Anesthesia coating spore as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses an anesthesia coating spore as well as a preparation method and application thereof. The coating spore comprises a spore and a ferric iron-propofol coating attached to the body surface of the spore, and the coating is formed through metal-phenol complexing and pi-pi stacking. The spores are dormant bodies of collagenase-producing bacteria. The coating spores can be selectively germinated and colonized in a tumor hypoxic microenvironment, Fe < 3 + > can promote bacterial proliferation, loaded propofol inhibits tumor metastasis by inhibiting migration and invasion of tumor cells, generated collagenase degrades tumor collagen, meanwhile, Fe < 3 + > is reduced into Fe < 2 + > by glutathione in the cells, and the coating spores can be selectively colonized in the tumor hypoxic microenvironment. Therefore, a powerful Fenton reaction is induced to trigger lipid peroxidation, and ferroptosis of tumor cells is initiated. The injection of the anesthetic coating spores in the single tumor not only effectively ablates the primary tumor and inhibits the growth of the tumor, but also significantly inhibits the distal pulmonary metastasis of the in-situ tumor model.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an anesthetic coated spore, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose a serious threat to human health, often recurring after surgery and threatening life due to their rapid growth. Collagen, the most abundant component of the tumor extracellular matrix (ECM), with type I collagen being the most abundant, determines the density and rigidity of the tumor, and is a significant factor in accelerating its progression. Furthermore, while increasing ECM density, collagen also creates a high interstitial pressure microenvironment, which is extremely detrimental to interventions such as radiotherapy, chemotherapy, and immunotherapy. Some studies have proposed that injecting collagenase to break down type I collagen in the tumor matrix can improve the interstitial transport of antibody drugs, and experimental results show that collagenase can enhance anti-tumor efficacy. However, enzyme preparations suffer from poor stability in vivo, short half-life, and the need for repeated administration leading to poor patient compliance. They also face challenges such as cumbersome production processes and difficult storage, limiting the application of collagenase in anti-tumor therapy. In addition, off-target effects cause serious toxicity, as non-specific degradation of systemic collagen can lead to potential bleeding in healthy organs, significantly hindering the utilization of collagenase in tumor treatment.

[0003] Bacteria, due to their anaerobic tendency, possess excellent tumor targeting and colonization capabilities, and can secrete toxins and compete for nutrients within tumors, making them highly promising anti-tumor materials. As living biological materials, bacteria can continuously secrete protease molecules at the tumor site, avoiding the need for repeated administration of proteases and their degradation and clearance in systemic circulation, thus improving anti-tumor efficacy and providing a strategy for long-term secretion of collagenases to inhibit tumor malignant development. However, it is important to note that after using trypsin to degrade type I collagen, cancer cells may invade the basement membrane, promoting metastasis. Currently, there is no research on the synergistic effect of collagen degradation in inhibiting tumor progression and metastasis. Summary of the Invention

[0004] To address the aforementioned problems, this invention utilizes the interaction between the bacterial interface and active substances to attach a coating to the surface of bacteria, thereby increasing their biological activity or other exogenous properties. It provides an anesthetic-coated spore, which, through metal-phenol complexation and π-π stacking interactions, enables Fe... 3+ - Propofol forms a network coating on the surface of the spores, constructing anesthesia-coated spores (Spore@PF), which utilize the spores to germinate and colonize in the hypoxic environment of the tumor to achieve precise tumor treatment.

[0005] The technical solution of the present invention is as follows:

[0006] An anesthetic coating spore of the present invention includes the spore and a ferric iron-propofol coating attached to its surface, the ferric iron-propofol coating being formed by metal-phenol complexation and π-π stacking.

[0007] In one embodiment of the present invention, preferably, the spores are dormant forms of collagenase-producing bacteria.

[0008] In one embodiment of the present invention, preferably, the bacteria is a collagenase-producing Clostridium bifermentans RJ-1, which was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on June 27, 2024, with accession number GDMCC 64809.

