Hybrid membrane coated BCZT nano material as well as preparation method and application thereof

Abstract

The invention discloses a hybrid membrane coated BCZT nano material as well as a preparation method and application thereof, and belongs to the technical field of biomedical treatment. The preparation method of the hybrid membrane coated BCZT nano material comprises the following steps: preparing a BCZT piezoelectric nano material; preparing hybrid membrane vesicles; and uniformly coating the hybrid membrane vesicles on the surface of the BCZT piezoelectric nano material by using a liposome extruder and an ultrasonic dispersion instrument to obtain the hybrid membrane coated BCZT nano material. The BCZT nano material disclosed by the invention can efficiently generate reactive oxygen species (ROS) under ultrasonic catalysis, has a killing effect on bacteria, has very strong tissue penetrability, and can go deep into infected parts to realize an effective antibacterial effect. And finally, the BCZT material is wrapped by a hybrid membrane wrapping technology, so that the stability and biocompatibility of the nano material can be improved, the nano material is more stable in an in-vivo environment, and possible side effects are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a hybrid membrane-wrapped BCZT nanomaterial, a preparation method and an application thereof. Background Art

[0002] With the widespread use of antibiotics, the emergence and spread of multidrug-resistant bacteria (MDR) has become a major challenge to global public health. These resistant bacteria are resistant to a variety of commonly used antibiotics, which greatly reduces the effectiveness of traditional antibacterial treatments. In particular, methicillin-resistant Staphylococcus aureus (MRSA), most of which infects patients in hospitals or other medical institutions, is a common and highly toxic bacterium in clinical practice. At present, the treatment options for resistant bacteria are relatively limited, so it is urgent to develop new and efficient antibacterial strategies.

[0003] In recent years, sonodynamic therapy (SDT) using nanomaterials has attracted widespread attention as a potential antibacterial technology. SDT involves exposing the target tissue to non-toxic sensitizing chemicals and low-intensity ultrasound, achieving site-specific cytotoxicity by generating reactive oxygen species (ROS) in response to ultrasound, and can be used to treat bacterial infections. This approach can potentially be used for both superficial and deep microbial infections. However, piezoelectric materials have problems with poor targeting and short in vivo existence in clinical antibacterial applications, which limits their clinical antibacterial effects. Therefore, constructing a therapeutic system that can target pathogens, prolong their in vivo existence time, and efficiently treat drug-resistant bacteria is an urgent problem that needs to be solved in the current nanomaterial sterilization.

[0004] To address these issues, researchers are exploring a variety of strategies. For example, targeted delivery agents such as nanoparticle composites, liposomes, and microbubbles are used to improve the bioavailability of sonosensitizers and reduce their in vivo clearance. In addition, researchers are also studying how to improve their in vivo circulation time and targeting through the design and surface modification of nanomaterials. By designing a double-layer polyethylene glycol polymer chain with a dynamic outer layer structure on the surface of nanoparticles, their circulation time in the blood can be greatly prolonged, thereby improving their effective accumulation at the site of infection. In summary, nanomaterials have broad application prospects in the field of antibacterial therapy, but they still face many challenges, such as improving targeting, prolonging in vivo circulation time, and how to effectively integrate them into clinical treatment. Summary of the invention

[0005] The present invention aims to provide a hybrid membrane-wrapped BCZT nanomaterial, its preparation method and application. By synthesizing BCZT nanomaterials with good piezoelectric properties and combining hybrid membrane wrapping technology, a therapeutic system that can accurately target pathogenic bacteria and reprogram immune cells in vivo is constructed. This system not only improves the stability and biocompatibility of nanomaterials, but also enhances the body's immune response to drug-resistant bacteria, providing a new strategy for the treatment of multidrug-resistant bacteria.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a hybrid membrane-wrapped BCZT nanomaterial comprises the following steps: S1. Preparation of BCZT piezoelectric nanomaterials; S2, preparing hybrid membrane vesicles; S3. Using a liposome extruder and an ultrasonic disperser, the hybrid membrane vesicles obtained in S2 are uniformly wrapped on the surface of the BCZT piezoelectric nanomaterial obtained in step S1 to obtain a hybrid membrane-wrapped BCZT nanomaterial.

