Chiral supramolecular nano assembly as well as preparation method and application thereof

Abstract

The invention discloses a chiral supramolecular nano assembly as well as a preparation method and application thereof, and belongs to the technical field of biomedical nano materials. The nano assembly (ZnO2 (at) CDP / Fc-DA) is constructed by taking zinc peroxide as a core, modifying a supramolecular ligand CDP through ligand exchange and combining D-alanine functionalized ferrocene guest molecules (Fc-DA) by virtue of interaction of a host and a guest. According to the assembly, the specific targeting effect of D-alanine on tumor tissues and gram-positive bacteria in tumors is utilized, Zn < 2 + > and H2O2 are decomposed and released in an acid lysosome environment of tumor cells, hydroxyl free radicals are generated under the catalysis of ferrocene, and the bacteria and the tumor cells are synchronously induced to generate ferroptosis. The microbial DNA released by bacterial ferroptosis can further activate the cGAS-STING innate immune pathway, reverse the immunosuppression microenvironment and synergistically enhance the anti-tumor immune response. According to the invention, the collaborative treatment of precise removal and immune activation of bacteria in the tumor is successfully realized, and a new effective strategy is provided for treating the breast tumor with bacteria colonization.

Description

Technical Field

[0001] This invention belongs to the field of chiral nanomaterials and medical technology, and relates to a chiral supramolecular nanoassembly, its preparation method, and its application in the treatment of bacterial-colonized breast tumors. Background Technology

[0002] The tumor microbiome, particularly the bacterial community colonizing tumor cells, has been shown in recent years to disrupt anti-tumor immunity and drive disease progression. These intratumoral bacteria primarily enhance the metastatic potential of tumors by nourishing the immunosuppressive environment, including inducing T cell inactivation, causing reactive oxygen species (ROS) dysregulation, and leading to systemic immunosuppression. Therefore, specifically eradicating bacteria within tumor cells holds promise as a novel therapeutic strategy to overcome bacterial-driven immunosuppression and tumor metastasis.

[0003] Current methods for eliminating intratumoral bacteria have many limitations. Traditional antibiotics suffer from insufficient cell penetration and indiscriminate damage to the human symbiotic microbiota, potentially leading to dysbiosis and secondary infections. While engineered bacterial therapies possess natural tumor-targeting properties, their colonization dynamics in vivo are difficult to predict, and they pose potential systemic toxicity risks. Photodynamic / photothermal therapy (PDT / PTT) is limited by insufficient tissue penetration depth and the potential for non-specific thermal damage to surrounding normal tissues. Crucially, these methods face challenges in achieving precise spatiotemporal targeting of intracellular bacteria within tumor cells and struggle to adequately or efficiently activate antitumor immune responses. Therefore, there is an urgent clinical need to develop antibacterial methods that are spatially precise, highly efficient, and synergistic with antitumor immunity.

[0004] Ferroprelation, an iron-dependent regulated cell death pathway driven by lethal lipid peroxidation (LPO), offers attractive therapeutic opportunities for antitumor and antibacterial applications. Notably, bacteria's primitive antioxidant defense mechanisms make them vulnerable to redox balance, making them more susceptible to ferroptosis than eukaryotic cells, which possess more sophisticated antioxidant systems, such as the glutathione system. Crucially, bacterial ferroptosis releases microbial DNA, which, as a potent agonist of the cyclic GMP-AMP synthase-interferon gene stimulator (cGAS-STING) pathway, can activate this key innate immune pathway. This provides a unique opportunity to reprogram the immunosuppressive tumor microenvironment into an immunostimulated state.