[0009] This invention also discloses a method for preparing the above-mentioned anesthetic-coated spores, comprising the following steps: taking 1×10 6 ~1×10 10 The CFU spores were suspended in ultrapure water, and 10 μL of a 25% glycerol ester solution of 0.5–45 mM propofol was added. Then, 10 μL of a 0.05–0.5 mM FeCl3 solution was added and mixed. The mixture was then washed with Tween-80 and ultrapure water in sequence, and centrifuged to obtain coated spores.

[0010] The present invention also discloses the application of the above-mentioned anesthetic-coated spores in the preparation of antitumor products for prevention, treatment and / or adjuvant therapy.

[0011] In one embodiment of the present invention, preferably, the tumor is breast cancer.

[0012] In one embodiment of the present invention, preferably, the tumor is an extracellular matrix-dense tumor.

[0013] The present invention also discloses a product for the prevention, treatment and / or adjuvant treatment of tumors, the product comprising the anesthetic-coated spores as described above.

[0014] In one embodiment of the present invention, preferably, the product is a drug.

[0015] In one embodiment of the present invention, preferably, the drug further includes pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention provides an anesthetic-coated spore, which can selectively germinate and colonize in the hypoxic microenvironment of tumors. The anesthetic coating contains Fe... 3+It can promote bacterial proliferation. Bacteria in tumors produce collagenase, which degrades tumor collagen, while propofol loaded onto coated spores inhibits tumor metastasis by suppressing tumor cell migration and invasion. Simultaneously, Fe... 3+ Reduced to Fe by intracellular glutathione 2+ This induces a potent Fenton response to trigger lipid peroxidation and ultimately induces ferroptosis in tumor cells. In addition to biosafety, a single intratumoral injection of anesthetic coated spores not only effectively ablates the growth of the primary tumor and slows tumor recurrence, but also significantly inhibits distant lung metastases in an in situ tumor model. This invention presents an attractive platform for developing advanced bacteria-based therapeutic strategies, providing a precise platform for the treatment and prevention of metastases in malignant tumors. Attached Figure Description

[0018] Figure 1 The image shows the characterization of the coated spore Spore@PF in Example 1 of the present invention, wherein (a) is a schematic diagram of the structure of the coated spore Spore@PF; (b) is a scanning electron microscope image of the spore and the coated spore Spore@PF (scale bar: 500 μm); and (c) is an X-ray energy dispersive spectroscopy (EDS) analysis image of the coated spore Spore@PF.

[0019] Figure 2 This is a statistical chart of bacterial counts for the proliferation or tumor colonization of coated spores Spore@PF in Example 2 of the present invention, wherein (a) OD 600nm (a) When the concentration is 0.01, the number of bacteria (CFU, n=3) in Spore and Spore@PF on DCA plates; (b) Under anaerobic conditions at 37℃, the OD 600nm (c) The growth curves of Spore and Spore@PF in BHI medium were monitored and fitted by Prism (GraphPad); (d) The bacterial count (CFU, n=3) of Spore and Spore@PF cultured in BHI medium for 24 hours under anaerobic conditions at 37℃ was obtained by DCA plate smearing; (e) When the tumor size was approximately 100 mm 3 At that time, inject 1×10 into the tumor. 8 After 24 hours of CFU Spore or Spore@PF (n=4), count the number of bacteria in the tumor; (e) when the tumor size is approximately 100 mm 3 At that time, inject 1×10 into the tumor. 8 After 24 hours of CFU Spore or Spore@PF (n=4), the number of bacteria in the organs was counted; all data are mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test, with P-values ​​(*P<0.05).

[0020] Figure 3To promote collagen degradation by the coating spores using Spore@PF in Example 3 of this invention, (a) after NIH-3T3 cells were co-incubated with Spore or Spore@PF for 24 h, the relative expression level of collagen was detected by Sirius red staining (n=4); (b) collagenase degraded collagen into hydroxyproline, 2×10 6 NIH-3T3 cells and 2×10 7 (c) Relative hydroxyproline concentrations after co-incubation with CFU Spore or Spore@PF (MOI:10) for different times (n=4); (c) When the tumor reaches 100 mm 3 At the same time, 50 μL of PBS, FeCl3 (24 μg), propofol (80 μg), and Spore (1×10⁻⁶) were injected intratumorally. 8 CFU), Spore@PF (1×10) 8 Schematic diagram of Sirius red staining on tumor fixed sections after 48 hours (CFU), scale bar, 200 μm. All data are mean ± standard deviation. Statistical analysis: Sirius red staining was analyzed using Turkey's test with one-way ANOVA, and hydroxyproline concentration was analyzed using multiple Student's tests. P-value (*P<0.001).

