Bioactive material for tumor treatment and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a bioactive material for tumor treatment and a preparation method and application thereof. The preparation method comprises the following steps: providing adriamycin-loaded magnesium-iron layered double-metal hydroxide nanoparticles; modifying the nanoparticles with glucose oxidase to obtain a drug-loaded enzyme compound; and dispersing the drug-loaded enzyme compound in water, mixing with shewanella, and carrying out centrifugal washing to obtain the bioactive material. The bioactive material disclosed by the invention has tumor targeting property, and in-vivo targeting can better utilize the metabolic function of bacteria to continuously consume metabolites at tumor parts; the apoptosis of tumor cells can be promoted by utilizing the generation of active oxygen and the release of adriamycin hydrochloride, and the ferroptosis of tumors can be realized by reducing glutathione and accumulating lipid peroxide.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a bioactive material for tumor treatment, its preparation method, and its application. Background Technology

[0002] Lactate in tumors plays a role far beyond being a byproduct; it is crucial in the occurrence and development of cancer and is closely associated with increased metastasis, tumor recurrence, and poor treatment efficacy. For example, it has been reported that lactate can promote the secretion of hepatocyte growth factor by cancer-associated fibroblasts, leading to chemotherapy resistance in liver cancer. Therefore, reducing lactate levels in the tumor microenvironment is a promising therapeutic strategy for enhancing chemotherapy efficacy and reversing multidrug resistance in tumors. To achieve this goal, strategies such as nanoencapsulation and targeted molecule grafting have been designed to increase the accumulation of metabolic modulatory drugs in tumors. Although some small molecule inhibitors and siRNAs can target lactate metabolism by inhibiting the lactate-proton cotransporter of monocarboxylic acid transporter 1 (MCT1), pyruvate carrier (MPC), or lactate dehydrogenase A (LDHA), thereby inhibiting tumor growth or enhancing sensitivity to conventional cancer treatments, drug-based metabolic modulation only provides temporary relief and requires frequent dosing, often leading to adverse side effects. Meanwhile, inhibiting MCT1 may accelerate the tumor's consumption of glucose and promote tumor development, while the toxic side effects of small molecule inhibitors of MPC on normal cells are unavoidable. Therefore, a feasible approach would be to eliminate lactic acid within the tumor while simultaneously accelerating the breakdown of glucose, an energy source. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a bioactive material for tumor therapy, its preparation method, and its application. The aim is to achieve tumor therapy by regulating the respiration of Shewanella bacteria. Specifically, a live biocatalytic therapy is constructed by combining microorganisms with nanomaterials. Layered double hydroxides (LDHs) are used to regulate the respiration of Shewanella (MR-1), inducing ferroptosis and apoptosis in tumor cells. This approach not only has targeted efficacy but also continuously consumes tumor metabolites. The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a method for preparing a bioactive material for tumor treatment, comprising the following steps: S1. Provide magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin (MgFe-LDH@DOX). S2. Glucose oxidase (GOx) is modified onto the above nanoparticles to obtain a drug-loaded enzyme complex (MgFe-LDH@DOX@GOx). S3. Disperse the above drug-loaded enzyme complex in water and mix it with Shewanella (MR-1), centrifuge and wash to obtain the bioactive material (LDGM).

[0004] Preferably, the method for preparing the doxorubicin-loaded magnesium-iron layered bimetallic hydroxide nanoparticles in step S1 includes the following steps: S1-1. Prepare magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH) and prepare doxorubicin hydrochloride (DOX) solution; S1-2. The magnesium-iron layered bimetallic hydroxide nanoparticles obtained above are dispersed in water, mixed and stirred with doxorubicin hydrochloride solution, and centrifuged and washed to obtain magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin (MgFe-LDH@DOX).

[0005] More preferably, the preparation method of the magnesium-iron layered bimetallic hydroxide nanoparticles in step S1-1 includes: under inert gas protection, according to Mg... 2+ with Fe 3+ Magnesium salt and iron salt were weighed in a molar ratio of 3:1, mixed, and then an alkaline solution was added. The mixture was stirred and centrifuged to obtain a precipitate, which was then resuspended in water. The mixture was heated to react, centrifuged, washed, and freeze-dried to obtain the magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH).

[0006] Specifically, the structural characteristics of MgFe-LDH itself allow it to load chemotherapeutic drugs and small molecules, while also coating the surface of bacteria, reducing the stress response of bacteria in the bloodstream, and promoting iron respiration of MR-1.

[0007] More preferably, in steps S1-2, the dispersion concentration of the magnesium-iron layered bimetallic hydroxide nanoparticles in water is 0.5~2 mg / mL.

[0008] More preferably, in steps S1-2, the concentration of the doxorubicin hydrochloride solution (DOX) is 0.5~2 mg / mL.