[0005] Achieving precise spatiotemporal targeting of intracellular bacteria in tumor cells remains a significant technological challenge. Chiral nanomaterials offer a promising solution, leveraging their stereochemical properties and biocompatibility to achieve selective delivery to tumor sites while minimizing off-target effects. Metagenomic analysis has confirmed the dominance of Gram-positive bacteria (such as Staphylococcus, Lactobacillus, Enterococcus, and Streptococcus) in the breast cancer microbiota. Utilizing this property, D-alanine—a key component of peptidoglycans in Gram-positive bacteria—can serve as an ideal targeting group, and studies have demonstrated its ability to specifically label intracellular bacteria in human breast tumors. Therefore, D-alanine-functionalized chiral nanomaterials exhibit significant potential for precise targeting of intratumoral bacteria.

[0006] In conclusion, developing a novel therapeutic platform that can simultaneously achieve precise targeting and efficient clearance of intratumoral bacteria and activate anti-tumor immune responses is of significant scientific and clinical value for advancing tumor microbiome-related therapeutic strategies. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a chiral supramolecular nanoassembly, its preparation method, and its application in the treatment of bacterial-colonized breast tumors.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The present invention first provides a chiral supramolecular nanoassembly, wherein the nanoassembly uses zinc peroxide nanoparticles as the core, and the surface of the nanoassembly is modified with supramolecular ligand CDP through ligand exchange, and binds guest molecule Fc-DA through host-guest interaction to form the chiral supramolecular nanoassembly ZnO2@CDP / Fc-DA.

[0009] The supramolecular ligand CDP is obtained by hydrolysis of mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin and diethyl 4-hydroxypyridine-2,6-dicarboxylic acid; the guest molecule Fc-DA is obtained by coupling N-succinimide ferrocene carboxylate with D-alanine.

[0010] This invention also provides a method for preparing the chiral supramolecular nanoassemblies, comprising the following steps: (1) Preparation of supramolecular ligand CDP: Mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin and anhydrous potassium carbonate were stirred evenly in dry N,N-dimethylformamide, and then 4-hydroxypyridine-2,6-dicarboxylic acid diethyl ester was added. The reaction was carried out at 75-85℃ for 10-14 hours under inert gas protection. After post-treatment, an intermediate was obtained. The intermediate was hydrolyzed under alkaline conditions, and then purified by acid neutralization and dialysis to obtain the final product CDP. The molar ratio of mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin, anhydrous potassium carbonate, and diethyl 4-hydroxypyridine-2,6-dicarboxylic acid is 1 : (4.5-5.5) : (4.5-5.5); the hydrolysis under alkaline conditions uses a 15-25% potassium hydroxide aqueous solution, and the reflux reaction time is 3-6 hours.

[0011] (2) Preparation of guest molecule Fc-DA: Ferrocene N-succinimide ester and D-alanine were dissolved in 60-70% ethanol aqueous solution, an alkaline catalyst was added, and the mixture was refluxed at 60-80℃ for 18-24 hours. After extraction, washing, drying and column chromatography purification, the product Fc-DA was obtained. The molar ratio of N-succinimide ferrocene to D-alanine is 1:(1.1-1.3), and the base catalyst is triethylamine.

[0012] (3) Preparation of ZnO2@PVP nanoparticles: Anhydrous zinc acetate and polyvinylpyrrolidone were dissolved in ultrapure water, and hydrogen peroxide solution was added under vigorous stirring. After reacting for 20-28 hours, ZnO2@PVP nanoparticles were obtained by centrifugation and freeze-drying. The mass ratio of anhydrous zinc acetate, polyvinylpyrrolidone, and hydrogen peroxide is 1:(0.8-1.2):(1.4-1.6), and the mass fraction of the hydrogen peroxide solution is 30%.

[0013] (4) Preparation of ZnO2@CDP nanoparticles: The ZnO2@PVP nanoparticles obtained in step (3) were dispersed in ultrapure water, CDP was added, and the mixture was stirred at room temperature for 60-84 hours to carry out ligand exchange. After the reaction was completed, the mixture was centrifuged and freeze-dried to obtain ZnO2@CDP nanoparticles. The mass ratio of ZnO2@PVP nanoparticles to CDP was 1: (1.8-2.2).