[0021] Figure 4 Example 4 of this invention describes the effect of coated spores (Spore@PF) on inhibiting tumor invasion and metastasis. (a) Microscopic images of 4T1 cells migrating from tumor spheres to the base of the 4T1 chambers after co-culturing with PBS, Spore, or Spore@PF; (b) Statistical analysis of the relative area of ​​4T1 cells below the chamber base (n=3). Scale bar: 50 μm. (c) H&E staining of lung tissue, with green lines representing metastatic tumor foci in the lungs. Scale bar: 2 mm. All data are mean ± SD. Statistical analysis was performed using Turkey's test with one-way ANOVA, and P-values ​​are given (*P<0.05, **P<0.01).

[0022] Figure 5 Example 5 of this invention describes how coated spores (Spore@PF) promote lipid peroxidation in tumor cells. (a) A photograph of the coating on Spore@PF after treatment with GSH solution for 10 minutes at room temperature; (b) Fe content of Spore and Spore@PF was measured using phenanthrene after exposure to 2 mm GSH for 24 h. 2+ Concentration (n=3); (c) 2mM GSH and 1×10 8After incubating CFU Spore or Spore@PF in BHI medium for 2 hours, GSH levels were detected (n=3); (d) the antioxidant activity of Spore or Spore@PF was determined by ABTS free radical scavenging assay (n=3); (e) and free radical scavenging assay (f) (n=3). (g) 4T1 cells were incubated with Spore or Spore@PF (MOI:10) for 2 hours, and then labeled with DCFH-DA probe (green) to quantify intracellular ROS by fluorescence intensity (n=3). (h) When the tumor reached 100 mm 3 Mice were randomly divided into 5 groups and injected intratumorally with 50 μL PBS, FeCl3 (24 μg), propofol (80 μg), and Spore (1 × 10⁻⁶), respectively. 8 CFU), Spore@PF (1×10) 8 CFU). Tumors were collected on day 19 post-treatment, and GPX-4 expression in 4T1 tumors was analyzed by Western blot. β-Actin was used as an internal control. Data are presented as mean ± SD. Two-tailed Student's t-tests were used for two groups, and Turkey's test for one-way ANOVA was used for multiple groups. P values ​​were *P<0.05 and **P<0.001, respectively.

[0023] Figure 6 Phylogenetic tree of 16S ribosomal RNA sequencing data of collagenase-producing Clostridium bifermentans RJ-1 in Example 1 of this invention. Detailed Implementation

[0024] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0025] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0026] Example 1: Preparation and Characterization of Coated Spores Spore@PF

[0027] Leachate was obtained from soil samples at the East Branch of Renji Hospital in Pudong New Area, Shanghai. The leachate was treated with a 60°C water bath for 30 min, and then 1 mL of the leachate was transferred to 50 mL of enhanced Clostridium tumefaciens liquid culture medium and incubated anaerobically at 37°C for 12 h. Subsequently, 100 μL of the liquid was spread onto Clostridium tumefaciens identification agar plates containing 3% skim milk and incubated at 37°C for 12 h until colonies formed. To prepare this plate, 3% sterilized skim milk was added to the liquid agar and solidified together.

[0028] Black colonies with the largest degradation loop were selected to isolate the Clostridium strain with the highest collagenase yield. The strain was then transferred to enhanced Clostridium broth for amplification for 12 hours. One mL of the bacterial culture was used for 16S ribosomal RNA sequencing. The strain was identified as *Clostridium bifermentans* by 16S ribosomal RNA sequencing and named RJ-1. The phylogenetic tree of its 16S ribosomal RNA sequencing data is shown below. Figure 6 As shown. This strain of Clostridium was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on June 27, 2024, with accession number GDMCC 64809.