[0009] Specifically, DOX, as a chemotherapy drug, can induce tumor cell apoptosis. However, the tumor site itself has multidrug resistance. MR-1's consumption of lactate can also reduce multidrug resistance and enhance the chemotherapy effect.

[0010] Preferably, step S2 further includes: S2-1. Prepare an aqueous solution of glucose oxidase (GOx). Disperse magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH@DOX) loaded with doxorubicin in water, mix and stir with the aqueous solution of glucose oxidase (GOx), and centrifuge to obtain the drug-loaded enzyme complex (MgFe-LDH@DOX@GOx). The concentration of the glucose oxidase aqueous solution is 1-4 mg / mL, and the concentration of the aqueous solution of the magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin is 1-4 mg / mL.

[0011] Specifically, GOx can catalyze the degradation of glucose at the tumor site, reduce the energy supply to the tumor, decrease the oxygen content at the tumor site, and promote the efflux of MR-1 electrons.

[0012] Preferably, in step S3, the concentration of Shewanella (MR-1) is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

[0013] Preferably, in step S3, the concentration of the drug-loaded enzyme complex in water is 0.5~2 mg / mL.

[0014] In a second aspect, the present invention provides a bioactive material obtained by the preparation method described in the first aspect.

[0015] Thirdly, the present invention provides the application of the bioactive material as described in the second aspect in tumor treatment.

[0016] Compared with the prior art, the advantages of the present invention are as follows: (1) In this invention, magnesium-iron layered double hydroxide (MgFe-LDH) was first prepared by hydrothermal method. Then, the interlayer structure and high specific surface area of ​​MgFe-LDH were used to load the chemotherapy drug doxorubicin hydrochloride (DOX) and glucose oxidase (GOx) respectively. Finally, the bioactive material (LDGM) was successfully constructed by utilizing the electrostatic interaction between MgFe-LDH and Shewanella (MR-1).

[0017] (2) The LDGM of the present invention has tumor targeting, and in vivo targeting can better utilize the metabolic function of bacteria to continuously consume the metabolites of the tumor site.

[0018] (3) On the one hand, the present invention utilizes the hamburger structure of MgFe-LDH to load the chemotherapy drug DOX in the interlayer and simultaneously consumes lactic acid through MR-1 respiration, thereby reducing the multidrug resistance of tumor cells; on the other hand, it also loads GOx on the outer layer of MgFe-LDH to consume glucose in the tumor site, enhance the hypoxia in the tumor site, promote electron transmembrane transfer, and promote MR-1 iron respiration.

[0019] (4) The LDGM of the present invention can not only promote the apoptosis of tumor cells by generating reactive oxygen species (ROS) and releasing doxorubicin hydrochloride (DOX), but also achieve tumor ferroptosis by reducing glutathione (GSH) and accumulating lipid peroxides (LPO). Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 (a) is the synthetic route of the bioactive material LDGM of the present invention; (b) is the process of ferroptosis / apoptosis of tumor cells achieved by regulating the respiration mode of bacteria.

[0022] Figure 2 In vitro activity verification and characterization of the bioactive material LDGM of the present invention; Figure 3 This invention validates the POD and GPx-like activities of the bioactive material LDGM. Figure 4 This invention demonstrates the application of the bioactive material LDGM in in vitro tumor cell ferroptosis. Figure 5 This invention provides a method for verifying the application of the bioactive material LDGM in in vitro tumor cell apoptosis. Figure 6 The antitumor effect of the bioactive material LDGM of this invention applied to 4T1 fed mice; Figure 7 This study aims to verify the in vivo tumor targeting ability of the bioactive material LDGM of this invention. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0024] Studies have shown that the facultative anaerobic bacterium Shewanella (MR-1) can selectively utilize lactic acid as an energy source for respiration and perform iron respiration under hypoxic conditions, using extracellular insoluble iron oxides as the final electron acceptor to reduce Fe by oxidizing electron donors. 3+This allows them to store the energy needed for life activities during the process. Nanozymes are nanomaterials with intrinsic enzyme-mimicking catalytic activity. They utilize the pathological characteristics of the tumor microenvironment to trigger relevant chemical reactions in situ, thereby initiating tumor nanocatalytic therapy (NCT). With the application of nanozymes in tumor NCT, various nanozymes have been developed and proven to be ideal candidates for cancer treatment. Similar to enzyme activity, nanozymes can be broadly classified into the following categories: nanozymes that mimic peroxidase (POD), catalase (CAT), oxidase, superoxide dismutase, and glutathione peroxidase (GPx). The active sites of nanozymes are usually metallic components. Metal elements can effectively mimic the electronic redox processes catalyzed by natural enzymes, which endows nanozymes with the advantages of continuous, controllable, and multiple enzyme activities. The abundant positive metal charges on the surface of layered double hydroxides (LDHs) can stably interact electrostatically with negatively charged bacteria and exhibit excellent catalytic performance. In addition, the acid-responsive degradation characteristics of LDHs can be used for intelligent protection of bacteria in targeted tumor therapy. Therefore, LDHs can be good candidate materials for modifying bacteria and utilizing their respective advantages to form biocatalytic reactors.