[0014] (5) Preparation of ZnO2@CDP / Fc-DA nanoassemblies: The ZnO2@CDP nanoparticles obtained in step (4) are dispersed in ultrapure water, Fc-DA is added under ultrasonic conditions, ultrasonic treatment is performed for 15-25 minutes, and then stirred at room temperature for 10-14 hours. The final product ZnO2@CDP / Fc-DA is obtained by centrifugation. The mass ratio of ZnO2@CDP nanoparticles to Fc-DA is 8:1~12:1.

[0015] The present invention further provides the application of the chiral supramolecular nanoassemblies in the preparation of drugs for treating bacterially colonized tumors, particularly in the treatment of breast cancer dominated by Gram-positive bacteria.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes the host-guest molecular recognition mechanism to prepare a chiral supramolecular nanoassembly. Based on a chiral-driven hierarchical targeting mechanism, it can specifically recognize Gram-positive bacteria colonizing tumors, effectively overcoming the significant limitations of traditional intratumoral bacterial elimination methods in terms of specificity, tissue penetration, and immune activation. The chiral nanoassemblies prepared in this invention utilize a dual ferroptosis mechanism that targets both intracellular bacteria and cancer cells colonizing tumors. Through an acidic microenvironment response, they release zinc ions and hydrogen peroxide, and generate reactive oxygen species under the catalysis of ferrocene, inducing lipid peroxidation, thereby achieving synergistic activation of innate immunity (cGAS-STING pathway) and adaptive immunity (immunogenic cell death). By precisely eliminating intracellular pathogens and inducing immunogenic death of tumor cells, this invention achieves synergistic regulation of bacterial clearance and immunosuppression reversal, providing new technical support and experimental evidence for the development of synergistic immunotherapies based on tumor microbiome regulation. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the immune response principle of the chiral supramolecular nanoassembly ZnO2@CDP / Fc-DA of the present invention.

[0018] Figure 2 This is a transmission electron microscope (TEM) image of the ZnO2@CDP / Fc-DA nanoassembly.

[0019] Figure 3 This is the X-ray diffraction (XRD) pattern of the ZnO2@CDP / Fc-DA nanoassembly.

[0020] Figure 4 This is the Fourier transform infrared (FTIR) spectrum of the ZnO2@CDP / Fc-DA nanoassembly.

[0021] Figure 5 These are circular dichroism (CD) spectra of the two enantiomers, ZnO2@CDP / Fc-LA and ZnO2@CDP / Fc-DA.

[0022] Figure 6 ZnO2@CDP / Fc-DA nanoassemblies after incubation under different pH conditions 2+ The release curves show its responsive release characteristics in an acidic environment.

[0023] Figure 7 The results of H2O2 release measurements after incubation of ZnO2@CDP / Fc-DA nanoassemblies under different pH conditions demonstrate its acid-triggered hydrogen peroxide release capability.

[0024] Figure 8 The images show the electron spin resonance (ESR) spectra of the ZnO2@CDP / Fc-DA nanoassemblies after incubation under different pH conditions, confirming the generation of hydroxyl radicals (•OH).

[0025] Figure 9 The images show colony plates and bacterial survival rate statistical analysis of Staphylococcus aureus treated with ZnO2@CDP / Fc-DA nanoassemblies, demonstrating their excellent antibacterial properties; *p<0.05, **p<0.01, ***p<0.001 VS control group.

[0026] Figure 10 C11 BODIPY is a bacterial and lipid peroxidation probe treated with ZnO2@CDP / Fc-DA nanoassemblies. 581 / 591 The fluorescence images after co-incubation visually demonstrate the degree of lipid peroxidation in the bacterial membrane.

[0027] Figure 11 The results of quantitative analysis of the amount of leaked DNA in the supernatant after bacterial treatment with ZnO2@CDP / Fc-DA nanoassemblies demonstrate its disruptive effect on bacterial membranes.

[0028] Figure 12 These are fluorescence images of rhodamine B-labeled ZnO2@CDP / Fc-DA nanoassemblies and their enantiomers after incubation with tumor cells for different times, showing the time- and configuration-dependent uptake by cells.