[0029] Collagenase-producing *Paraclostridium bifermentans* RJ-1 was selected from DCA agar plates. It was transferred to brain heart extract (BHI, 5 mL) containing 0.4 g / L D-cycloserine and cultured overnight. Then, it was transferred to 1 L of fresh BHI medium and cultured for 10 days to obtain dormant bacterial spores producing collagenase. The mixture was then centrifuged at 69400 g for 50 minutes and resuspended in 50 mL of sterile phosphate-buffered saline (PBS). The mixture was then boiled at 70 °C for 20 minutes and centrifuged at 69400 g for 50 minutes to obtain the precipitate. After incubation overnight at 4°C, the precipitate was resuspended in 50 mL of sterile ultrapure water and then purified by sucrose density gradient centrifugation. Equal volumes of 60%, 40%, 20%, and 10% sucrose were added sequentially to 50 mL centrifuge tubes, and an equal volume of the mixture was slowly added to the top. The prepared sample was centrifuged at 800 g for 45 minutes at 4°C under gentle acceleration and deceleration conditions. The spores in the middle layer were collected and washed three times with PBS to obtain spores, which were designated as Spores.

[0030] Take 1×10 6 ~1×10 10CFU spores (Spore) were washed with ultrapure water and suspended in 1 mL of ultrapure water. Then, 10 μL of propofol (0.5–45 mM, 25% glycerol ester solution, sonicated) was added to the spore suspension and vortexed for 1 minute. Subsequently, 10 μL of FeCl3 (0.05–5 mM) was added and stirred for 1 minute, followed by washing three times with 0.01% Tween-80, then three times with ultrapure water. The mixture was centrifuged at 13000 g for 3 minutes to obtain coated spores, denoted as Spore@PF. A schematic diagram of its structure is shown below. Figure 1 As shown in (a).

[0031] The morphology and composition of spores and coated spores@PF were observed using scanning electron microscopy (SEM, SU3800, HITACH). Figure 1 As shown in (b), the surface of Spore@PF is rougher than that of Spore spores. Iron element localization was performed using field emission transmission electron microscopy (FE-TEM, Talos). TM F200i, Thermo Scientific TM ) to conduct an evaluation, such as Figure 1 As shown in (c), energy-dispersive X-ray spectroscopy (EDS) mapping analysis confirmed the formation of complete Fe on Spore@PF. 3+ - Propofol coating, the surface of which shows an iron-based coating, forms coating spores Spore@PF through metal-phenol complexation and π-π stacking.

[0032] Example 2: Coating Promotes Bacterial Growth: Survival Rate and Growth Curve of Spore@PF

[0033] To assess whether the coating affects spore germination and promotes bacterial proliferation, 1×10⁻⁶ spores were applied to each spore. 7 CFU-containing Spore spores and coated spores Spore@PF were suspended in 1 mL of BHI medium containing 0.4 g / L D-cycloserine and incubated at 37°C. The optical density (OD) value of the bacterial suspension at 600 nm was measured and recorded hourly using a microplate reader. Simultaneously, serial dilutions were performed, with 100 μL of the bacterial suspension spread onto DCA plates. These plates were then incubated overnight under anaerobic conditions at 37°C, and the bacterial count was statistically analyzed.

[0034] When OD 600nm When the concentration is 0.01, after anaerobic incubation at 37°C for 24 hours, the number of Spores and Spore@PF (CFU, n=3) is counted on Clostridium difficile differential agar (DCA) plates. Figure 2As shown in (a), the bacterial plate count results confirm that the number of important bacteria is comparable between the Spore group and the Spore@PF group, which means that the effect of the coating on spore germination is negligible.

[0035] When OD 600nm When the concentration was 0.01, the cells were cultured under anaerobic conditions at 37°C for 24 hours in BHI medium, and the OD was measured. 600nm To monitor bacterial growth curves, the curves were obtained by fitting Prism (GraphPad), such as... Figure 2 As shown in (b), compared with Spore, the experiment found that the growth of Spore@PF bacteria was significantly improved, such as Figure 2 As shown in b.

