Bionic nanocomposite and application thereof

Abstract

The invention provides a bionic nano-composite and application thereof. The bionic nano-composite comprises nano-particles, a medicine and a cell membrane, the nano-particles are used as carriers to load the medicine, and the cell membrane is coated on the surfaces of the nano-particles; the particle size of the bionic nano composite is 80 to 130 nm; the nano-particles are meso-porous silicon nano-particles, the medicine is bee toxin, and the cell membrane is a cancer cell membrane. The bionic nano-composite disclosed by the invention has high selectivity, low toxicity and a strong anti-tumor effect, and a mechanism of an anti-tumor effect is exerted by relieving an Fn-induced tumor inhibition microenvironment and activating tumor specific immunity; the effect of the targeted nucleic acid bacillus in colorectal cancer treatment and the synergistic treatment effect of a strategy for simultaneously targeted removal of Fn and tumor cells are further clarified, so that a zoology basis and related data are provided for further human body clinical application research; an important reference basis is provided for subsequent pathogenic bacterium killing, immunosuppression microenvironment improvement and immunotherapy, the immunotherapy effect of the colorectal cancer patient can be improved, and a very high clinical transformation value is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a bionic nanocomposite and application thereof. Background Art

[0002] Colorectal cancer (CRC) is a malignant tumor originating from the intestinal epithelium. Its high morbidity and mortality have put significant pressure on health and medical resources. Fusobacterium nucleatum (Fn), a core pathogen in tumors, is enriched in colorectal cancer tissues, promoting the proliferation and metastasis of CRC and affecting the efficacy of chemotherapy and other treatments. However, there is currently a lack of specific clearance methods for Fn. Therefore, targeted treatment for patients with Fn-infected colorectal cancer has become a key scientific issue that needs to be addressed.

[0003] At present, most studies on the effect of eliminating intestinal pathogens on colorectal cancer efficacy use antibiotics, such as metronidazole or amphotericin. These antibiotics will affect other flora while eliminating pathogens, which may lead to intestinal microecological imbalance. [1] Only a few researchers have attempted targeted treatment of a single pathogen, such as the research team led by Professor Zhang Xianzheng, a Chinese scholar. [2] A phage-guided nanocomplex system loaded with irinotecan was designed. After phages eliminated Fusobacterium nucleatum (Fn), the chemotherapy effect of irinotecan was improved, but phages still have potential biosafety issues. Therefore, if the characteristics of Fn enrichment in tumor tissues and homologous tumor cell adhesion can be utilized, developing a therapeutic strategy that targets and eliminates intratumoral Fn and tumor cells without destroying the balance of intestinal microecology is considered to be a promising experimental exploration direction 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] The present invention provides a bionic nanocomposite and application thereof. The bionic nanocomposite Mel-SiO2@CCM inherits the homologous adhesion characteristics of tumor cells and the targeting ability of Fusobacterium nucleatum, so that it can achieve targeted enrichment in a CRC model infected with Fusobacterium nucleatum and exhibit excellent enhanced anti-tumor and bactericidal dual therapeutic effects.

[0008] The technical solution adopted by the present invention to achieve the above purpose is: a bionic nanocomposite, which includes nanoparticles, drugs and cell membranes; the nanoparticles are used as carriers to load the drugs, and the cell membranes are coated on the surfaces of the nanoparticles.

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

[0010] Furthermore, the nanoparticles are any one of mesoporous silicon nanoparticles, gold nanoparticles, magnetic nanoparticles and polymer nanoparticles, the drug is any one of bee toxin, 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 silicon nanoparticles, the drug is bee toxin, and the cell membrane is a cancer cell membrane.

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

[0013] The invention also discloses the application of the bionic nanocomposite in preparing drugs for treating colorectal cancer.

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

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

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

[0017] Second, 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 also showed significant anti-tumor effect in various tumor models.

