A biomimetic nanocomposite and its application

Abstract

The application provides a kind of bionic nanocomposite and its application, the bionic nanocomposite includes nanoparticle, drug and cell membrane;The nanoparticle is loaded as carrier The drug, the cell membrane is coated on the surface of nanoparticle;The particle size of the bionic nanocomposite is 80-130nm;The nanoparticle is mesoporous silica nanoparticle, the drug is bee toxin, and the cell membrane is cancer cell membrane.The bionic nanocomposite of the application has high selectivity, low toxicity and strong antitumor effect, through the mechanism of relieving Fn-induced tumor suppression microenvironment and activating tumor-specific immunity to exert antitumor effect, further elucidate the role of targeted nucleic acid bacillus in the treatment of colorectal cancer and the synergistic therapeutic effect of simultaneous targeting and removing Fn and tumor cell strategy, thereby providing zoological basis and related data for further human clinical application research, providing an important reference for subsequent pathogenic bacteria killing and immune suppression microenvironment improvement combined immunotherapy, which helps to improve the immunotherapy effect of patients with colorectal cancer, and has strong clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic nanocomposite and its applications. Background Technology

[0002] Colorectal cancer (CRC) is a malignant tumor originating from the intestinal epithelium, and its high incidence and mortality rates place significant pressure on health and medical resources. The core pathogenic bacterium within the tumor, *Fusobacterium nucleatum* (Fn), accumulates in colorectal cancer tissue, promoting CRC proliferation and metastasis, and affecting the efficacy of treatments such as chemotherapy. However, there is currently a lack of specific methods to eliminate Fn; therefore, targeted therapy for Fn-infected colorectal cancer patients has become a key scientific problem that needs to be solved.

[0003] Currently, most studies on the impact of eliminating pathogenic bacteria in the gut on the treatment of colorectal cancer use antibiotics, such as metronidazole or amphotericin B. While these antibiotics eliminate pathogenic bacteria, they can also affect other gut microbiota, potentially leading to an imbalance in the gut microbiota. [1] Only a very small number of researchers have attempted targeted therapy against a single pathogen, such as Professor Zhang Xianzheng's team in China. [2] A phage-guided nanocomposite system loaded with irinotecan was designed. Phage clearance of Fusobacterium nucleatum (Fn) improved the efficacy of irinotecan chemotherapy; however, phage still presents potential biosafety concerns. Therefore, developing a therapeutic strategy that targets and eliminates intratumoral Fn and tumor cells without disrupting the gut microbiota balance, leveraging the properties of Fn's accumulation in tumor tissue and adhesion to homologous tumor cells, is considered a promising direction for experimental research in this field.

[0004] References

[0005] [1] SONG W, ANSELMO AC, HUANG L. Nanotechnology intervention of the microbiome for cancer therapy [J]. Nat Nanotechnol, 2019, 14(12): 1093-1103.

[0006] [2]ZHENG DW,DONG Summary of the Invention

[0007] This invention provides a biomimetic nanocomposite and its application. The biomimetic nanocomposite Mel-SiO2@CCM inherits the homologous adhesion properties of tumor cells and the targeting ability of Fusobacterium nucleatum, enabling it to achieve targeted enrichment in a CRC model infected with Fusobacterium nucleatum and exhibiting excellent dual therapeutic effects of enhanced anti-tumor and bactericidal effects.

[0008] The technical solution adopted by the present invention to achieve the above objectives is: a biomimetic nanocomposite, the biomimetic nanocomposite comprising nanoparticles, a drug, and a cell membrane; the nanoparticles serve as a carrier for loading the drug, and the cell membrane is coated on the surface of the nanoparticles.

[0009] Furthermore, the particle size of the biomimetic nanocomposite is 80-130 nm.

[0010] Furthermore, the nanoparticles are any one of mesoporous silica nanoparticles, gold nanoparticles, magnetic nanoparticles, and polymer nanoparticles; the drug is any one of bee venom, docetaxel, cisplatin, and antibiotics; and the cell membrane is any one of cancer cell membrane, normal cell membrane, stem cell membrane, and engineered cell membrane.