[0036] Under anaerobic conditions at 37℃, when OD 600nm At a concentration of 0.01, Spore and Spore@PF were cultured in BHI medium for 24 hours, and the bacterial count (CFU, n=3) was performed on DCA plates. The plate count results showed that Spore@PF exhibited a 3-fold increase in bacterial proliferation compared to Spore. Figure 2 As shown in c.

[0037] Cell culture: Mouse breast cancer cells 4T1 were cultured in high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin (100 U / mL penicillin and 100 μg / mL streptomycin) in a 37°C incubator with 5% carbon dioxide.

[0038] The experimental animals were 6-8 week old female BALB / c nude mice, purchased from Spiefol (Beijing) Biotechnology Co., Ltd. All animal experiments were conducted in an environment free of specific pathogens. All animal experiments were carried out in accordance with the "Guidelines for Laboratory Animal Care and Use" issued by the Shanghai Science and Technology Commission.

[0039] Inoculate 2×10⁻⁶ mice mammary fat pads 5 / mL (50μL) 4T1 cells, when the tumor reaches 100mm 3 Mice were randomly divided into two groups, and 50 μL of Spore spores (1 × 10⁻⁶) were injected into the tumor of each group. 8 CFU) and coated spores Spore@PF (1×10) 8 CFU). Tumor tissue was collected 24 hours later, homogenized using a tissue homogenizer (60 Hz, 180 seconds), serially diluted, and 100 μL of bacterial suspension was plated on DCA plates. The plates were then incubated overnight at 37°C under anaerobic conditions. The number of collagenase-producing bacteria was then counted. Results showed a significant increase in bacterial count in the Spore@PF group tumor tissue, approximately 25 times that of the Spore group. Figure 2As shown in d. It is noteworthy that, comparing the number of viable bacteria in the major organs of mice (including the heart, liver, spleen, lungs, and kidneys), approximately 99% of the detected bacteria were concentrated in the tumor site, indicating that the bacteria have a characteristic of colonizing only in tumors, such as... Figure 2 As shown in e.

[0040] Example 3: Coating promotes collagen degradation after Spore@PF germination.

[0041] NIH 3T3 cells were seeded in 12-well plates (2 × 10⁶ cells per well). 5 (cells), and respectively with 2×10 6 CFU cells containing Spore and Spore@PF were co-cultured under anaerobic conditions for 24 hours. Cells were washed with PBS, fixed with 4% PFA for 20 minutes at room temperature, washed three times with water, and then stained with Sirius red dye at room temperature (RT) for 1 hour. After three washes with water, collagen in the cells was dissolved in a weakly alkaline solution (0.5% NaOH), and the absorbance at 540 nm was recorded using a microplate reader (BioTek, USA). After co-incubating NIH-3T3 cells with Spore and Spore@PF for 24 hours, the relative expression level of collagen was detected by Sirius red staining (n=4). The collagen degradation capacity of spores was evaluated using NIH-3T3 fibroblasts, the main collagen-producing cells. After co-incubation of NIH-3T3 cells with Spore or Spore@PF for 24 hours, Sirius red staining showed that the collagen degradation rates of Spore and Spore@PF were ~50% and ~80%, respectively, indicating that the coating increased the collagen degradation rate of the bacteria. Figure 3 As shown in a.

[0042] Simultaneously, a more sensitive hydroxyproline assay kit was used to quantify the efficiency of collagen degradation, as collagen can be degraded into hydroxyproline by collagenase. The hydroxyproline assay kit was used to detect collagen degradation products. The specific steps are as follows: 2×10 6 NIH-3T3 cells were respectively combined with 2×10 7 The relative hydroxyproline concentrations (n=3) after co-incubation of CFU Spore and Spore@PF (MOI:10) for different times were then analyzed. Cells were then collected, lysed, and analyzed according to the manufacturer's instructions. As expected, a significant increase in hydroxyproline levels over time was observed in the Spore@PF group compared to Spore, such as... Figure 3 As shown in b.