[0018] Third, the bionic nanocomplex of the present invention exerts its anti-tumor effect by relieving the tumor suppressive microenvironment induced by Fusobacterium nucleatum Fn and activating tumor-specific immunity, further clarifying the role of targeted Fn in the treatment of colorectal cancer and the synergistic therapeutic effect of the strategy of simultaneously targeting and clearing Fn and tumor cells, thereby providing an zoological basis and relevant data for further human clinical application research, and providing an important reference basis for subsequent killing of pathogenic bacteria and improving the immunosuppressive microenvironment combined with immunotherapy, which is helpful to improve the immunotherapy effect of colorectal cancer patients and has a strong clinical transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without paying creative work.

[0020] Figure 1 The high-resolution transmission electron microscopy analysis diagram of SiO2@CCM prepared in Example 1, wherein (A) is a transmission electron microscopy scanning diagram and (B) is an elemental analysis diagram;

[0021] Figure 2 The transmission electron microscope scanning images of Mel-SiO2@CCM NPs prepared in Example 1, wherein (A) is a transmission electron microscope scanning image and (B) is an enlarged image;

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

[0023] Figure 4 The membrane protein SDS-PAGE gel electrophoresis patterns of the marker, cell membrane lysate, SiO2@CCM and Mel-SiO2@CCM NPs prepared in Example 1;

[0024] Figure 5The drug loading and encapsulation efficiency of Mel-SiO2@CCM NPs prepared in Example 1 are analyzed, wherein (A) is the drug loading and (B) is the encapsulation efficiency;

[0025] Figure 6 The drug release analysis diagram of the biomimetic nanocomposite prepared in Example 1, wherein (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 in different pH solutions of Mel-SiO2@CCM NPs;

[0026] Figure 7 This is a stability analysis diagram of the bionic nanocomposite prepared in Example 1;

[0027] Figure 8 This is a diagram of the hemolytic activity assay of the biomimetic nanocomposite prepared in Example 1, in which 1 is the PBS group, 2 is the SiO2@CCM group, 3 is the Mel group, 4 is the Mel-SiO2 group, and 5 is the Mel-SiO2@CCM group;

[0028] Fig. 9 The in vitro targeted antibacterial effect analysis diagram of the bionic nanocomposite prepared in Example 1, wherein (A) is an enrichment diagram of the bionic nanocomposite on the Fn surface under a scanning electron microscope, (B) is an antibacterial effect analysis diagram of the bionic nanocomposite under a scanning electron microscope, (C) is a diagram of the enhanced antibacterial effect of the nanocomposite loaded with bee venom peptide, (D) is a diagram of the competitive antibacterial effect of the nanocomposite through Gal-GalNAc, (E) is an antibacterial curve diagram of different formula drugs at equal bee venom peptide concentrations, (F) is an anti-Fn effect analysis diagram under a microscope, (G) is an antibacterial effect diagram of live-dead staining, (H) is an antibacterial effect diagram of colony counts, and (I) is a statistical diagram of colony counts;

[0029] Fig.10 The metabolic diagram of nanoparticles enriched in the tumor site of mice over time after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with intestinal cancer orthotopic tumors via the tail vein. (A) is the fluorescent in vivo imaging of nanoparticles in the tumor site at different time points, and (B) is the corresponding average fluorescence intensity statistical diagram;

[0030] Fig.11 The fluorescence in vivo imaging of isolated organs and tumors at different times after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with orthotopic tumors via the tail vein;

[0031] Fig.12The fluorescence statistics of the isolated organs and tumors over time were obtained after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with orthotopic tumors via the tail vein.

[0032] Fig.13 The in vitro fluorescence distribution statistics of the two fluorescent nanoparticles in various ex vivo organs and tumors at 8h (A), 24h (B) and 48h (C) after SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs were injected into mice with orthotopic tumors via the tail vein.