[0011] Furthermore, the nanoparticles are mesoporous silica nanoparticles, the drug is bee venom, and the cell membrane is a cancer cell membrane.

[0012] Furthermore, the drug loading rate of the biomimetic nanocomposite is 7.96%.

[0013] The present invention also discloses the application of the above-mentioned biomimetic nanocomposite in the preparation of drugs for treating colorectal cancer.

[0014] Furthermore, the dosage of the biomimetic nanocomposite is 12.56 mg / kg.

[0015] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least:

[0016] First, the biomimetic nanocomposite of the present invention has high selectivity, can specifically recognize and bind to tumor cells, and reduce damage to normal cells;

[0017] Secondly, the biomimetic nanocomposite of the present invention has low toxicity and strong anti-tumor effect. Both in vitro and in vivo experiments have confirmed its good biosafety, and it has also shown significant anti-tumor effect in a variety of tumor models.

[0018] Third, the biomimetic nanocomposite of this invention exerts its anti-tumor effect by relieving the tumor suppressor microenvironment induced by Fusobacterium nucleatum Fn and activating tumor-specific immunity. This further elucidates the role of targeting Fn in the treatment of colorectal cancer and the synergistic therapeutic effect of the strategy of simultaneously targeting and clearing Fn and tumor cells. This provides an animal basis and related data for further human clinical application research, and provides an important reference for subsequent combined immunotherapy to kill pathogenic bacteria and improve the immunosuppressive microenvironment. It helps to improve the immunotherapy effect of colorectal cancer patients and has strong clinical translational value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0020] Figure 1 The images shown are high-resolution transmission electron microscopy (TEM) images of the SiO2@CCM prepared in Example 1, where (A) is a TEM scan image and (B) is an elemental analysis image.

[0021] Figure 2 The images shown are transmission electron microscope (TEM) scans of the Mel-SiO2@CCM NPs prepared in Example 1, where (A) is a TEM scan and (B) is a magnified view.

[0022] Figure 3 The particle size analysis diagram and potential analysis diagram of Mel-SiO2@CCM NPs prepared in Example 1 are shown, where (A) is the particle size analysis diagram and (B) is the potential analysis diagram.

[0023] Figure 4 SDS-PAGE gel electrophoresis patterns of membrane proteins of marker, cell membrane lysis buffer, SiO2@CCM and Mel-SiO2@CCM NPs prepared in Example 1;

[0024] Figure 5The image shows the drug loading and encapsulation efficiency of the Mel-SiO2@CCM NPs prepared in Example 1, where (A) represents the drug loading and (B) represents the encapsulation efficiency.

[0025] Figure 6 The image shows the drug release analysis of the biomimetic nanocomposite prepared in Example 1. In the image, (A) is the release curve of Mel in pH 7.5 solution before and after CCM modification, and (B) is the release curve of Mel-SiO2@CCM NPs in different pH solutions.

[0026] Figure 7 The graph shows the stability analysis of the biomimetic nanocomposite prepared in Example 1.

[0027] Figure 8 The graph shows the hemolytic activity assay of the biomimetic nanocomposite prepared in Example 1. In the graph, 1 represents the PBS group, 2 represents the SiO2@CCM group, 3 represents the Mel group, 4 represents the Mel-SiO2 group, and 5 represents the Mel-SiO2@CCM group.

[0028] Figure 9 The images show the in vitro targeted antibacterial effect analysis of the biomimetic nanocomposite prepared in Example 1. Among them, (A) is the enrichment map of the biomimetic nanocomposite on the Fn surface under scanning electron microscopy, (B) is the antibacterial effect analysis map of the biomimetic nanocomposite under scanning electron microscopy, (C) is the antibacterial effect enhanced by loading bee venom peptide-enabled bacteria with nanocomposite, (D) is the competitive antibacterial effect of nanocomposite through Gal-GalNAc, (E) is the antibacterial curve of different formulation drugs at the same concentration of bee venom peptide, (F) is the anti-Fn effect analysis map under microscope, (G) is the antibacterial effect map of live and dead staining, (H) is the antibacterial effect map of colony counting, and (I) is the colony counting statistics map.