[0025] Drug-based tumor microenvironment (TME) metabolic modulation only provides temporary relief and requires frequent administration, often leading to adverse side effects. Here, the present invention constructs a self-driven, tumor-tendency-oriented bacterial biocatalytic reactor (LDGM) comprising Shewanella (MR-1) and layered double hydroxides (LDHs), such as... Figure 1 As shown, it can induce ferroptosis and apoptosis by in-situ consuming lactate for respiration and iron respiration under hypoxic conditions. The LDGM bioreactor is constructed by depositing layered double hydroxide (LDH) nanosheets loaded with doxorubicin / glucose oxidase on Shewanella (MR-1), which can specifically target hypoxic tumor regions and release doxorubicin, glucose oxidase, and Fe. 3+ Furthermore, MR-1 can continuously utilize intratumoral lactate for respiration, thereby enhancing the effectiveness of chemotherapy and reversing multidrug resistance in tumors. Moreover, MR-1 undergoes iron respiration via GOx under hypoxic conditions, promoting Fe production through electron transfer. 3+ The reduction of [the tumor microenvironment] was observed. In a mouse tumor model, LDGM significantly inhibited tumor growth and enhanced ferroptosis and apoptosis. Overall, the developed in vivo biomaterial provides a promising strategy for enhancing cancer therapy by continuously modulating the intratumoral lactate / glucose metabolic microenvironment.

[0026] In summary, this invention primarily promotes electron transfer by regulating bacterial respiration, thereby inducing ferroptosis and apoptosis in tumor cells. The core mechanisms are: ① LDGM attacks tumor cells and consumes lactate to improve the tumor microenvironment and overcome tumor drug resistance. ② LDGM catalyzes glucose breakdown, reducing the energy supply to tumor cells and decreasing oxygen content at the tumor site, thus promoting bacterial iron respiration and stimulating Fe production. 3+ The reduction of hydroxyl radicals (·OH) occurs during the Fenton reaction. These radicals, in conjunction with chemotherapy drugs, can promote apoptosis and reduce ferritin expression, thereby leading to ferroptosis in tumor cells.

[0027] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0028] In this document, the terms “containing,” “comprising,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0029] In this document, the terms “optional,” “optionally,” or “optional” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0031] The following examples and comparative examples further illustrate the specific implementation methods and beneficial effects of the present invention in detail. All materials used in this invention were purchased commercially, specifically: MgCl2·6H2O and FeCl3·6H2O were purchased from Sinopharm Chemical Reagent; Doxorubicin hydrochloride (DOX) and glucose oxidase (GOx) were purchased from Beijing Innocare Reagent Co., Ltd.; Shewanella (MR-1) was obtained from the China Center for Type Culture Collection, Wuhan University, with accession number ATCC 700550.

[0032] Example 1

[0033] This embodiment provides a bioactive material for tumor treatment and its preparation method, including the following steps: (1) Preparation of MgFe-LDH(L): Synthesized using a hydrothermal method; based on Mg 2+ / Fe 3+ Magnesium chloride (MgCl2·6H2O, 609.9 mg) and ferric chloride (FeCl3·6H2O, 240.0 mg) were weighed in a molar ratio of 3:1 and prepared into a 10 mL mixed metal salt solution. An alkaline solution was added at 37 °C under a N2 atmosphere, and the pH was adjusted to 10 ± 0.01 with NaOH. The mixture was then stirred in a water bath for 0.5 h, centrifuged, and the supernatant was discarded to obtain the initial product. The initial precipitate was resuspended in 40 mL of deionized water and reacted in a hydrothermal reactor at 100 °C for 16 h. After cooling, the mixture was centrifuged and washed twice, and finally freeze-dried to obtain magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH), abbreviated as L. (2) Loading DOX (LD): 10 mg of MgFe-LDH nanoparticles were uniformly dispersed in 10 mL of ultrapure water (1 mg / mL), and then 5 mL of doxorubicin hydrochloride (DOX) (1 mg / mL) solution was added. After stirring for 24 h, the DOX-loaded MgFe-LDH nanoparticles were collected by centrifugation (10,000 rpm). Finally, excess DOX was washed by centrifugation until the supernatant was colorless, and the supernatant was lyophilized to obtain doxorubicin-loaded magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH@DOX), abbreviated as LD. (3) GOx load (LDG): First, dissolve 1 mg of glucose oxidase (GOx) in 500 μL of water to prepare an aqueous solution of 2 mg / mL. Then, mix 200 μL of the GOx aqueous solution with 20 mL of MgFe-LDH@DOX (2 mg / mL) and stir overnight. Then, centrifuge at 10000 rpm for 10 min to obtain the drug-loaded enzyme complex (MgFe-LDH@DOX@GOx), abbreviated as LDG. (4) Preparation of bioactive materials (LDGM): Add 1 mL of MR-1 (10 8 The active biocatalytic material was prepared by mixing CFU / mL with 2 mL of LDG aqueous solution (1 mg / mL) and gently shaking for 60 s. The resulting mixture was centrifuged (6000 rpm, 3 min) and washed three times with PBS to obtain the MgFe-LDH@DOX@GOx / MR-1 bioactive material, abbreviated as LDGM.