[0033] Fig.14 The figures are the analysis of the in vivo tumor inhibition effect of tumor-bearing BALB / c nude mice after different treatments, where (A) is the growth curve of tumor volume, (B) is the tumor weight, and (C) is a photo of the isolated tumor;

[0034] Fig.15 This is a tumor growth curve after treatment in Example 5. DETAILED DESCRIPTION

[0035] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0036] The following describes the implementation methods of the present application through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0037] Example 1

[0038] A bionic nanocomposite, the preparation method of which is as follows:

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

[0040] RPMI-1640 medium was used for culture, and its formula contained 1% non-essential amino acids and L-glutamine. The medium was additionally supplemented with penicillin (10000U / mL) and streptomycin (10mg / mL) as dual antibiotics, and contained 10% fetal bovine serum as a nutritional supplement. The culture conditions were set in an environment of 5% CO2, maintaining 90% relative humidity, and kept in an incubator at 37°C. After the CT26 cancer cells were fully grown in a T-75 culture flask, the cells were washed with PBS to remove impurities such as serum and dead cells in the culture medium, and then the cells were collected and treated with PBS or 1mM EDTA solution, centrifuged at 500g for 10min, the supernatant was removed, the cell pellet was collected, and rinsed with PBS three times.

[0041] Resuspend the lysed cells with 10 mL of hypotonic lysis buffer (containing 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl and a solution of EDTA-free protease inhibitors), freeze the cells in liquid nitrogen for 10 min, thaw them in a 37°C water bath for 10 min, and then sonicate on ice (frequency 40 kHz, power 100 W) for 5 min, repeat 6-10 cycles. Centrifuge at 3200 g for 5 min, discard the unbroken cell and cell nucleus precipitate, save the supernatant, centrifuge the supernatant at 4°C, 20,000 g for 30 min, discard the precipitate, and transfer the supernatant 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 (80000g, 1.5h) to obtain a membrane precipitate, and the precipitate containing the plasma membrane was washed once with 10mMTris-HCl and 1mM EDTA, and then the purified cell membrane was collected. The protein content in the purified membrane was determined by the BCA method, so that vesicles could be further prepared. The particles were physically squeezed 10 times on a microliposome extruder to pass through a 400nm polycarbonate porous membrane to obtain CCM vesicles.

[0043] (2) Synthesis of SiO2 NPs

[0044] Preparation of SiO2 precursor: In a constant temperature water bath at 30°C, mix 3.14 mL of ammonia water, 71.4 mL of anhydrous ethanol and 10 mL of deionized water in a beaker and stir with a magnetic stirrer until uniform. Quickly add 6 mL of TEOS and stir rapidly at 30°C for 2 h to obtain a white solution. Centrifuge to separate the white solid, wash with anhydrous ethanol and deionized water and centrifuge for 3 times, then air dry naturally to obtain SiO2 powder.

[0045] Preparation of SiO2 suspension: Take 0.5g SiO2 powder, add 100mL deionized water, and ultrasonically treat for 20min to disperse to obtain SiO2 suspension; prepare a mixed solution containing 0.75g CTAB, 150mL ethanol, 150mL deionized water and 2.75mL ammonia water, and mix it with the SiO2 suspension. After continuing ultrasonic treatment for 2h, quickly add 1.5mL TEOS, stir evenly, and ultrasonicate for another 6h. Collect the solid after centrifugation, wash with deionized water and centrifuge 3 times, then redisperse in anhydrous ethanol and ultrasonicate for 10min, and 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 Na2CO3 solution with a concentration of 0.6 M. The white solid powder was added to the Na2CO3 solution, stirred thoroughly and dispersed by ultrasound, and then stirred in a water bath at 80°C for 6 h. After cooling to room temperature, the mixture was centrifuged and washed with deionized water for 2-3 times, and then 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, which was thoroughly washed with anhydrous ethanol and freeze-dried. This step was repeated 3 times to ensure that CTAB was completely removed to form a mesoporous structure. The final product was 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 with an ultrafiltration membrane to remove excess unreacted reagents, and the obtained SiO2 NPs were divided into 3 groups for subsequent coupling experiments. Melittin and SiO2 NPs were added to the suspension of SiO2 NPs at a mass ratio of 1:5, 1:10, 1:20 and 1:40, and after stirring for 2 hours, 3 mg of EDC was added to each group for reaction for 12 hours. After the reaction was completed, the mixture was subjected to solid-liquid separation at a speed of 1000 rpm for 10 minutes. The obtained sample was then rinsed with deionized water 3 times to remove residual incomplete reaction reagents. Finally, the obtained product was freeze-dried 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) was first passed through a micro-extruder equipped with a 400 nm filter membrane to form uniform membrane fragments, and then mixed with the SiO2 NPs obtained in step (2) or the Mel-SiO2 NPs suspension obtained in step (3), and then dispersed on ice by ultrasound (frequency of 40 kHz, power of 100 W) for 5 min. Subsequently, the obtained solution was extruded through an extruder equipped with a polycarbonate filter membrane with a pore size of 200 nm for 10 consecutive times to obtain CCM-encapsulated SiO2@CCM and melittin-loaded and CCM-encapsulated Mel-SiO2@CCM NPs, respectively.