[0029] Figure 10 Metabolic images of nanoparticles enriched in the tumor site of mice after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with in situ colorectal cancer via the tail vein. Among them, (A) is the fluorescence in vivo imaging image of nanoparticles in the tumor site at different time points, and (B) is the statistical graph of the corresponding average fluorescence intensity.

[0030] Figure 11 Fluorescence in vivo imaging of ex vivo organs and tumors at different time points after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with orthotopic tumors via the tail vein;

[0031] Figure 12Fluorescence statistics of various isolated organs and tumors over time after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with orthotopic tumors via the tail vein;

[0032] Figure 13 Statistical distribution of in vitro fluorescence of SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs in mice with orthotopic tumors via tail vein injection. At 8h (A), 24h (B), and 48h (C), the two fluorescent nanoparticles were observed in various ex vivo organs and tumors.

[0033] Figure 14 The image shows the in vivo tumor suppression effect of BALB / c nude mice after different treatments. In the image, (A) is the growth curve of tumor volume, (B) is the tumor weight, and (C) is a photograph of the tumor in vitro.

[0034] Figure 15 This is a tumor growth curve after treatment in Example 5. Detailed Implementation

[0035] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0036] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] Example 1

[0038] A biomimetic nanocomposite is prepared by the following method:

[0039] (1) Extraction of cancer cell membrane vesicles (CCM vesicles)

[0040] CT26 cancer cells were cultured using RPMI-1640 medium, which contains 1% non-essential amino acids and L-glutamine. The medium was supplemented with penicillin (10000 U / mL) and streptomycin (10 mg / mL) as dual antibiotics and contained 10% fetal bovine serum as a nutrient supplement. Culture conditions were set at 5% CO2, 90% relative humidity, and maintained in a 37°C incubator. After the CT26 cancer cells were confluent in T-75 culture flasks, the cells were washed with PBS to remove serum and dead cells from the medium. The cells were then collected and treated with PBS or 1 mM EDTA solution, centrifuged at 500g for 10 min, the supernatant was removed, and the cell pellet was collected and washed three times with PBS.

[0041] Cells were resuspended in 10 mL of hypotonic lysis buffer (containing 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl, and an EDTA-free protease inhibitor). Cells were frozen in liquid nitrogen for 10 min, then thawed in a 37°C water bath for 10 min, and finally sonicated on ice (40 kHz, 100 W) for 5 min. This cycle was repeated 6-10 times. Cells were centrifuged at 3200 g for 5 min, discarding the precipitate containing unbroken cells and nuclei, and the supernatant was retained. The supernatant was centrifuged at 4°C and 20000 g for 30 min, the precipitate was discarded, and the supernatant was transferred to a high-speed centrifuge tube to obtain a supernatant containing cancer cell membrane fragments.

[0042] The supernatant containing cancer cell membrane fragments was ultracentrifuged at 4°C (80,000 g, 1.5 h) to obtain a membrane precipitate. The precipitate containing the plasma membrane was washed once with 10 mM Tris-HCl and 1 mM EDTA, and then the purified cell membrane was collected. The protein content in the purified membrane was determined by the BCA method for further vesicle preparation. CCM vesicles were obtained by physically extruding the particles 10 times on a microliposome extruder through a 400 nm polycarbonate porous membrane.

[0043] (2) Synthesis of SiO2 NPs

[0044] Preparation of SiO2 precursor: In a constant temperature water bath at 30℃, 3.14 mL of ammonia water, 71.4 mL of anhydrous ethanol, and 10 mL of deionized water were mixed in a beaker and stirred with a magnetic stirrer until homogeneous. 6 mL of TEOS was quickly added, and the mixture was stirred rapidly at 30℃ for 2 hours to obtain a white solution. The white solid was separated by centrifugation, washed with anhydrous ethanol and deionized water three times each, and then air-dried to obtain SiO2 powder.