[0034] Example 2

[0035] This embodiment provides a bioactive material for tumor treatment and its preparation method, including the following steps: (1) Preparation of MgFe-LDH(L): Synthesized using a hydrothermal method; based on Mg 2+ / Fe 3+ Magnesium chloride (MgCl2·6H2O, 609.9 mg) and ferric chloride (FeCl3·6H2O, 240.0 mg) were weighed in a molar ratio of 3:1 and prepared into a 10 mL mixed metal salt solution. An alkaline solution was added at 37 °C under a N2 atmosphere, and the pH was adjusted to 10 ± 0.01 with NaOH. The mixture was then stirred in a water bath for 0.5 h, centrifuged, and the supernatant was discarded to obtain the initial product. The initial precipitate was resuspended in 40 mL of deionized water and reacted in a hydrothermal reactor at 100 °C for 16 h. After cooling, the mixture was centrifuged and washed twice, and finally freeze-dried to obtain magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH), abbreviated as L. (2) Loading DOX (LD): 5 mg of MgFe-LDH nanoparticles were uniformly dispersed in 10 mL of ultrapure water (0.5 mg / mL), and then 5 mL of doxorubicin hydrochloride (DOX) solution (0.5 mg / mL) was added. After stirring for 24 h, the DOX-loaded MgFe-LDH nanoparticles were collected by centrifugation (10,000 rpm). Finally, excess DOX was washed by centrifugation until the supernatant was colorless, and the supernatant was lyophilized to obtain doxorubicin-loaded magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH@DOX), abbreviated as LD. (3) GOx load (LDG): First, 0.5 mg of glucose oxidase (GOx) was dissolved in 500 μL of water to prepare an aqueous solution of 1 mg / mL. Then, 200 μL of the GOx aqueous solution was mixed with 20 mL of MgFe-LDH@DOX (1 mg / mL) and stirred overnight. After centrifugation at 10000 rpm for 10 min, the drug-loaded enzyme complex (MgFe-LDH@DOX@GOx), abbreviated as LDG, was obtained. (4) Preparation of bioactive materials (LDGM): Add 1 mL of MR-1 (10 7 The active biocatalytic material was prepared by mixing CFU / mL with 2 mL of LDG aqueous solution (0.5 mg / mL) and gently shaking for 60 s. The resulting mixture was centrifuged (6000 rpm, 3 min) and washed three times with PBS to obtain MgFe-LDH@DOX@GOx / MR-1 bioactive material, abbreviated as LDGM.

[0036] Example 3

[0037] This embodiment provides a bioactive material for tumor treatment and its preparation method, including the following steps: (1) Preparation of MgFe-LDH(L): Synthesized using a hydrothermal method; based on Mg 2+ / Fe 3+ Magnesium chloride (MgCl2·6H2O, 609.9 mg) and ferric chloride (FeCl3·6H2O, 240.0 mg) were weighed in a molar ratio of 3:1 and prepared into a 10 mL mixed metal salt solution. An alkaline solution was added at 37 °C under a N2 atmosphere, and the pH was adjusted to 10 ± 0.01 with NaOH. The mixture was then stirred in a water bath for 0.5 h, centrifuged, and the supernatant was discarded to obtain the initial product. The initial precipitate was resuspended in 40 mL of deionized water and reacted in a hydrothermal reactor at 100 °C for 16 h. After cooling, the mixture was centrifuged and washed twice, and finally freeze-dried to obtain magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH), abbreviated as L. (2) Loading DOX (LD): 20 mg of MgFe-LDH nanoparticles were uniformly dispersed in 10 mL of ultrapure water (2 mg / mL), and then 5 mL of doxorubicin hydrochloride (DOX) (2 mg / mL) solution was added. After stirring for 24 h, the DOX-loaded MgFe-LDH nanoparticles were collected by centrifugation (10,000 rpm). Finally, excess DOX was washed by centrifugation until the supernatant was colorless, and the supernatant was lyophilized to obtain doxorubicin-loaded magnesium-iron layered bimetallic hydroxide nanoparticles (MgFe-LDH@DOX), abbreviated as LD. (3) GOx load (LDG): First, 2 mg of glucose oxidase (GOx) was dissolved in 500 μL of water to prepare an aqueous solution of 4 mg / mL. Then, 200 μL of the GOx aqueous solution was mixed with 20 mL of MgFe-LDH@DOX (4 mg / mL) and stirred overnight. After centrifugation at 10000 rpm for 10 min, the drug-loaded enzyme complex (MgFe-LDH@DOX@GOx), abbreviated as LDG, was obtained. (4) Preparation of bioactive materials (LDGM): Add 1 mL of MR-1 (10 9 The active biocatalytic material was prepared by mixing CFU / mL with 2 mL of LDG aqueous solution (2 mg / mL) and gently shaking for 60 s. The resulting mixture was centrifuged (6000 rpm, 3 min) and washed three times with PBS to obtain MgFe-LDH@DOX@GOx / MR-1 bioactive material, abbreviated as LDGM.