[0029] Figure 13 The results of Western blot analysis of GPX4 protein expression after treatment of tumor cells with ZnO2@CDP / Fc-DA nanoassemblies demonstrate the activation of the ferroptosis pathway.

[0030] Figure 14 The results of treating Staphylococcus aureus-infected tumor cells with ZnO2@CDP / Fc-DA nanoassemblies showed the intracellular bacterial colony plate diagram and survival rate analysis, demonstrating its highly efficient intracellular bactericidal ability.

[0031] Figure 15 The Western blot analysis of cGAS-STING pathway-related protein expression after treatment of infected cells with ZnO2@CDP / Fc-DA nanoassemblies demonstrated the effective activation of the innate immune pathway.

[0032] Figure 16 This is a graph showing the change in tumor volume over time in tumor-bearing mice after treatment with ZnO2@CDP / Fc-DA nanoassemblies, demonstrating its significant in vivo anti-tumor effect.

[0033] Figure 17 The curves showing the change in mouse body weight during treatment demonstrate the good biocompatibility of the ZnO2@CDP / Fc-DA nanoassemblies.

[0034] Figure 18 This is an immunofluorescence staining image of a tumor tissue section after treatment, showing CD4. + and CD8 + The infiltration of T cells confirmed the activation of the anti-tumor immune response. Detailed Implementation

[0035] The present invention will be further explained and described below with reference to specific embodiments. Example

[0036] The preparation of ZnO2@CDP / Fc-DA nanoassemblies includes the following steps: Step 1: Preparation of supramolecular ligands (CDP): Mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin (1.0 eq, 500 mg) and anhydrous potassium carbonate (5.0 eq, 270 mg) were stirred thoroughly in dry N,N-dimethylformamide (20 mL), followed by the addition of diethyl 4-hydroxypyridine-2,6-dicarboxylic acid (5.0 eq, 466.5 mg). The mixture was stirred at 80 °C under a nitrogen atmosphere for 12 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure to 2-3 mL. The concentrate was then precipitated dropwise in ethyl acetate (200 mL). The crude product was collected by filtration and repeatedly extracted with water and ethyl acetate. Finally, it was purified by C18 reversed-phase column chromatography. The resulting product was dissolved in a water / ethanol mixture (2:1), and 20% potassium hydroxide (1 mL) aqueous solution was added. The reaction mixture was refluxed for more than 3 hours, the pH was adjusted to 5 with 1 M HCl, the mixture was concentrated under reduced pressure, and dialyzed for 2 days to obtain the final product (CDP).

[0037] Step 2: Preparation of guest molecules (Fc-DA): N-succinimide ferrocene (1.0 eq, 100 mg) and D-alanine (1.2 eq, 32.68 mg) were dissolved in 4 mL of 65% ethanol. Triethylamine (2.1 eq, 89.2 μL) was added, and the mixture was refluxed at 60–80 °C for 20 hours. After concentration under reduced pressure, 5 mL of ethyl acetate was added to the residue. The organic layer was washed successively with cold hydrochloric acid (1 M, 6 mL × 2) and saturated brine (5 mL × 2). The organic layer was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain the crude product. The final product was purified by silica gel column chromatography using dichloromethane / methanol (v / v ratio: 10:1).