[0045] Preparation of SiO2 suspension: Take 0.5g of SiO2 powder, add 100mL of deionized water, and sonicate for 20min to disperse, obtaining a SiO2 suspension; prepare a mixed solution containing 0.75g of CTAB, 150mL of ethanol, 150mL of deionized water and 2.75mL of ammonia, and mix it with the SiO2 suspension. Continue sonication for 2h, then quickly add 1.5mL of TEOS, stir evenly, sonicate for another 6h, centrifuge, collect the solid, wash with deionized water and centrifuge 3 times, then redisperse in anhydrous ethanol and sonicate for 10min, freeze overnight to obtain a white solid powder.

[0046] Preparation of hollow SiO2 nanoparticles: 3.18 g of Na2CO3 was dissolved in 50 mL of deionized water to prepare a 0.6 M Na2CO3 solution. The white solid powder was added to the Na2CO3 solution, stirred thoroughly, and ultrasonically dispersed. Then, the mixture was stirred in a water bath at 80 °C for 6 h. After cooling to room temperature, the mixture was centrifuged, washed 2-3 times with deionized water, and dried to obtain hollow SiO2 nanoparticles containing CTAB.

[0047] Removal of template agent CTAB: 1 g of hollow SiO2 nanoparticles containing CTAB and 1 mL of concentrated hydrochloric acid were added to 180 mL of anhydrous ethanol and stirred at 50 °C for 8 h. The solid precipitate was obtained by centrifugation, thoroughly washed with anhydrous ethanol, and then freeze-dried. This step was repeated three times to ensure complete removal of CTAB, forming a mesoporous structure. The final product is hollow silica nanoparticles (SiO2 NPs) with a mesoporous structure.

[0048] (3) Synthesis of Mel-SiO2 NPs

[0049] The aqueous solution of SiO2 NPs prepared in step (2) was ultrafiltered using an ultrafiltration membrane to remove excess unreacted reagents. The resulting SiO2 NPs were divided into three groups for subsequent coupling experiments. Meliostein and SiO2 NPs were added to the SiO2 NPs suspension at mass ratios of 1:5, 1:10, 1:20, and 1:40. After stirring for 2 hours, 3 mg of EDC was added to each group and reacted for 12 hours. After the reaction was complete, the mixture was subjected to solid-liquid separation at 1000 rpm for 10 minutes. The resulting samples were then rinsed three times with deionized water to remove residual unreacted reagents. Finally, the obtained product was lyophilized to obtain Mel-SiO2 NPs.

[0050] (4) Synthesis of SiO2@CCM and Mel-SiO2@CCM NPs

[0051] The CCM vesicle solution obtained in step (1) is first passed through a micro extruder equipped with a 400nm filter membrane to form a uniform membrane fragment, and then mixed with the SiO2 NPs obtained in step (2) or the Mel-SiO2 NPs suspension obtained in step (3). The mixture is then dispersed by sonication on ice (frequency 40kHz, power 100W) for 5 minutes. Subsequently, the resulting solution is extruded 10 times through an extruder equipped with a polycarbonate filter membrane with a pore size of 200nm to obtain CCM-encapsulated SiO2@CCM and Mel-SiO2@CCM NPs loaded with bee venom peptide and encapsulated by CCM.

[0052] The prepared SiO2 NPs were observed using high-resolution transmission electron microscopy. Figure 1 A indicates that SiO2 NPs have a typical crystal morphology, with a regular and ordered pore structure, resulting in a high specific surface area, smooth surface, and uniform dispersion. Figure 1 The elemental analysis diagram of B also confirmed the elemental composition of SiO2 NPs. The above results demonstrate that the SiO2 NPs prepared in this invention have uniform size and morphology, and are evenly dispersed, which is the basis for further drug loading and CCM biomimetic encapsulation modification.