[0038] Comparative Example 1 The difference between this comparative example and Example 2 is that the dispersion concentration of the LDG aqueous solution in step (4) is further reduced to 0.2 mg / mL, while the rest is the same as in Example 2. As a carrier, insufficient dispersion concentration of LDG leads to a decrease in the activity of the obtained LDGM; this is because the low concentration of LDG reduces the total amount of doxorubicin (DOX) loaded, resulting in insufficient release of chemotherapy drugs; the loading of glucose oxidase (GOx) is also reduced, and there is less hydrogen peroxide (H2O2) and gluconic acid; moreover, the surface coating of MR-1 bacteria is incomplete, making MR-1 bacteria more easily recognized and cleared by the body's immune system (such as macrophages), resulting in decreased stability; in addition, insufficient iron leads to insufficient formation of hydroxyl radicals (·OH).

[0039] Comparative Example 2 The difference between this comparative example and Example 3 is that the concentration of the LDG aqueous solution in step (4) is further increased to 3 mg / mL, while the rest is the same as in Example 3. The activity of LDGM obtained by LDG with too high a concentration of LDG also decreases; this is because when the concentration is too high, the distance between the particles is too close, which easily leads to aggregation and flocculation, forming large, irregular clumps, which hinders the exchange between MR-1 and the external environment, affecting its respiration and metabolism.

[0040] Test Example 1 LDGM was synthesized and characterized in vitro. Due to electrostatic interaction, positively charged MgFe-LDH(L) was adsorbed onto the surface of negatively charged MR-1 bacteria, thus constructing a bio-hybrid. Scanning electron microscopy (SEM) revealed a clear nanosheet structure between L and MR-1. Figure 2 (b) of the text, while naked bacteria have a smooth surface ( Figure 2 (a)). Dynamic light scattering (DLS) was used to measure the changes in size and surface charge between L and LDGM. It can be seen that the diameter of LDGM increases significantly, while the Zeta potential decreases in the opposite direction. This is beneficial for achieving long-term circulation in the human body. Figure 2 (c), (d) and (f)).

[0041] Meanwhile, the bioactivity of MR-1 was further measured on agar plates. Figure 2 As shown in (e), the bacterial colonies in pure bacteria and LDGM biohybrids were almost identical, indicating that the integration of MR-1 and L nanosheets did not affect bacterial activity. To determine whether LDGM retained its glucose (Glu) catalytic and lactate (Lac) consumption capabilities, the oxygen content in glucose solution mediated by GOx and its lactate consumption capacity were investigated sequentially. Figure 2As shown in (g), after 24 hours of treatment, the oxygen content in both the LDG and LDGM groups was significantly reduced, indicating that LDG and LDGM containing GOx can undergo glucose-catalyzed reactions in the presence of glucose. Furthermore, the final lactate concentration was significantly reduced in the LDGM+Lac and LDGM+Lac+Glu (O2 excluded) groups, suggesting that MR-1 can effectively break down lactate regardless of the presence or absence of oxygen. Figure 2 In the middle (h), that is, electrons produced by excretion during anaerobic respiration and electrons generated by bacteria consuming lactic acid can both lead to Fe. 3+ Converted to Fe 2+ .