[0038] Step 3: Preparation of chiral supramolecular nanoassemblies (ZnO2@CDP / Fc-DA): 1) Preparation of ZnO2@PVP nanoparticles: Anhydrous zinc acetate (100 mg) and polyvinylpyrrolidone (100 mg) were dissolved in 5 mL of ultrapure water. 0.5 mL of 30% hydrogen peroxide was added rapidly under vigorous stirring. After reacting for 24 hours, the ZnO2@PVP nanoparticles were collected by centrifugation and lyophilized for later use. 2) Preparation of ZnO2@CDP nanoparticles: ZnO2@CDP was prepared by ligand exchange method: ZnO2@PVP (20 mg) was dispersed in 40 mL of ultrapure water, and then CDP (40 mg) was added to the solution. The mixture was stirred at room temperature for 72 hours. The resulting product was collected by centrifugation and then lyophilized for later use. 3) Preparation of ZnO2@CDP / Fc-DA nanoassemblies: ZnO2@CDP (40 mg) was dispersed in ultrapure water, and Fc-DA (4 mg) was added under ultrasonic conditions. The mixture was continuously sonicated at room temperature for 20 minutes, then stirred overnight at room temperature, and the final product ZnO2@CDP / Fc-DA was collected by centrifugation.

[0039] Structural characterization and performance evaluation of the prepared products in the examples: 1. Structural characterization 1.1 Morphological Characterization The morphology of the ZnO2@CDP / Fc-DA nanoassemblies was observed using transmission electron microscopy (TEM). Figure 2 As shown, the nanoassemblies are irregularly spherical with good dispersion and an average particle size of approximately 50 nm. Elemental mapping analysis revealed a uniform distribution of Zn, O, and Fe elements, confirming the successful modification of the nanoparticle surface with Fc-DA.

[0040] 1.2 Crystal Structure Analysis The crystal structure of the nanoassemblies was characterized by X-ray diffraction (XRD). The results are as follows: Figure 3 As shown, ZnO2@CDP / Fc-DA retains the characteristic diffraction peaks of ZnO2, indicating that the ligand exchange and host-guest recognition processes do not change the crystal structure of the ZnO2 core.

[0041] 1.3 Chemical Structure Characterization The chemical structure of the nanoassemblies was characterized using Fourier transform infrared spectroscopy (FTIR). Figure 4 As shown, at 1670 cm -1(C=O stretching vibration) and 1285 cm -1 The disappearance of the characteristic absorption peak of PVP at (CN stretching vibration), and at 1155 cm⁻¹ -1 (CDP's COC stretching vibration) and 1617 cm -1 The appearance of the characteristic absorption peak of CDP at (C=N stretching vibration of the pyridine ring in CDP) indicates successful ligand exchange; at 1720 cm⁻¹ -1 The disappearance of the characteristic absorption peak of the carboxyl group confirms that the carboxyl group of CDP interacts with Zn. 2+ Matching successful.

[0042] 1.4 Chiral Characterization The chiral properties of nanoassemblies were characterized using circular dichroism (CD) chromatography. For example... Figure 5 As shown, ZnO2@CDP / Fc-LA and ZnO2@CDP / Fc-DA exhibit obvious mirror-symmetric circular dichroism signals, confirming the successful construction of chiral supramolecular nanoassemblies.

[0043] 2. Performance Evaluation 2.1 pH-responsive release performance ZnO2@CDP / Fc-DA aqueous solution was dialyzed in phosphate-buffered saline (PBS) at pH 7.4 and pH 5.5, respectively, and samples were taken at different time points. The Zn concentration in the dialysate was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). 2+ concentration. Figure 6 The test results showed that under pH 5.5 conditions, Zn... 2+ The cumulative release rate reached over 80%, and its release rate was significantly higher than that under pH 7.4 conditions.

[0044] 1 mg ZnO2@CDP was dispersed in PBS at different pH values ​​(7.4 and 5.5). The solutions were incubated at 37°C with shaking. After different incubation times, the supernatant was collected by centrifugation and mixed with titanium sulfate (1 mL, 1 mg / mL). The absorbance of the mixture at 410 nm was recorded using a UV-Vis spectrophotometer. Figure 7 The results showed that the amount of H2O2 generated at pH 5.5 was significantly higher than that at pH 7.4.