[0053] Morphological examination of Mel-SiO2@CCM NPs was performed using transmission electron microscopy. Figure 2 The results show that the typical spherical core-shell structure of SiO2 NPs is coated with a low-density halo-like thin coat with a diameter of about 9 nm, which directly proves that CCM was successfully applied to the core structure surface of SiO2 NPs.

[0054] In this invention, the particle size and particle size distribution of SiO2 NPs were determined using a particle size analyzer via dynamic light scattering (DLS) technology. Figure 3 A revealed that the average particle size of SiO2 NPs was 92.8 ± 4.4 nm. When they were encapsulated by CCM to form Mel-SiO2@CCM NPs, the average particle size increased to 102.2 ± 4.0 nm, indicating an increase of approximately 9 nm. The particle size distribution of both types of particles was within the range of 80 to 130 nm and exhibited a normal distribution, which is consistent with the observation results of electron microscopy. The SiO2 NPs and Mel-SiO2@CCM NPs prepared in this invention have particle sizes close to 100 nm, which belongs to a very small size range and is highly suitable as a carrier for nanomedicines. Figure 3The results of the Zeta potentiometer measurements of nanoparticle surface potential showed that the surface potential of SiO2 NPs was -36.9±3.3mV, the cell membrane potential was -24.6±2.5mV, and the potential of Mel-SiO2@CCM NPs after drug loading and cell membrane encapsulation was -27.4±4.7mV, which tends to the cell membrane potential, which also indirectly indicates the successful encapsulation by the cell membrane.

[0055] Membrane proteins are fundamental to the function of subsequent nanocomposites. After confirming successful cell membrane encapsulation through electron microscopy, particle size analysis, and potential measurement, the retention of functional proteins on the CCM surface of Mel-SiO2@CCM NPs was characterized. SDS-PAGE gel electrophoresis was performed on membrane proteins from markers, cell membrane lysis buffer, SiO2@CCM, and Mel-SiO2@CCM NPs at specific concentrations. Figure 4 As shown, the membrane protein bands of SiO2@CCM and Mel-SiO2@CCM NPs are extremely similar to the protein bands of the CCM in CT26 cells, indicating that Mel-SiO2@CCM NPs successfully retains most of the functional proteins on the cell membrane. This is also the basis for the targeting effect of the biomimetic nanocomposite Mel-SiO2@CCM NPs.

[0056] Example 2: Determination of the properties of biomimetic nanocomposites

[0057] The biomimetic nanocomposite prepared in Example 1 was subjected to drug loading and encapsulation efficiency determination, drug release determination, stability determination and hemolytic activity determination.

[0058] The drug loading and encapsulation efficiency of the biomimetic nanocomposites prepared in Example 1 were determined. The BCA protein assay was used to quantify the Mel encapsulated in Mel-SiO2@CCM NPs. Experiments were conducted using Mel / SiO2 mass ratios of 1:5, 1:10, 1:20, and 1:40. The results (…) Figure 5 A and B show that when the initial Mel dosage was low, the encapsulation efficiency of Mel remained stable at over 80%, which also proves the high drug loading capacity of the carrier SiO2. As the Mel dosage increased, when the Mel / SiO2 mass ratio increased to 1:10, the Mel encapsulation efficiency and the rate of increase in Mel drug loading also decreased. Considering the utilization rate of both the drug and the material, we selected Mel-SiO2@CCM NPs with a Mel / SiO2 mass ratio of 1:10 for subsequent release experiments. At this time, the drug loading capacity of melittin in Mel-SiO2@CCM NPs was 7.96%, and the encapsulation efficiency was 79.6%.