[0043] Spore@PF-mediated collagen degradation in vivo:

[0044] Inoculate 2×10⁻⁶ mice mammary fat pads 5 / mL (50μL) 4T1 cells, when the tumor reaches 100mm3 Mice were randomly divided into five groups and injected intratumorally with 50 μL of PBS, FeCl3 (24 μg), propofol (80 μg), and Spore (1 × 10⁻⁶). 8 CFU) or Spore@PF (1×10) 8 CFU). Tumors were collected at 48 hours, followed by fixation in 4% paraformaldehyde (PFA), paraffin embedding, and sectioning for Sirius red staining to indicate collagen expression in the tumor. 48 hours after drug administration, Sirius red staining was used to analyze collagen expression in the tumor tissue, such as... Figure 3 As shown in Figure c, the results indicate that Spore@PF significantly reduced collagen expression in tumors.

[0045] Example 4: Coated spores inhibit tumor metastasis

[0046] To verify that Spore@PF coated spores can inhibit tumor metastasis, an in vitro 4T1 tumor sphere invasion assay was used to demonstrate that Spore@PF has an inhibitory effect on tumor metastasis. 4T1 tumor spheres were prepared using the hanging drop technique, consisting of 20 μL of 4T1 cells (2 × 10⁻⁶ cells). 5 Mix with 0.24% carboxymethyl cellulose and drop onto the lid of a 100mm culture dish. Invert the lid onto a culture dish containing 10mL of sterile water. Incubate at 37°C with 5% CO2 for approximately 5 to 7 days, allowing sufficient gravity sedimentation to form tumor spheres. Add cell culture medium containing 1% FBS to the upper chamber and cell culture medium containing 10% FBS to the basal chamber. Collect 10 tumor spheres and place them in the upper chamber with Spore and Spore@PF (1×10⁻⁶) respectively. 7 CFU (cells containing 3 FU) were co-cultured under anaerobic conditions for 24 hours (n=3). The chambers were fixed with 4% paraformaldehyde for 30 minutes, and the cells in the upper chambers were gently wiped with a cotton swab and stained with 0.1% crystal violet for 20 minutes at room temperature. Then, the cells were washed three times with water and imaged under a microscope. The imaging results of the invasion experiment are shown below. Figure 4 As shown in a, and the statistical results are as follows: Figure 4 As shown in b, the experiment showed that, compared with the PBS and Spore groups, the Spore@PF group of cells at the bottom of the Transwell chamber had fewer 4T1 tumor cells invading.

[0047] In vivo metastasis inhibition experiments were demonstrated using a mouse orthotopic breast cancer model. Specifically, 2 × 10⁶ cells were inoculated onto the mammary fat pads of mice. 5 / mL (50μL) 4T1 cells, when the tumor reaches 100mm 3 Mice were randomly divided into five groups and injected intratumorally with 50 μL of PBS, FeCl3 (24 μg), propofol (80 μg), and Spore (1 × 10⁻⁶). 8CFU) and Spore@PF (1×10) 8 CFU). Lung tissue from mice was collected on day 19 after treatment, followed by fixation in 4% paraformaldehyde (PFA), paraffin embedding, and sectioning for hematoxylin and eosin (H&E) staining to analyze lung metastasis. Results showed that Spore treatment indeed promoted lung metastasis, while Spore@PF inhibited its occurrence. Figure 4 As shown in c.

[0048] Example 5: Coated spores induce Fenton effect, leading to ferroptosis in tumor cells.

[0049] Spore and Spore@PF (1×10) respectively 10 CFU spores (1 mL) were incubated with 2 mM GSH at room temperature for 10 minutes. The experiment observed that the Spore@PF coating decomposed after 10 minutes of treatment with 2 mM GSH. Figure 5 As shown in a.