[0045] ZnO2@CDP / Fc-DA nanoassemblies were dispersed in PBS at pH 7.4 and pH 5.5, respectively. A •OH scavenger, 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO), was added to each dispersion, and the mixtures were incubated at 37°C with shaking for 12 hours. The mixtures were filtered, and the filtrates were analyzed using electron spin resonance spectroscopy (ESR). The results are as follows: Figure 8 As shown, a significant DMPO-•OH adduct characteristic signal was detected at pH 5.5, confirming that the chiral nanoassemblies in this invention have a strong pH-dependent •OH generation ability.

[0046] 2.2 Evaluation of antibacterial properties The antibacterial properties were evaluated using the agar plate dilution method. Staphylococcus aureus (S. aureus) was used as the target strain. S. aureus ) as the model bacteria, the bacterial suspension (10 4 CFU / mL was mixed with PBS (control group), ZnO2@CDP / Fc-DA (experimental group, 60 μg / mL), and other control materials (ZnO2@CDP, Fc-LA, Fc-DA, ZnO2@CDP / Fc-LA, 60 μg / mL), and incubated on a shaker (37°C, 180 rpm) for 6 hours. 100 μL of each mixture was plated on agar plates and incubated at 37°C for 24 hours. Colony forming units (CFU) were then counted. Results are as follows: Figure 9 As shown, the bacterial survival rate of the ZnO2@CDP / Fc-DA treatment group (60 μg / mL) was <1%, demonstrating an almost complete bactericidal effect, which was significantly better than other control groups.

[0047] C11 Body 581 / 591 Fluorescent probes were used to detect bacterial lipid peroxidation levels. Bacterial suspensions treated with different materials were collected by centrifugation, and the bacterial pellets were resuspended in PBS and then reacted with C11 BODIPY. 581 / 591 Mix with (10 μM) fluorescent dye and incubate at room temperature in the dark for 30 minutes. Wash to remove free dye, resuspend the bacterial pellet in a smear, and observe immediately under an inverted fluorescence microscope. Results are as follows. Figure 10 As shown, the green fluorescence intensity of the ZnO2@CDP / Fc-DA treatment group was significantly enhanced, indicating that severe lipid peroxidation damage was induced.

[0048] Bacterial DNA leakage can be assessed by detecting absorbance values ​​at 260 nm. Figure 11 As shown, the ZnO2@CDP / Fc-DA treatment group had the highest absorbance value, indicating that it caused the most severe bacterial membrane damage and DNA leakage.

[0049] 2.3 Cellular uptake and intracellular bactericidal effect Rhodamine B-labeled nanoassemblies were used to study cellular uptake behavior. 4T1 cells were cultured at 5 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 24-well plates and cultured for 12 hours. Subsequently, 40 µg / mL of Rhodamine B (RhB)-labeled ZnO2@CDP / Fc-LA or ZnO2@CDP / Fc-DA was added, and incubation was performed for 30 minutes, 1 hour, 2 hours, or 4 hours, respectively. After washing three times with PBS, the cell nuclei were stained with DAPI for 5 minutes. Residual dye was removed by washing, and the cells were immediately observed using an inverted fluorescence microscope. Results are as follows: Figure 12 As shown, the cell uptake efficiency of the ZnO2@CDP / Fc-DA group was significantly higher than that of its enantiomer, the ZnO2@CDP / Fc-LA group, indicating that the D-configuration assembly has a stronger affinity for the cell membrane.

[0050] GPX4 protein expression levels were detected by Western blot. After co-incubating 4T1 cells with different materials for 24 hours, cells were collected by trypsin digestion, washed twice with PBS, and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. The supernatant was collected by centrifugation, and the total protein content was determined using a BCA protein quantification kit. GPX4 protein expression levels were detected by Western blotting (WB). Results are as follows: Figure 13 As shown, the expression level of GPX4 was significantly reduced in the ZnO2@CDP / Fc-DA treatment group, indicating that ferroptosis was successfully induced.