[0007] Preferably, in step S1, the method for preparing the BCZT piezoelectric nanomaterial comprises the following steps: A1. TiCl 4 Dissolve in anhydrous ethanol to obtain solution A; A2. BaCl 2 ·2H 2 O, CaCl 2 ·2H 2 O and ZrCl 4 Dissolve in deionized water to obtain solution B; A3. Mix solution A and solution B evenly, add NaOH and PVP, mix well and transfer to a stainless steel autoclave lined with tetrafluoroethylene for reaction. After the reaction, wash the precipitate repeatedly with deionized water and anhydrous ethanol, and dry to obtain BCZT piezoelectric nanomaterials.

[0008] Preferably, in step S2, the method for preparing the hybrid membrane vesicles comprises the following steps: B1. Co-incubate macrophages with pathogenic bacteria to activate the targeted proteins on the surface, and extract the pre-activated macrophage membranes; B2. Take blood to separate platelets and extract platelet membranes; B3, extracting exosome membranes from M1 macrophages; B4. The above-mentioned macrophage membrane, platelet membrane and M1 macrophage exosome membrane are fused by ultrasound to form hybrid membrane vesicles.

[0009] The invention also discloses a hybrid membrane-wrapped BCZT nano material prepared by the preparation method.

[0010] The invention also discloses a hybrid membrane-wrapped BCZT nanomaterial prepared by the preparation method or an application of the hybrid membrane-wrapped BCZT nanomaterial in treating drug-resistant bacterial infection.

[0011] The present invention also discloses a method for treating drug-resistant bacteria by using a hybrid membrane-wrapped BCZT nanomaterial system, comprising the following steps: T1, targeting TLR2 and TLR6 on the membrane surface of preactivated macrophages to Gram-positive pathogens or targeting TLR4 to Gram-negative pathogens; T2, using M1 macrophage exosome membranes to reprogram macrophages, converting M2 immunosuppressive macrophages into M1 macrophages that resist pathogenic microorganisms; T3. Ultrasonic therapy is used to activate the hybrid membrane wrapped with BCZT nanomaterials to produce ROS, which effectively kills drug-resistant bacteria.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention discloses a nanomaterial BCZT with excellent piezoelectric properties, and combines hybrid membrane wrapping technology to provide a new strategy for the treatment of multi-drug resistant bacteria. First, by using pre-activated macrophage membranes, BCZT nanomaterials can accurately target the inflammatory site where the pathogens are located, significantly improving the targeting of the treatment. Secondly, through the CD47 "don't eat me" protein on the surface of the platelet membrane, the BCZT nanomaterial can effectively escape the phagocytic clearance of immune cells in the body, so that it exists in the body for a longer time, improving the therapeutic effect. In addition, the platelet membrane can bind to α-hemolysin on the surface of pathogenic bacteria, protect other immune cells in the inflammatory site, and improve the immune effect. At the same time, the M1 macrophage exosome membrane can convert immunosuppressive M2 macrophages into M1 macrophages that resist pathogenic microorganisms, further enhancing the body's immune response to drug-resistant bacteria. Sonodynamic therapy (SDT) using BCZT nanomaterials has better tissue penetration, spatiotemporal controllability, and non-invasiveness, which makes SDT have significant advantages in the treatment of bacterial disease infections. BCZT nanomaterials can efficiently generate reactive oxygen species (ROS) under ultrasonic catalysis, which have a killing effect on bacteria. They have strong tissue penetration and can penetrate deep into the infected site to achieve effective antibacterial effects. Finally, by encapsulating BCZT materials with hybrid membrane encapsulation technology, the stability and biocompatibility of nanomaterials can be improved, making them more stable in the in vivo environment and reducing possible side effects. In summary, the present invention provides a new method for efficient, targeted, and immune-enhancing treatment of multidrug-resistant bacteria, which is expected to play an important role in clinical treatment.