[0050] Fe 3+ Reduced to Fe 2+ This is the first step in triggering the Fenton reaction. Fe was quantitatively determined using a colorimetric method based on phenanthroline (Ferrozine). 2+ 0.5% 1,10-o-phenanthroline dissolved in 0.1 mM hydrochloric acid solution was used to determine Fe in Spore@PF after exposure to GSH. 3+ Converted to Fe 2+ The process involves applying Spore and Spore@PF (1×10⁻⁶) to the appropriate pores. 10 CFU was suspended in 2 mM GSH at room temperature for 10 minutes (n=3). After centrifugation at 10000g for 3 minutes, the supernatant was collected and then incubated with the detection solution (100 μL, containing 10 μL of FeSO4 and 10 μL of fresh Ferrozine solution) at 37°C for 10 minutes. The absorbance was measured at 570 nm using a microplate reader. O-phenanthroline colorimetric analysis showed that GSH accelerated coating degradation and promoted Fe... 3+ Converted to Fe 2+ ,like Figure 5 As shown in b, the analysis of o-phenanthroline indicates that this is due to Fe. 3+ Converted to Fe 2+ Caused by.

[0051] The concentration of GSH was assessed using a detection kit to evaluate the ability of Spore@PF to consume GSH. In brief, Spore and Spore@PF (1×10⁻⁶) were used to separately detect GSH concentrations. 8CFU was incubated with 2 mM GSH for 10 minutes, and then the supernatant was collected (n=3). Subsequently, 150 μL of the GSH detection working solution containing GSH reductase and DNTB solution was added to 50 μL of the result supernatant, mixed, and incubated at room temperature for 25 minutes. After adding 50 μL of 0.5 mg / mL NADPH solution, the absorbance at 412 nm was measured using a microplate reader. In the total glutathione assay, the GSH concentration decreased after incubation in the Spore@PF group, while no change in GSH concentration was observed in the non-Spore group. Figure 5 As shown in c, this indicates that GSH in Fe 3+ The restoration process is consumed.

[0052] 2,2′-N-bis(3-ethylbenzothiazoline-6-sulfonic acid)ABTS radical scavenging assay: Spore@PF consumes GSH, leading to a decrease in antioxidant activity. ABTS radical scavenging assay is based on the formation of ABTS· by free radical cations. + The colorimetric method was used for determination. In brief, it involves mixing ABTS and K₂S₂O₈ to form ABTS· + Free radical cation solutions were mixed and then placed in the dark at room temperature for 16 hours. Spore and Spore@PF (1×10⁻⁶) were then separately added. 8 Incubate with 2 mM GSH for 10 minutes, then collect the supernatant (n=3). Add ABTS· + Add 200 μL of the solution to 10 μL of the result supernatant, mix, and incubate in the dark at room temperature for 5 minutes. Measure the absorbance at 734 nm using a microplate reader. ABTS assay further confirms the ABTS effect of the Spore@PF group. + A high concentration indicates a decrease in the overall antioxidant capacity of the solution, corresponding to a low concentration of GSH, such as... Figure 5 As shown in d.

[0053] To detect the GSH level of Spore@PF in cells, 4T1 cells were seeded into 6-well plates (5 × 10⁶ cells per well). 5 Incubate overnight in Spore or Spore@PF (5×10⁻⁶) solution. Add Spore or Spore@PF to each well. 6 The cells were co-incubated at 37°C under anaerobic conditions for 2 hours (n=3). After washing three times with cold PBS, 4T1 cells were collected and lysed in 200 μL PBS. Samples were prepared by centrifugation at 13000g for 10 minutes at 4°C, and the supernatant was separated. GSH detection was performed using the same method. The results showed that Spore@PF-treated 4T1 tumor cells effectively consumed GSH, such as... Figure 5 As shown in e.

[0054] To test the ability of Spore@PF to scavenge free radicals in cells, 4T1 cells were seeded into 6-well plates (5 × 10⁶ cells per well). 5 Incubate overnight in Spore or Spore@PF (5×10⁻⁶) 6 The cells were co-cultured at 37°C for 2 hours (n=3). ABTS free radical scavenging was performed as above. The results showed that Spore@PF treatment effectively consumed GSH and reduced total antioxidant capacity in 4T1 tumor cells. Figure 5 As shown in e-5f.