[0042] To further assess the changes in ions during the above process, X-ray photoelectron spectroscopy (XPS) was used to monitor the valence state of iron ions. For example... Figure 2 As shown in (i, j, k, l), the iron valence state in the LDGM+Glu+Lac group significantly changed from 3+ to 2+, and after 24 hours of treatment, Fe... 3+ The area of ​​2p3 / 2 is significantly reduced, while Fe 2+ The area of ​​2p3 / 2 is significantly increased compared to LDGM, indicating that bacterial-mediated bioactivity can promote Fe 3+ Converted to Fe 2+ Naturally, only glucose was consumed in the "LDGM+Glu" group, compared to the LDGM group, from Fe... 3+ To Fe 2+ The amount of Fe transferred accounts for only a small percentage. 3+ A portion of the total. During metabolism, electrons produced can be transferred to Fe. 3+ This allows the "LDGM+Lac" group to continuously consume lactate. Taken together, these results indicate that MR-1 indeed utilizes the consumption of lactate and glucose, aided by high-valence metal ions (such as Fe). 3+ As an electron acceptor, it is consumed, resulting in a significant reduction in the amount of lactic acid and glucose in the environment, as well as a decrease in the content of metal ions.

[0043] Application Example 1 This application example validates the POD (peroxidase) and GPx (glutathione peroxidase) activities of LDGM biohybrids under in vitro conditions. Electron transfer is promoted by subjecting tumor cells to dual starvation, thereby increasing Fe... 3+ Converted to Fe 2+ Then, H2O2 needs to be converted into highly toxic ·OH via a Fenton-like reaction, which is crucial for targeting CDT. Otherwise, the high glutathione levels in tumor cells compared to normal cells make Fe... 3+The reduction reaction can then occur. Therefore, methylene blue (MB) is used to assess the ·OH generation capacity of LDGM biohybrids, since ·OH can decompose methylene blue. Figure 3 As shown in (a), LDGM combines the functions of both POD and GPx nanozymes on a single platform, thus becoming a competitive multifunctional nanozyme. Figure 3 As shown in (b), the control experiment without glucose and / or lactate had no significant effect on the absorbance of MB at 664 nm. In contrast, at pH 5.5, after co-incubating LDGM with glucose and lactate for 60 min, we observed a significant decrease in the absorbance of MB, indicating that... Figure 3 Effective degradation of MB in (b). These results undoubtedly demonstrate the effective generation of ·OH as a degrading agent and lead to significant MB decomposition.

[0044] Furthermore, electron spin resonance (ESR) spectroscopy using a spin trapping agent (5,5-dimethyl-1-pyrrolidine-NOxide, DMPO) has been identified as a simpler tool for determining the type of reactive oxygen species (ROS), such as ·OH. We observed that the control group, which did not contain glucose and / or lactate, produced no signal. Figure 3 (c) Conversely, after incubating LDGM with glucose and / or lactate at pH 5.5 for 15 min, a specific spectrum of the DMPO / ·OH adduct was observed, showing a distinct 1:2:2:1 quadruple signal pattern, which undoubtedly demonstrates the effective generation of extracellular ·OH. Simultaneously, DTNB experiments showed that GSH levels decreased after 1 h of LDGM treatment, which is beneficial for enhancing ·OH generation. Figure 3 Oxidative stress effects in (d). From the perspective of in vitro biomedical applications, smart nanomedicines for tumor therapy should be able to release sensitive drug carriers in response to specific stimuli (such as pH). In LDGM biohybrids, doxorubicin is disrupted by low pH and high glucose and lactate levels in the tumor microenvironment (TME), thereby achieving responsive drug release, which is activated after internalization in tumor cells.

[0045] Subsequently, the drug release behavior of LDGM was investigated by dialysis under Glu, Lac, and Glu+Lac conditions. Compared with the release rates of LDGM+Glu and LDGM+Lac (58.3% and 63.2% within 48 hours, respectively), the drug release rate of LDGM was slower (31.3% within 48 hours), because some energy sources were retained. As expected, a burst of drug release was indeed observed with the addition of Glu+Lac (90.8% within 48 hours, see...). Figure 3(e) This behavior is certainly induced by electrons generated by the dual starvation at the tumor site. Given the relatively high pH (7.4) and low glucose or lactate concentrations in the blood, LDGM remains intact and inactive during blood circulation, thus avoiding accidental leakage and side effects. In contrast, the release of glucose and lactate in response can be effectively achieved within tumor cells, characterized by a relatively low pH (5.0–5.5) and high glucose or lactate concentrations. Therefore, these data clearly demonstrate that LDGM biohybrids can be used as a conductive, stimulus-responsive nanomedicine for tumor therapy.

[0046] Application Example 2 This application example validates ferroptosis in in vitro tumor cells. Reactive oxygen species (ROS), primarily ·OH, are considered major factors capable of simultaneously inducing apoptosis and ferroptosis in tumor cells. Intracellular ROS levels after various treatments were assessed using the 2,7-dichlorofluorescein (DCFH) assay. In all treatment groups, DCFH exhibited significant green fluorescence, indicating a substantial increase in ·OH generation. Furthermore, in the LDGM+Glu, LDGM+Lac, and LDGM+Glu+Lac groups, ROS production was even higher due to the catalytic effect of GOx and the consumption of lactate by MR-1. Figure 4 (b) and (c) in the text.