[0059] The drug release rate of the biomimetic nanocomposite prepared in Example 1 was determined. To evaluate the response of Mel-SiO2@CCMNPs to pH changes, drug release rate was measured under different pH conditions. The results are as follows: Figure 6 As shown in Figure A, Mel-SiO2@CCM NPs exhibited a low Mel release rate in PBS buffer at pH 7.4, releasing only 23.4% within 24 hours. In contrast, unencapsulated Mel-SiO2 showed a release rate of 82.9% under the same conditions. This indicates that CCM-modified Mel-SiO2@CCM NPs demonstrated good stability in a neutral environment simulating physiological conditions, maintaining structural integrity, effectively encapsulating Mel, and preventing premature drug leakage that could lead to toxicity to normal tissues, thereby improving biosafety. When the ambient pH decreased to 6.5 and 5.5 (simulating the acidic conditions of the tumor microenvironment and endosomes / lysosomes), the Mel release rate significantly increased, reaching 78.4% and 88.7% respectively after 24 hours. Figure 6 B). This phenomenon indicates that the nanocarrier material becomes unstable under acidic conditions, and after reaching the tumor site and being taken up by cells, it can disintegrate in a slightly acidic environment, effectively releasing the encapsulated melitoxin and exerting its therapeutic effect, thus achieving specific and controllable drug release targeting the tumor site.

[0060] The stability of the biomimetic nanocomposite prepared in Example 1 was determined. The stability of the biomimetic nanocomposite is a prerequisite for the drug to reach its target organ and exert its effect after entering the body. Good stability of Mel-SiO2@CCM NPs is crucial for maintaining long blood circulation time, preventing drug leakage, and improving drug bioavailability. Conversely, if the NPs exhibit instability during circulation in the body, it may lead to low drug utilization efficiency and poor therapeutic efficacy. Therefore, the stability of Mel-SiO2@CCM NPs was evaluated in deionized water at room temperature and under physiological conditions (PBS buffer) at 37°C. The results are as follows: Figure 7 As shown, the experimental results indicate that Mel-SiO2@CCM NPs exhibit minimal changes in particle size and maintain good dispersibility when stored in aqueous solution or PBS solution for 24 hours. This demonstrates that Mel-SiO2@CCM NPs demonstrate excellent dimensional stability under both environments. This stability is crucial for the application, storage, and transportation of nanoparticles and will benefit the large-scale production required for subsequent conversion.

[0061] The hemolytic activity of the biomimetic nanocomposite prepared in Example 1 was determined. Although the above results demonstrate that the biomimetic nanocomposite Mel-SiO2@CCM NPs has advantages such as high drug loading, release in the acidic environment of tumors, and good stability under physiological conditions, Mel is a membrane-dissolving peptide that can disrupt cell membranes, leading to leakage of cell contents and ultimately cell death. This action has poor selectivity and can also cause some damage to normal cells, with hemolytic activity being particularly prominent. Therefore, for safe application in animals and even potential clinical translation, we further investigated whether Mel-SiO2@CCM NPs could reduce the hemolytic activity of Mel. We extracted blood from BALB / c mice, centrifuged it to obtain red blood cells (RBCs), and performed an in vitro hemolysis test, comparing the hemolytic activity of free Mel and Mel encapsulated in Mel-SiO2@CCM NPs under the same conditions. The results are as follows: Figure 8 As shown, free Mel itself has a significant hemolytic effect, a phenomenon confirmed in our experiments: free Mel at a concentration of 8 μg / mL can cause hemolysis in approximately 79.8% of erythrocytes. In stark contrast, Mel-SiO2@CCM NPs containing the same amount of Mel did not exhibit significant hemolytic behavior, and even when the Mel concentration in Mel-SiO2@CCM NPs reached as high as 100 μg / mL, the hemolysis rate remained at an extremely low level, only about 5%. This result indicates that Mel loaded with SiO2 NPs and then encapsulated in CCM significantly reduces hemolytic toxicity and effectively avoids non-specific cytotoxicity issues, which is of great significance for its future clinical applications.