[0052] The prepared SiO2 NPs were observed using high-resolution transmission electron microscopy. Figure 1 A shows that SiO2 NPs have a typical crystal morphology, which can be seen to be full of regular and ordered pore structures, thus having a high specific surface area, a smooth surface, and uniform dispersion. Figure 1 The elemental analysis diagram of B also confirms the elemental composition of SiO2 NPs. The above results show that the SiO2 NPs prepared by the present invention have uniform size and morphology and are evenly dispersed, which is the basis for further loading of drugs and CCM biomimetic encapsulation modification.

[0053] The morphology of Mel-SiO2@CCM NPs was examined using transmission electron microscopy. Figure 2 The results showed that the surface of the typical spherical core-shell structure of SiO2 NPs was coated with a low-density halo-like thin layer with a diameter of about 9 nm, which directly proved that CCM was successfully encapsulated and modified on the surface of the SiO2 NPs core structure.

[0054] In the present invention, the particle size and particle size distribution of SiO2 NPs were measured by dynamic light scattering (DLS) technique using a particle size analyzer. Figure 3 A reveals that the average particle size of SiO2 NPs is 92.8±4.4nm. When they are encapsulated by CCM to form Mel-SiO2@CCM NPs, the average particle size increases to 102.2±4.0nm, indicating that the particle size increases by about 9nm. The particle size distribution of both particles is in the range of 80 to 130nm and presents a normal distribution morphology, which is consistent with the observation results of electron microscopy. The particle sizes of SiO2 NPs and Mel-SiO2@CCM NPs prepared by the present invention are both close to 100nm, which belongs to the very small size range and is very suitable as a carrier of nanomedicine. Figure 3The results of measuring the surface potential of nanoparticles using B's Zeta potential instrument showed that the surface potential of SiO2NPs was -36.9±3.3mV, and the cell membrane potential was -24.6±2.5mV. After drug loading and cell membrane wrapping, the potential of Mel-SiO2@CCM NPs was -27.4±4.7mV, which was close to the cell membrane potential, which indirectly indicated the successful wrapping of the cell membrane.

[0055] Membrane proteins are the basis for the subsequent nanocomplexes to function. After electron microscopy, particle size and potential have demonstrated the successful coating of the cell membrane, the surface of Mel-SiO2@CCM NPs was then characterized to determine whether the functional proteins on CCM were successfully retained. SDS-PAGE gel electrophoresis was performed on the membrane proteins of certain concentrations of Marker, cell membrane lysate, SiO2@CCM and Mel-SiO2@CCM NPs, respectively. Figure 4 As shown in the figure, the membrane protein bands of SiO2@CCM and Mel-SiO2@CCM NPs are very similar to the protein bands of CCM of CT26 cells, which also shows that Mel-SiO2@CCM NPs successfully retain most of the functional proteins on the cell membrane. This is also the basis for the biomimetic nanocomposite Mel-SiO2@CCM NPs to play a targeting role.

[0056] Example 2 Determination of properties of biomimetic nanocomposites

[0057] The bionic 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 nanocomposite prepared in Example 1 were measured, and the BCA protein assay was used to quantify the Mel encapsulated in the Mel-SiO2@CCM NPs. Experiments were conducted with Mel / SiO2 mass ratios of 1:5, 1:10, 1:20, and 1:40. The results were ( Figure 5 A and B) show that at the beginning, when the Mel input amount is low, the encapsulation efficiency of Mel is stably maintained at more than 80%, which also proves the high drug loading capacity of the carrier SiO2. With the increase of Mel input amount, when the Mel / SiO2 mass ratio increases to 1:10, the encapsulation efficiency of Mel decreases significantly and the increase rate of Mel's drug loading rate also decreases. Considering the utilization rate of drugs and materials, 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 bee venom peptide in Mel-SiO2@CCM NPs is 7.96%, and the encapsulation efficiency is 79.6%.