[0051] To evaluate the clearance effect of nanoassemblies on intracellular bacteria, 4T1 cells were cultured at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 μL in 24-well plates and cultured for 12 hours. The medium was removed, and Staphylococcus aureus suspension (MOI = 5:1) was added for infection for 30 minutes. The medium was discarded, and the cells were washed 2-3 times with PBS. Then, 500 μL of 1640 medium containing 10% FBS and gentamicin (Gen, 50 μg / mL) was added to each well, and the cells were incubated for 2 hours to remove extracellular bacteria. Next, each well was replaced with 500 μL of 1640 medium containing 10% FBS and Gen (10 μg / mL). PBS (control group), ZnO2@CDP / Fc-DA (experimental group), and other control materials (ZnO2@CDP, Fc-LA, Fc-DA, ZnO2@CDP / Fc-LA) were added to each well, and the cells were cultured for another 12 hours. The culture medium was discarded, and the cells were washed three times with PBS. Cell lysis was performed by adding PBS solution containing 1% Triton X-100 to each well. The lysis buffer was serially diluted with PBS, and 100 μL was spread onto agar plates. After incubation at 37°C for 24 hours, colony forming units (CFU) were counted. Results are as follows: Figure 14 As shown: Among all the tested materials, ZnO2@CDP / Fc-DA showed the most significant effect in killing intracellular bacteria.

[0052] 2.4 Evaluation of immune activation effect The expression of proteins related to the cGAS-STING pathway was analyzed using Western blot. For example... Figure 15 As shown, in the Staphylococcus aureus infection + ZnO2@CDP / Fc-DA treatment group, the expression levels of p-STING, p-TBK1 and p-IRF3 were upregulated by 1.6-fold, 1.7-fold and 1.4-fold, respectively, compared with the uninfected + ZnO2@CDP / Fc-DA treatment group, indicating that ZnO2@CDP / Fc-DA can specifically activate the cGAS-STING pathway in infected cells.

[0053] 2.5 Evaluation of in vivo antitumor effects A 4T1 breast cancer-bearing mouse model was established to evaluate the in vivo antitumor effect of the nanoassemblies. Six- to eight-week-old female Balb / c mice (weighing 18-22g) were subcutaneously inoculated with 1×102 mouse breast cancer 4T1 cells in the right abdomen. 6 A mouse subcutaneous breast cancer tumor model was established (each mouse had one tumor). When the tumor volume reached approximately 100 mm... 3 Mice bearing tumors were randomly divided into 5 groups (n=5): I) saline-Staphylococcus aureus (uninfected control group), II) saline + Staphylococcus aureus (infected control group), III) ZnO2@CDP + Staphylococcus aureus, IV) ZnO2@CDP / Fc-LA + Staphylococcus aureus, and V) ZnO2@CDP / Fc-DA + Staphylococcus aureus. Staphylococcus aureus was injected intratumorally 24 hours before the first administration. The injection dose of ZnO2@CDP, ZnO2@CDP / Fc-LA, and ZnO2@CDP / Fc-DA was 10 mg / kg. Injections were administered via tail vein every two days, and tumor volume and body weight were measured. After 16 days, the mice were euthanized, and tumor tissue was collected. The results are shown in Figure 16: After 16 days of treatment, ZnO2@CDP / Fc-DA showed a particularly significant inhibitory effect on tumor growth, which was significantly better than the ZnO2@CDP / Fc-LA and ZnO2@CDP groups. Figure 17 This indicates that the body weight of mice in each group did not decrease significantly throughout the treatment period.

[0054] Immunofluorescence analysis was used to assess T cell infiltration within tumor tissue. For example... Figure 18 As shown, CD4+ levels in tumor tissues treated with ZnO2@CDP / Fc-DA were significantly higher than those in the control group. + and CD8 + The significant increase in the number of T cells indicates that the anti-tumor immune response has been successfully activated.