[0062] Example 3: Analysis of the in vitro targeted antibacterial effect of biomimetic nanocomposites

[0063] The targeting and binding ability of Mel-SiO2@CCM prepared in Example 1 to Fn was measured. Images obtained by scanning electron microscopy (SEM) showed that, compared with SiO2, SiO2@CCM had stronger enrichment on the Fn surface. Figure 9 Similarly, in the co-culture system of Fn and Escherichia coli (E. coli), selective enrichment of SiO2@CCM on the Fn surface was observed relative to that on the E. coli surface, confirming the targeting binding ability of SiO2@CCM to Fn. Figure 9 C). Subsequently, we selected two Mel concentrations (5 μg / mL and 10 μg / mL) previously confirmed to have antibacterial effects to test their antibacterial efficacy. Compared with the Mel-only group, the Mel-SiO2@CCM group showed improved antibacterial efficacy at both concentrations, as assessed by the reduction rate of colony forming units (CFU). Figure 9D). Meanwhile, SEM images revealed several distinct changes in the bacteria, including twisting, perforation, and dissolution. Figure 9 B). In summary, both the CFU reduction rate and morphological changes confirmed that Mel-SiO2@CCM had enhanced targeted bactericidal effects compared to Mel. Furthermore, the anti-Fn activity of various formulations, including control (PBS), SiO2@CCM, Mel, Mel-SiO2, and Mel-SiO2@CCM, was further investigated. Fn suspensions were inoculated into LB medium, and the above formulations were added accordingly. OD600 was measured at different time points. The upward trend of growth curves in the Mel-containing treatment groups was inhibited to varying degrees after 4 hours of treatment. Notably, the Mel-SiO2@CCM group completely inhibited bacterial replication within 24 hours. Figure 9 E). Antimicrobial efficiency was quantitatively assessed by colony counting. After 8 hours of treatment, the Mel group and the Mel-SiO2 group showed a reduction in bacterial colonies with 3-log10 and 5-log10 CFU counts, respectively, while all bacteria treated with Mel-SiO2@CCM were killed. Figure 9 H and 9I). The antibacterial activity of Mel-SiO2@CCM was visualized and validated by observation using optical microscopy and the LIVE / DEAD bacterial viability kit, as well as further application of confocal laser scanning microscopy. Figure 9 When treated with PBS (F and 9G), most bacteria exhibited green fluorescence (FL) (live), while increased red FL (dead) was observed in the Mel and Mel-SiO2 groups, consistent with previous growth curves and colony counts. All bacteria treated with Mel-SiO2@CCM showed red FL (dead). In summary, these results not only demonstrate the excellent antibacterial properties of Mel-SiO2@CCM, but also suggest that this function is mainly attributed to its targeting ability for Fn and membrane disruption properties.

[0064] Example 4: Analysis of the in vivo targeted antitumor effect of biomimetic nanocomposites

[0065] In vivo imaging techniques were used to evaluate the biodistribution characteristics of the biomimetic nanocomposite prepared in Example 1 in BALB / c mice with Fn-infected colorectal cancer in situ. Cy7.5-labeled SiO2 NPs (SiO2 / Cy7 NPs) and SiO2@CCM NPs (SiO2@CCM / Cy7 NPs) were injected into mice via the tail vein. Subsequent in vivo imaging monitoring showed that the accumulation of SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs in the tumor region gradually increased over time, reaching peak values ​​at 4 h and 8 h post-injection, respectively. Figure 10(A and B). Careful observation revealed that at 2h, 4h, 8h, 12h, and 24h post-injection, the mean fluorescence intensity (MFL) of the SiO2@CCM / Cy7.5 group was 1.26 times, 1.12 times, 1.53 times, 2.52 times, and 3.83 times that of the SiO2 / Cy7.5 group, respectively. Notably, at 48h, the mean fluorescence intensity of the tumor in the SiO2 / Cy7.5 group was still close to one-third of its peak value, while the mean fluorescence intensity (MFL) of the SiO2@CCM / Cy7.5 group was undetectable. Compared to the SiO2 / Cy7.5 group alone, SiO2@CCM / Cy7.5 NPs exhibited significantly enhanced tumor targeting and more persistent retention, resulting in a stronger MFL signal and a longer duration of MFL in the tumor region. This is based on the typical EPR effect of nanoparticles and the dual targeting capability of CCM.