[0013] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The preparation process of the hybrid membrane-wrapped BCZT nanomaterial system of the present invention; Figure 2 A transmission electron microscope image of the BCZT piezoelectric nanomaterial provided in Example 1 of the present invention; Figure 3 The lattice structure of the BCZT piezoelectric nanomaterial provided in Example 1 of the present invention; Figure 4 This is a statistical graph of changes in the gene content of macrophage membrane-targeted Gram-positive bacterial membrane proteins provided in Example 2 of the present invention, wherein Figure 4 A in the figure is the statistical graph of the change of TLR2 gene expression in macrophages over time. Figure 4 B in the figure is a statistical graph showing changes in TLR4 gene expression in macrophages over time; Figure 5 A transmission electron micrograph of a hybrid membrane encapsulating BCZT nanomaterial provided in Example 3 of the present invention; Figure 6 This is a comparison of the electron microscope before and after the hybrid membrane of the present invention wraps the BCZT nanomaterial, where: Figure 6 A in the figure is a transmission electron microscope image of BCZT. Figure 6 B in the figure is the transmission electron microscope image of BCZT@HM; Figure 7 The flow chart of the hybrid membrane of the present invention before and after wrapping the BCZT nanomaterial; Figure 8 It is a statistical diagram of protein blotting detection before and after the hybrid membrane of the present invention encapsulates BCZT nanomaterials; Fig. 9 The relative fluorescence amount of ROS generated by BCZT ultrasound before and after the hybrid membrane of the present invention wraps the BCZT nanomaterial; Fig.10 This is an electron microscopic image of the hybrid membrane-wrapped BCZT nanomaterial system combined with pathogenic bacteria; Fig.11 This is a graph showing the number of pathogenic bacterial colonies treated by the hybrid membrane-encapsulated BCZT nanomaterial system and immune cells of the present invention. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0016] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0017] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0018] Embodiment 1 This embodiment provides a BCZT piezoelectric nanomaterial, and the preparation method includes the following steps: A1. 8 mmol TiCl 4 Dissolve in 10 ml of anhydrous ethanol to obtain solution A; A2, 12 mmol BaCl 2 ·2H 2 O, 3 mmol CaCl 2 ·2H 2 O and 2 mmol ZrCl 4 Dissolve in 30 ml of deionized water to obtain solution B; A3. Mix solution A and solution B evenly, add 80 mmol NaOH and 1.0 g PVP, mix well and transfer to a 50 ml tetrafluoroethylene-lined stainless steel autoclave for reaction at 180 ° C for 24 h. After the reaction, wash the precipitate repeatedly with deionized water and anhydrous ethanol until the pH is 7, and dry at 60 ° C for 24 h to obtain BCZT piezoelectric nanomaterials.

[0019] Example 2 This example provides a hybrid membrane vesicle, and the preparation method includes the following steps: B1. After culturing methicillin-resistant Staphylococcus aureus overnight, measure the OD to 0.8, co-incubate macrophages with methicillin-resistant Staphylococcus aureus at a ratio of 1:20, add 100ug / ml gentamicin sulfate after culturing for 1h, and incubate for 24h. Then, wash the macrophages with PBS for 3 times, destroy the macrophage membrane with hypotonic sucrose solution, grind with a homogenizer for at least 20 times, centrifuge at 600xg for 30min, and take the supernatant. Centrifuge the supernatant at 15000xg for 60min to obtain the precipitate, which is the macrophage membrane that activates the targeting protein; B2. After adding anticoagulant to whole blood, centrifuge at 100 x g for 20 min to obtain the supernatant, centrifuge the supernatant at 800 x g for 20 min, resuspend the resulting precipitate in PBS, and then freeze-thaw repeatedly to obtain lysed platelets, and centrifuge at 12,000 x g for 15 min at 4°C to obtain platelet membranes; B3. Treat macrophages with LPS to obtain M1 macrophages, culture M1 macrophages with exosome-free serum medium for 24 hours, take the supernatant medium and centrifuge at 200×g for 20 minutes, take the supernatant and centrifuge at 10,000×g for 30 minutes, filter the supernatant with a 0.22 μm filter membrane, and centrifuge at 100,000×g for 70 minutes. Repeatedly freeze and thaw the resulting precipitate to obtain M1 macrophage exosome membranes; B4. The above macrophage membrane, platelet membrane and M1 macrophage exosome membrane were fused by ultrasound at 42kHZ and 100W for 5 minutes to form hybrid membrane vesicles.