[0047] Furthermore, Western blotting results showed that the expression level of GPX4 (glutathione peroxidase 4) was lowest after LDGM+Glu+Lac treatment, indicating increased glutathione consumption. Figure 4 In (a) of the study, LDGM+Glu+Lac treatment induced the highest Cas-3 expression, indicating that abundant ROS enhanced apoptosis. It is well known that GSH (reduced glutathione), as a cofactor of GPX4, catalyzes the lipid repair system, and GSH depletion inhibits GPX4, thereby promoting ferroptosis. Therefore, this invention measured intracellular GSH and GPX4 as important antioxidant defense mechanisms protecting tumor cells from ferroptosis. The DTNB assay kit showed significant GSH depletion in all treatment groups, and glucose and lactate further exacerbated the reduction of intracellular GSH. Figure 4(f)). In addition to oxidative stress and redox balance disturbances, ferroptosis is also associated with iron metabolism disorders. Increased iron uptake and decreased iron storage increase the unstable iron pool (LIP), in which ferrous ions promote effective ferroptosis more effectively than ferric ions. Since ferritin is the main intracellular iron storage protein, ferritin expression levels were also examined after various treatments. Ferritin degradation was most severe in the LDGM+Glu+Lac group, indicating that enhanced oxidative stress led to the release of reactive iron from ferritin to replenish LIP, thereby amplifying ferroptosis. Lethal accumulation of lipid peroxides is considered the gold standard marker of ferroptosis. Intracellular lipid peroxide levels were assessed after various treatments using a fluorescent probe of C11-BODIPY. As expected, significant lipid peroxide accumulation in the LDGM+Glu+Lac group indicated the most severe ferroptosis ( Figure 4 (d) and (e) in the figure. These results indicate that LDGM-mediated glucose catalysis and lactate consumption can accelerate electron transfer, exacerbate oxidative stress, and aggravate glutathione deficiency, thereby enhancing a mixture of cell death mechanisms, including apoptosis, ferroptosis, and necroptosis, to optimize antitumor effects.

[0048] Application Example 3 This application example validates in vitro tumor cell apoptosis: doxorubicin (DOX)-induced chemotherapy and reactive oxygen species (ROS) generated through the Fenton reaction may lead to a loss of mitochondrial membrane potential (MMP) and increased mitochondrial membrane permeability. Furthermore, JC-1 staining, as an indicator of MMP changes, was used to describe these alterations. Figure 5 As shown in (a) and (b), specifically ①L, ②LD, ③LDG, ④LDGM+Glu, ⑤LDGM+Lac, ⑥LDGM+Glu+Lac; the LD and LDG groups (similar to the L group) showed strong red fluorescence, indicating minimal changes in MMPs, attributed to incomplete DOX release and only a small amount of ROS production. However, the LDGM+Glu+Lac treatment group exhibited the highest green fluorescence, indicating effective destruction of a large number of mitochondria. The results indicate that LDGM can not only achieve chemotherapy by releasing DOX but also enhance ROS production by utilizing bacterial electron transport. Flow cytometry experiments also showed that the apoptosis and necrosis rates in the experimental groups reached 65.8%, higher than LDGM+Glu (48.8%) and LDGM+Lac (54.1%). Figure 5 (c) and (d) in the data. These data strongly demonstrate that LDGM can simultaneously induce apoptosis and ferroptosis, and that the biohybrid effect of LDGM has an irreplaceable advantage in promoting tumor cell death.

[0049] Application Example 4 This application example evaluated the antitumor effect of LDGM in 4T1 tumor-fed mice: such as Figure 6 As shown in (a), the treatment lasted for 21 days. Consistent with the results of in vitro anti-proliferative assays, LDGM treatment significantly inhibited the primary tumor after 21 days. Figure 6 (b, c, d) (where I is the control group; II is L; III is LD; IV is DOX; V is LDG; VI is LDGM). Notably, during the 21-day treatment period, each treatment group experienced a slight decrease in body weight, indicating that LDGM has no short-term systemic toxicity in mice. Figure 6 (e)

[0050] In addition, LDG also inhibited the growth of the primary tumor. However, on day 21, the tumor inhibition effect in the LD treatment group was not as significant as that in the LDGM group, possibly due to the lack of electron transport capacity in the bacteria. Chemotherapy also showed a less significant growth inhibition effect in the LD treatment group. On day 21, the mice were sacrificed to collect the primary tumor and major organs for further H&E staining and immunohistochemical (IHC) examination. Hematoxylin-eosin staining of the tumors showed large areas of necrosis in the LDGM group. Figure 6 (f, first row) indicates that the combination of ferroptosis and apoptosis may lead to severe tumor tissue damage and the formation of intercellular gaps, which may further promote the deep penetration of the nanocomposite. Immunohistochemical results of Ki67 showed that the LDGM group inhibited the proliferation of tumor cells ( Figure 6 (f, second row) in the text, while TUNEL staining showed that, under the influence of ferroptosis and apoptosis, a large number of cells underwent apoptosis after LDGM ( Figure 6 (f in the third line).