[0059] The drug release amount of the biomimetic nanocomposite prepared in Example 1 was measured. In order to evaluate the responsiveness of Mel-SiO2@CCMNPs to pH changes, the drug release amount was measured under different pH environments. The results are shown in Figure 2. Figure 6 As shown in A, the release rate of Mel from Mel-SiO2@CCM NPs in PBS buffer at pH 7.4 is low, with only 23.4% released within 24 hours. In contrast, the release rate of unencapsulated Mel-SiO2 under the same conditions reached 82.9%. This indicates that CCM-modified Mel-SiO2@CCM NPs exhibit good stability in a neutral environment simulating physiological conditions, can maintain structural integrity, effectively seal Mel, and prevent premature drug leakage from causing toxicity to normal tissues, thereby improving biosafety. When the environmental pH value is reduced to 6.5 and 5.5 (simulating the acidic conditions of the tumor microenvironment and endosomes / lysosomes), the release rate of Mel increases significantly, with the cumulative release rates reaching 78.4% and 88.7% (24 hours) respectively. Figure 6 B). This phenomenon indicates that the nanocarrier material becomes unstable under acidic conditions and can disintegrate in a slightly acidic environment after reaching the tumor site and being taken up by cells, effectively releasing the encapsulated bee venom peptide to exert its efficacy, thus achieving specific and controllable drug release at the tumor site.

[0060] The stability of the bionic nanocomposite prepared in Example 1 was measured. The stability of the bionic nanocomposite is the prerequisite for the drug to take effect after entering the body and finally reaching the target organ. The good stability of Mel-SiO2@CCM NPs is the key to maintaining a long blood circulation time, preventing drug leakage, and improving the bioavailability of the drug. On the contrary, if the NPs show instability during the circulation process in the body, it may lead to drawbacks such as low drug utilization efficiency and poor treatment effect. Therefore, the stability of Mel-SiO2@CCM NPs was evaluated in a deionized water solution at room temperature and under simulated physiological conditions (PBS buffer) at 37°C. The results are as follows: Figure 7 As shown, the experimental results indicate that the particle size of Mel-SiO2@CCM NPs changes little and maintains good dispersibility whether stored in aqueous solution or PBS solution for 24 h, which indicates that Mel-SiO2@CCM NPs exhibit excellent dimensional stability in both environments. This stability is crucial for the application, storage and transportation of nanoparticles, and will be beneficial to the mass production required for subsequent transformation.

[0061] The hemolytic activity of the biomimetic nanocomposite prepared in Example 1 was measured. Although the above results demonstrate that the biomimetic nanocomposite Mel-SiO2@CCM NPs has the advantages of high drug loading, release in the acidic environment of tumors and good stability under physiological conditions, however, given that Mel is a membrane-dissolving peptide, it can destroy the cell membrane, causing leakage of cell contents and ultimately cell death. This action has poor selectivity and will also cause certain damage to normal cells, among which the hemolytic activity is particularly prominent. Therefore, for the subsequent safe application in animals and even potential clinical transformation, we then investigated whether Mel-SiO2@CCM NPs can reduce the hemolytic activity of Mel. We extracted blood from BALB / c mice, obtained red blood cells (RBC) after centrifugation, and performed an in vitro hemolysis test to compare 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 in the figure, free Mel itself has a significant hemolytic effect, which has been confirmed in our experiment: free Mel at a concentration of 8 μg / mL can cause hemolysis of about 79.8% of red blood cells. In sharp contrast, Mel-SiO2@CCM NPs containing the same amount of Mel did not show obvious hemolytic behavior, and even when the Mel concentration in Mel-SiO2@CCM NPs was as high as 100 μg / mL, its hemolysis rate remained at an extremely low level of only about 5%. This result shows that Mel loaded into SiO2 NPs and then encapsulated in CCM significantly reduced hemolytic toxicity and effectively avoided the problem of nonspecific cytotoxicity, which is of great significance for its future clinical application.