[0055] The embodiments described above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A chiral supramolecular nanoassembly, characterized in that, The nano-assembly uses ZnO2 nanoparticles as the core, and its surface is modified with supramolecular ligand CDP through ligand exchange. It also binds guest molecule Fc-DA through host-guest interaction to form the chiral supramolecular nano-assembly ZnO2@CDP / Fc-DA. The supramolecular ligand CDP is obtained by hydrolysis following a reaction of mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin with diethyl 4-hydroxypyridine-2,6-dicarboxylate. The guest molecule Fc-DA is obtained by the reaction coupling of N-succinimide ferrocene with D-alanine.

2. A method for preparing the chiral supramolecular nanoassembly as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of supramolecular ligand CDP: Mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin and anhydrous potassium carbonate were stirred evenly in dry N,N-dimethylformamide, and then 4-hydroxypyridine-2,6-dicarboxylic acid diethyl ester was added. The reaction was carried out at 75-85℃ for 10-14 hours under inert gas protection. After post-treatment, an intermediate was obtained. The intermediate was hydrolyzed under alkaline conditions, and then purified by acid neutralization and dialysis to obtain the final product CDP. (2) Preparation of guest molecule Fc-DA: Ferrocene N-succinimide ester and D-alanine were dissolved in 60-70% ethanol aqueous solution, an alkaline catalyst was added, and the mixture was refluxed at 60-80℃ for 18-24 hours. After extraction, washing, drying and column chromatography purification, the product Fc-DA was obtained. (3) Preparation of ZnO2@PVP nanoparticles: Anhydrous zinc acetate and polyvinylpyrrolidone were dissolved in ultrapure water, and hydrogen peroxide solution was added under vigorous stirring. After reacting for 20-28 hours, ZnO2@PVP nanoparticles were obtained by centrifugation and freeze-drying. (4) Preparation of ZnO2@CDP nanoparticles: The ZnO2@PVP nanoparticles obtained in step (3) were dispersed in ultrapure water, CDP was added, and the mixture was stirred at room temperature for 60-84 hours to carry out ligand exchange. After the reaction was completed, the mixture was centrifuged and freeze-dried to obtain ZnO2@CDP nanoparticles. (5) Preparation of ZnO2@CDP / Fc-DA nanoassemblies: The ZnO2@CDP nanoparticles obtained in step (4) are dispersed in ultrapure water, Fc-DA is added under ultrasonic conditions, ultrasonic treatment is performed for 15-25 minutes, and then stirred at room temperature for 10-14 hours. The final product ZnO2@CDP / Fc-DA is obtained by centrifugation.

3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin, anhydrous potassium carbonate, and diethyl 4-hydroxypyridine-2,6-dicarboxylic acid is 1:(4.5-5.5):(4.5-5.5); the hydrolysis under alkaline conditions uses a 15-25% potassium hydroxide aqueous solution, and the reflux reaction time is 3-6 hours.

4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of N-succinimide ferrocene to D-alanine is 1: (1.1-1.3), and the alkaline catalyst is triethylamine.

5. The preparation method according to claim 2, characterized in that, In step (3), the mass ratio of anhydrous zinc acetate, polyvinylpyrrolidone and hydrogen peroxide is 1: (0.8-1.2): (1.4-1.6), and the mass fraction of the hydrogen peroxide solution is 30%.

6. The preparation method according to claim 2, characterized in that, The mass ratio of ZnO2@PVP nanoparticles to CDP in step (4) is 1: (1.8-2.2).

7. The preparation method according to claim 2, characterized in that, The mass ratio of ZnO2@CDP nanoparticles to Fc-DA in step (5) is 8:1 to 12:

1.

8. The use of the chiral supramolecular nanoassembly as described in claim 1 in the preparation of a medicament for treating bacterially colonized tumors.

9. The application according to claim 8, characterized in that, The bacteria are Gram-positive, preferably Staphylococcus, Lactobacillus, Enterococcus, or Streptococcus; the tumor is breast cancer; the mechanism of action of the drug includes: inducing ferroptosis in bacteria and tumor cells; and / or activating the cGAS-STING innate immune pathway.

10. A pharmaceutical composition, characterized in that, It contains the chiral supramolecular nanoassembly as described in claim 1 as an active ingredient.

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