[0051] Application Example 5 This application example validates the tumor-targeting ability of the LDGM biohybrid in vivo. The tumor-targeting ability of the LDGM biohybrid was detected using an in vivo imaging system, with Cy5 as a marker to label L and MR-1 for in vivo fluorescence observation. Specifically, Cy5-labeled L, Cy5-labeled MR-1, and Cy5-labeled L+MR-1 were intravenously injected into 4T1 tumor-bearing mice. Figure 7As shown in (a) and (b), only weak fluorescence was observed in tumor tissue in the Cy5-labeled L group, indicating that L itself could not target and accumulate in the tumor. After injection of Cy5-labeled MR-1, strong fluorescence was detected at the tumor site, and the fluorescence intensity gradually increased over time, indicating that MR-1 bacteria exhibit tumor tropism and can target and colonize specific tumor areas. Unsurprisingly, when the surface of MR-1 bacteria was modified with Cy5-labeled L, the fluorescence intensity of this Cy5-labeled L+MR-1 biocomplex in tumor tissue was significantly enhanced compared to unlabeled Cy5-labeled L, demonstrating that MR-1 bacteria can deliver L to the tumor site and generate efficient accumulation of Cy5-labeled L+MR-1 at the tumor site. Furthermore, optical images of the removed tumor and major organs showed that after 4 hours, Cy5-labeled L+MR-1 had the ability to target and accumulate at the tumor site. Figure 7 (c) and (d) in the figure). After injection of Cy5-labeled L+MR-1, the strong fluorescence intensity of the tumor tissue was maintained for the next 24 hours, and even after 54 hours, indicating that Cy5-labeled L+MR-1 has good tumor targeting and preservation capabilities. Figure 7 (e, f, g, h)). Taken together, these findings suggest that LDGM, with the aid of live MR-1, holds promise for targeted and therapeutic purposes against tumors, thereby combining metabolic therapy and enhancing chemotherapy within local tumor tissue and reducing off-target side effects.

[0052] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a bioactive material for tumor treatment, characterized in that, Includes the following steps: S1. Provides magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin; S2. Glucose oxidase is modified onto the above nanoparticles to obtain a drug-loaded enzyme complex. S3. Disperse the above-mentioned drug-loaded enzyme complex in water and mix it with Shewanella bacteria, then centrifuge and wash to obtain the bioactive material.

2. The preparation method according to claim 1, characterized in that, The preparation method of the magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin in step S1 includes the following steps: S1-1. Prepare magnesium-iron layered bimetallic hydroxide nanoparticles and prepare doxorubicin hydrochloride solution; S1-2. The magnesium-iron layered bimetallic hydroxide nanoparticles obtained above are dispersed in water, mixed and stirred with doxorubicin hydrochloride solution, and centrifuged and washed to obtain magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin.

3. The preparation method according to claim 2, characterized in that, The preparation method of the magnesium-iron layered bimetallic hydroxide nanoparticles in step S1-1 includes: under the protection of an inert gas, according to Mg... 2+ with Fe 3+ Magnesium salt and iron salt were weighed in a molar ratio of 3:1, mixed, and then an alkaline solution was added. The mixture was stirred and centrifuged to obtain a precipitate, which was then resuspended in water. The mixture was heated to react, centrifuged, washed, and freeze-dried to obtain the magnesium-iron layered bimetallic hydroxide nanoparticles.

4. The preparation method according to claim 2, characterized in that, In steps S1-2, the dispersion concentration of the magnesium-iron layered bimetallic hydroxide nanoparticles in water is 0.5~2 mg / mL.

5. The preparation method according to claim 2, characterized in that, In steps S1-2, the concentration of the doxorubicin hydrochloride solution is 0.5~2 mg / mL.

6. The preparation method according to claim 1, characterized in that, Step S2 also includes: S2-1. Prepare glucose oxidase aqueous solution by dispersing magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin in water, mixing and stirring with glucose oxidase aqueous solution, and centrifuging to obtain drug-loaded enzyme complex. The concentration of the glucose oxidase aqueous solution is 1-4 mg / mL, and the concentration of the aqueous solution of the magnesium-iron layered bimetallic hydroxide nanoparticles loaded with doxorubicin is 1-4 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step S3, the concentration of Shewanella is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

8. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the drug-loaded enzyme complex in water is 0.5~2 mg / mL.

9. A bioactive material obtained by the preparation method according to any one of claims 1 to 8.

10. The application of the bioactive material as described in claim 9 in tumor treatment.

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