[0055] Fe 2+ The Fenton reaction between tumor-endogenous peroxides and reactive oxygen species (ROS) can generate reactive oxygen species (ROS), which can induce lipid peroxidation and ferroptosis. ROS detection: 4T1 cells (5×10⁻⁶) 5 The cells were inoculated overnight in 3.5 mm glass-bottomed culture dishes and incubated in 37°C basal cell culture medium with Spore or Spore@PF (5 × 10⁻⁶). 6 Cells were co-cultured for 2 hours, washed three times with PBS, and then intracellular ROS levels were detected using a DCFH-DA (2,7-dichloro-diacetic acid fluorescein) fluorescent probe (n=3). Cells were incubated with 5 μM DCFH-DA working solution at 37°C for 30 minutes. After washing three more times with PBS, the fluorescence of DCFH-DA in the cells was detected using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The results showed that after co-incubation with Spore@PF for 2 hours, the intracellular ROS level of 4T1 cells was significantly higher than that of the Spore group cells. Figure 5 As shown in g.

[0056] The effects of Spore@PF on tumor ferroptosis were evaluated using lipid peroxidation inhibitory molecules. Downregulation of glutathione peroxidase 4 (GPX4, a key lipid peroxidation inhibitory molecule) levels suggested increased lipid peroxidation. 2 × 10⁶ cells were injected into the mammary fat pads of mice. 5 / mL (50μL) 4T1 cells, when the tumor reaches 100mm 3 Mice were randomly divided into five groups and injected intratumorally with 50 μL of PBS, FeCl3 (24 μg), propofol (80 μg), and Spore (1 × 10⁻⁶). 8 CFU) or Spore@PF (1×10) 8CFU). Tumors were collected after treatment on day 19. Tissue proteins were extracted using a kit, and all tissue proteins were separated by 4–12% SDS-PAGE and transferred to a membrane. The protein membrane was washed with 0.1% Tween 20 TBST. Non-specific proteins were blocked by incubation with rapid blocking buffer at room temperature for 10 minutes. Then, the membrane was incubated overnight at 4°C with primary antibody GPX4 (1:1000), COL1A (1:200), or β-Actin (1:5000). Subsequently, the membrane was washed three times with 0.1% Tween 20 TBST for 10 minutes each time. The membrane was then incubated with secondary antibody at room temperature for 2 hours, and finally washed three times with 0.1% Tween 20 TBST for 10 minutes each time. Protein bands were imaged using a chemiluminescence (ECL) system (GE & Amersham Imager 680R). Western blot results showed that GPX4 was significantly reduced in Spore@PF treated mouse tumor tissues, such as... Figure 5 The results, shown in h, indicate that Spore@PF promotes ROS production and lipid peroxidation in tumor cells.

[0057] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An anesthetic-coated spore, characterized in that, It includes the spores and a ferric-propofol coating attached to their surface, the ferric-propofol coating being formed by metal-phenol complexation and π-π stacking.

2. The anesthetic coated spores according to claim 1, characterized in that, The spores are dormant forms of collagenase-producing bacteria.

3. The anesthetic-coated spores according to claim 2, characterized in that, The bacteria in question is a collagenase-producing clostridium bifermentans RJ-1, which was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on June 27, 2024, with accession number GDMCC 64809.

4. A method for preparing anesthetized coated spores as described in any one of claims 1-3, characterized in that, Includes the following steps: Take 1×10 6 ~1×10 10 The CFU spores were suspended in ultrapure water, and 10 μL of a 25% glycerol ester solution of 0.5–45 mM propofol was added. Then, 10 μL of a 0.05–0.5 mM FeCl3 solution was added and mixed. The mixture was then washed with Tween-80 and ultrapure water in sequence, and centrifuged to obtain coated spores.

5. The use of the anesthetic-coated spores as described in any one of claims 1-3 in the preparation of antitumor products for prevention, treatment and / or adjuvant therapy.

6. The application according to claim 5, characterized in that, The tumor in question is breast cancer.

7. The application according to claim 6, characterized in that, The tumor in question is a tumor with dense extracellular matrix.

8. A product for the prevention, treatment, and / or adjuvant therapy of tumors, characterized in that, The product comprises the anesthetic coated spores as described in any one of claims 1-3.

9. The product according to claim 8, characterized in that, The product in question is a medicine.

10. The product according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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