillus rhamnosus grown to the logarithmic phase, centrifuge and wash for later use; S2, mixing the dopamine hydrochloride solution with Lactobacillus rhamnosus, stirring for reaction, centrifuging the reaction product, and separating the dopamine-coated Lactobacillus rhamnosus; When the dopamine hydrochloride solution is mixed with the Lactobacillus rhamnosus, 2 mg / ml of dopamine hydrochloride and the Lactobacillus rhamnosus solution with an OD600 of 1 are mixed in a volume ratio of 1:

1.

7. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 2, characterized in that: The copper-irinotecan nano-coordination polymer was combined with dopamine-coated Lactobacillus rhamnosus through electrostatic adsorption to form the final nano-drug.

8. The method for preparing the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 2, characterized in that: When the copper-irinotecan nano-coordination polymer is combined with dopamine-coated Lactobacillus rhamnosus, the mass ratio of the copper-irinotecan nano-coordination polymer to the dopamine-coated Lactobacillus rhamnosus is 4:

1.

9. The use of the copper-irinotecan metal nanodrug delivered by live bacteria with anti-tumor effect according to claim 1, characterized in that: The drug is used in preparing a drug for treating breast cancer.

10. The use according to claim 9, characterized in that: The drug is used in preparing a drug for treating triple-negative breast cancer.

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