[0020] Example 3 This example provides a hybrid membrane-wrapped BCZT nanomaterial, and the preparation method includes the following steps: The BCZT piezoelectric nanomaterial prepared in Example 1 and the hybrid membrane vesicles prepared in Example 2 were mixed in a ratio of 1:1, and mixed by ultrasonic treatment at 42 kHz and 100 W for 5 min. The mixed solution was extruded 20 times through 400 nm and 200 nm polycarbonate membranes respectively using a liposome extruder. The solution obtained after extrusion contained hybrid membrane-encapsulated BCZT nanomaterial BCZT@HM.

[0021] The physicochemical properties of Examples 1-3 were measured by the following tests.

[0022] 1. The BCZT piezoelectric nanomaterial provided in Example 1 was subjected to a transmission electron microscope (TEM) test. The results are as follows: Figure 2 and Figure 3 .

[0023] Depend on Figure 2 and Figure 3 It can be seen that the morphology of BCZT nanomaterials was characterized by transmission electron microscopy, and high resolution was used to observe the lattice of the nanomaterials, thereby inferring their crystal form.

[0024] 2. The macrophages in Example 2 were co-incubated with methicillin-resistant Staphylococcus aureus, and the RNA level of the macrophages was detected. The results were as follows: Figure 4 .

[0025] Depend on Figure 4 It can be seen that the expression levels of TLR2 and TLR6 genes targeting Gram-positive bacteria increased with time.

[0026] 3. The hybrid membrane wrapped BCZT nanomaterial provided in Example 3 was scanned by electron microscope, and the results were as follows: Figure 5-6 .

[0027] Depend on Figure 5 It can be seen that the surface of the nanomaterial is covered with a film structure.

[0028] Depend on Figure 6 It can be seen that the nanomaterials were successfully covered with a layer of film after physical extrusion.

[0029] 4. The materials provided in Example 1 and Example 3 were subjected to fluorescence detection. The test method was as follows: 10 uM DIO dye was added to the hybrid membrane vesicles obtained in Example 2, incubated at room temperature for 10 min, then centrifuged at 12000 x g for 10 min at 4°C, the supernatant was discarded, 0.5 ml PBS was added, and the centrifugation was repeated 3 times. Then, fluorescent BCZT@HM was obtained by the method of Example 3. The fluorescence of the obtained BCZT@HM and BCZT was detected by flow cytometry. The results are shown in FIG. Figure 7 .

[0030] Depend on Figure 7 It can be seen that the hybrid membrane in Example 3 is successfully combined with the BCZT nanomaterial.

[0031] 5. The materials provided in Example 1 and Example 3 were subjected to Western blotting detection. The test method was as follows: BCZT and BCZT@HM obtained in Example 1 and Example 3 were centrifuged at 3000×g, the supernatant was discarded, 200 ul of cell lysis solution (containing protease inhibitors) was added, lysed in an ice bath for 30 min, centrifuged at 10000×g for 10 min at 4°C, the supernatant was collected, 50 ul of 5×loding buffer was added, and the mixture was boiled in a metal bath at 95°C for 10 min, followed by Western blotting. The results are shown in FIG. Figure 8 .