[0066] Furthermore, this invention also tracked the distribution of SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs in ex vivo tumors and major organs at 8h, 24h, and 48h. Figure 11 , Figure 12 and Figure 13 The results showed that at 8h, 24h, and 48h, the tumor MFL intensity in the SiO2@CCM / Cy7.5 group was higher than that in the SiO2 / Cy7.5 group, further demonstrating the better tumor targeting and persistence of SiO2@CCM / Cy7.5 NPs. Furthermore, a small amount of SiO2@CCM / Cy7.5 NPs was observed in the liver and kidneys at 8h and 24h, but completely disappeared at 48h. This may be because the CCM coating, to some extent, prevents clearance by the immune system, thus prolonging the in vivo circulation time, while still retaining a certain degree of immunogenicity.

[64] Overall, the SiO2@CCM / Cy7.5 nanocarrier exhibited good biocompatibility and did not cause significant toxicity in in vitro and in vivo studies, indicating its potential for future clinical applications.

[0067] Example 5: Analysis of the therapeutic effect of biomimetic nanocomposite on mice with subcutaneous tumors

[0068] Mice were treated with PBS, SiO2@CCM NPs, Mel, Mel-SiO2 NPs, and Mel-SiO2@CCM NPs on days 0, 2, 4, and 6, respectively, for a total of four treatments. The Mel dose was consistently 1 mg / kg. Tumor volume and body weight were measured every other day. After two weeks, the mice were sacrificed, and the tumors were removed for photography and weighing. Figure 14As shown in Figure A, both the Mel group and the Mel-SiO2 group showed some degree of tumor growth inhibition in mice, with relative tumor volumes (V / V0) of 2.71 and 2.36, respectively. Unlike the in vitro results, the relative tumor volume in the Mel-SiO2 group was lower than that in the Mel group. This may be attributed to the enhanced permeation and retention effect (EPR) induced by the nanomaterials, which allows the concentration of Mel-SiO2 at the tumor site to be maintained, improving the bioavailability of Mel compared to the rapid clearance of free Mel. Of particular note is that the Mel-SiO2@CCM group exhibited the strongest inhibitory effect on Fn-infected tumors, significantly superior to both the Mel group and the Mel-SiO2 group (V / V0 = 0.39, ****, P < 0.0001). The tumor tissue weight and gross imaging results were also consistent with the tumor volume results, further demonstrating that Mel-SiO2@CCM has the best anti-tumor effect against Fn-infected tumors. Figure 14 (B and C), this is due to its dual-targeting properties. Furthermore, during the 14-day observation period, there was no significant difference in body weight among the treatment groups (B and C). Figure 15 This indicates that at the current dose, Mel-SiO2@CCM NPs do not cause significant systemic toxicity in mice with subcutaneous tumors, and their tumor growth inhibition effect is significantly better than that of free Mel and Mel-SiO2 NPs.

[0069] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A biomimetic nanocomposite, characterized in that, The biomimetic nanocomplex comprises nanoparticles, a drug and a cell membrane; the nanoparticles load the drug as a carrier, and the cell membrane is coated on the surface of the nanoparticles; The nanoparticles are mesoporous silica nanoparticles, the drug is bee venom, and the cell membrane is a cancer cell membrane. The biomimetic nanocomplex is used for treating colorectal cancer related to Fusobacterium nucleatum infection.

2. The biomimetic nanocomplex of claim 1, wherein, The particle size of the biomimetic nanocomplex is 80-130 nm.

3. The biomimetic nanocomplex of claim 1, wherein, The cancer cell membrane is a colorectal cancer cell membrane.

4. The biomimetic nanocomplex of any one of claims 1-3, wherein, The drug loading rate of the biomimetic nanocomplex is 7.96%.

5. Use of the biomimetic nanocomplex according to any one of claims 1-4 in the preparation of a medicament for treating colorectal cancer related to Fusobacterium nucleatum infection.

6. The use according to claim 5, wherein the compound is ###0002### The administration dose of the biomimetic nanocomplex is 12.56 mg / kg.

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