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

[0063] The targeting binding ability of Mel-SiO2@CCM prepared in Example 1 to Fn was measured, and the images obtained by scanning electron microscopy (SEM) showed that compared with SiO2, SiO2@CCM was more strongly enriched on the Fn surface ( Fig. 9 A). Similarly, in the co-culture system of Fn and Escherichia coli (E. coli), the selective enrichment of SiO2@CCM on the Fn surface relative to the E. coli surface was observed, confirming the targeting binding ability of SiO2@CCM to Fn ( Fig. 9 C). Subsequently, we selected two Mel concentrations (5ug / mL and 10ug / mL) confirmed by previous studies to test their antibacterial effects. Compared with the Mel group alone, the Mel-SiO2@CCM group showed an improved antibacterial effect at these two concentrations as assessed by the colony forming unit (CFU) reduction ratio ( Fig. 9D). Meanwhile, SEM images revealed a variety of obvious changes in the bacteria, including distortion, perforation, and dissolution ( Fig. 9 B). In summary, both the CFU reduction ratio and morphological changes confirmed that the targeted bactericidal effect of Mel-SiO2@CCM was enhanced compared with Mel. In addition, the anti-Fn activity of various formulations including control (PBS), SiO2@CCM, Mel, Mel-SiO2 and Mel-SiO2@CCM was further studied. The Fn suspension was inoculated in LB medium and the above formulations were added respectively. The OD600 was measured at different time points. The rising trend of the growth curve of the treatment group containing Mel was inhibited to varying degrees after 4h of treatment. It is worth noting that the Mel-SiO2@CCM group completely inhibited bacterial replication within 24h ( Fig. 9 E). The antibacterial efficiency was quantitatively evaluated by colony counts, and the Mel and Mel-SiO2 groups showed a 3-log10 and 5-log10 CFU reduction in the number of bacterial colonies, respectively, after 8 h of treatment, while all the bacteria treated with Mel-SiO2@CCM were killed ( Fig. 9 H and 9I). The antibacterial activity of Mel-SiO2@CCM was further visualized and verified by optical microscopy and LIVE / DEAD bacterial viability kit observation and confocal laser scanning microscopy ( Fig. 9 F and 9G), when treated with PBS, most bacteria showed green fluorescence (FL) (live), while increased red FL (dead) was found in the Mel group and Mel-SiO2 group, which was consistent with the previous growth curve and colony counts. All bacteria under Mel-SiO2@CCM treatment showed red FL (dead). In summary, the above results not only demonstrated the excellent antibacterial property of Mel-SiO2@CCM, but also inferred that this function was mainly attributed to its targeting ability to Fn and membrane disruption properties.

[0064] Example 4 Analysis of the in vivo targeted anti-tumor effect of biomimetic nanocomposites

[0065] The biodistribution characteristics of the biomimetic nanocomplex prepared in Example 1 in BALB / c mice with Fn-infected intestinal cancer in situ tumors were evaluated using in vivo imaging technology. Cy7.5-labeled SiO2 NPs (SiO2 / Cy7 NPs) and SiO2@CCM NPs (SiO2@CCM / Cy7 NPs) were injected into mice through the tail vein, and subsequent in vivo imaging monitoring showed that the accumulation of SiO2 / Cy7.5 NPs and SiO2@CCM / Cy7.5 NPs in the tumor area gradually increased over time and reached peak values ​​at 4h and 8h after injection, respectively ( Fig.10A and B). After careful observation, it was found that 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 at 2h, 4h, 8h, 12h and 24h after injection, respectively. It is worth noting that at the 48h time point, the tumor mean fluorescence intensity (MFL) of the SiO2 / Cy7.5 group was still close to 1 / 3 of its peak value, while the tumor mean fluorescence intensity (MFL) of the SiO2@CCM / Cy7.5 group was undetectable. Compared with the SiO2 / Cy7.5 group alone, the SiO2@CCM / Cy7.5 NPs showed significantly enhanced tumor targeting and more persistent retention ability, which resulted in a stronger MFL signal and a longer duration in the tumor area, which was based on the typical EPR effect of nanoparticles and the dual targeting ability of CCM.

[0066] In addition, the present 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 ( Fig.11 , Fig.12 and Fig.13 ). The results showed that at 8h, 24h and 48h, the tumor MFL intensity of the SiO2@CCM / Cy7.5 group was higher than that of the SiO2 / Cy7.5 group, which once again demonstrated the better tumor targeting and persistence of SiO2@CCM / Cy7.5NPs. In addition, it was observed that a small amount of SiO2@CCM / Cy7.5NPs was still present in the liver and kidneys at 8h and 24h, and completely disappeared at 48h. This may be because the CCM coating avoided the clearance of the immune system to a certain extent, thereby prolonging the circulation time in the body, but still had a certain immunogenicity

[64] But in general, the SiO2@CCM / Cy7.5 nanocarrier showed good biocompatibility and did not cause obvious toxicity in in vitro and in vivo studies, indicating that it has good potential for clinical application in the future.

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

[0068] The 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 times, with a uniform Mel dose of 1 mg / kg. The tumor volume and body weight of the mice were measured every other day. After two weeks, the mice were killed, and the tumors were removed for photographing and weighing. Fig.14As shown in A, mice in the Mel group and Mel-SiO2 group showed a certain degree of tumor growth inhibition, with relative tumor volumes (V / V0) of 2.71 and 2.36, respectively. Different from the results of in vitro experiments, the relative tumor volume of mice in the Mel-SiO2 group was lower than that in the Mel group, which may be attributed to the enhanced permeation and retention effect (EPR) caused by the nanomaterials, which enables the concentration of Mel-SiO2 in the tumor site to be maintained, and improves the bioavailability of Mel compared with the rapid clearance of free Mel. It is particularly noteworthy that the Mel-SiO2@CCM group showed the strongest inhibitory effect on Fn-infected tumors, which was significantly better than the Mel group and Mel-SiO2 group (V / V0 value = 0.39, ****, P < 0.0001). The results of tumor tissue weight and macroscopic photos were also consistent with the results of tumor volume, further proving that Mel-SiO2@CCM has the best anti-tumor effect on Fn-infected tumors ( Fig.14 B and C), which is due to its dual targeting properties. In addition, no significant difference was observed in the body weight of mice in the treatment groups during the 14-day observation period ( Fig.15 ), which indicates that at the current dose, Mel-SiO2@CCM NPs do not cause obvious systemic toxicity when used for the treatment of subcutaneous tumor mice, and their inhibitory effect on tumor growth is significantly better than that of free Mel and Mel-SiO2 NPs.

[0069] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A bionic nanocomposite, characterized in that: The bionic nanocomposite comprises nanoparticles, drugs and cell membranes; the nanoparticles are used as carriers to load the drugs, and the cell membranes are coated on the surfaces of the nanoparticles.

2. The biomimetic nanocomposite according to claim 1, characterized in that: The particle size of the bionic nanocomposite is 80-130 nm.

3. The biomimetic nanocomposite according to claim 1, characterized in that: The nanoparticles are any one of mesoporous silicon nanoparticles, gold nanoparticles, magnetic nanoparticles and polymer nanoparticles; the drug is any one of bee toxin, 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.

4. The biomimetic nanocomposite according to claim 3, characterized in that: The nanoparticles are mesoporous silicon nanoparticles, the drug is bee toxin, and the cell membrane is a cancer cell membrane.

5. The biomimetic nanocomposite according to any one of claims 1 to 4, characterized in that: The drug loading rate of the bionic nanocomposite is 7.96%.

6. Use of the bionic nanocomposite according to any one of claims 1 to 5 in the preparation of a drug for treating colorectal cancer.

7. The use according to claim 6, characterized in that The dosage of the bionic nanocomposite is 12.56 mg / kg.

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