[0032] Depend on Figure 8 It can be seen that the proteins on the membrane surface still exist after the hybrid membrane is wrapped.

[0033] 6. Ultrasonic testing was performed on the materials provided in Example 1 and Example 3. The test method was as follows: 0.15 mM 9,10-anthracenyl-bis(methylene) was added to the materials obtained in Example 1 and Example 3, and a fluorescence change graph was obtained using an ultrasonic therapy instrument with 1.5 W ultrasound and an ultraviolet spectrophotometer. The results were as follows: Fig. 9 .

[0034] Depend on Fig. 9 It can be seen that the amount of ROS generated by ultrasound before and after BCZT hybrid membrane wrapping is not affected by the membrane.

[0035] 7. The hybrid membrane-wrapped BCZT nanomaterial provided in Example 3 is used to detect drug-resistant bacteria, comprising the following steps: T1, targeting TLR2 and TLR6 on the surface of the macrophage membrane pre-activated in Example 2 to Gram-positive pathogens; T2, achieves immune escape through CD47 protein on the surface of platelet membrane, and binds to α-hemolysin on the surface of pathogenic bacteria to protect immune cells; T3, using M1 macrophage exosome membranes to reprogram macrophages, converting M2-type immunosuppressive macrophages into M1-type macrophages that resist pathogenic microorganisms; T4, using an ultrasonic physiotherapy device to ultrasonically activate the hybrid membrane-wrapped BCZT nanomaterial provided in Example 3 to produce ROS, effectively killing drug-resistant bacteria. Figure 10-11 .

[0036] Depend on Fig.10 It can be seen that Fig.10 This is an electron microscopic image of the drug-resistant bacteria and the hybrid membrane-wrapped BCZT nanomaterial system, proving that the hybrid membrane can bind to bacteria and adhere to their surface.

[0037] Fig.11 The piezoelectric material and the hybrid membrane wrapped BCZT nanomaterial were co-incubated with pathogenic bacteria in the presence of macrophages. The number of pathogenic bacteria was detected through ultrasonic treatment, proving that the hybrid membrane wrapping system can not only effectively kill drug-resistant bacteria, but also play a certain protective role on immune cells and enhance the immune response.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a hybrid membrane-wrapped BCZT nanomaterial, characterized in that: The steps include: S1. Preparation of BCZT piezoelectric nanomaterials; S2, preparing hybrid membrane vesicles; S3. Using a liposome extruder and an ultrasonic disperser, the hybrid membrane vesicles obtained in S2 are uniformly wrapped on the surface of the BCZT piezoelectric nanomaterial obtained in step S1 to obtain a hybrid membrane-wrapped BCZT nanomaterial.

2. The preparation method according to claim 1, characterized in that: In step S1, the method for preparing the BCZT piezoelectric nanomaterial comprises the following steps: A1. Dissolve TiCl4 in anhydrous ethanol to obtain solution A; A2, dissolving BaCl2·2H2O, CaCl2·2H2O and ZrCl4 in deionized water to obtain solution B; A3. Mix solution A and solution B evenly, add NaOH and PVP, mix well and transfer to a stainless steel autoclave lined with tetrafluoroethylene for reaction. After the reaction, wash the precipitate repeatedly with deionized water and anhydrous ethanol, and dry to obtain BCZT piezoelectric nanomaterials.

3. The preparation method according to claim 1, characterized in that: In step S2, the method for preparing the hybrid membrane vesicles comprises the following steps: B1. Co-incubate macrophages with pathogenic bacteria to activate the targeted proteins on the surface, and extract the pre-activated macrophage membranes; B2. Take blood to separate platelets and extract platelet membranes; B3, extracting exosome membranes from M1 macrophages; B4. The above-mentioned macrophage membrane, platelet membrane and M1 macrophage exosome membrane are fused by ultrasound to form hybrid membrane vesicles.

4. The hybrid membrane-wrapped BCZT nanomaterial prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the hybrid membrane-encapsulated BCZT nanomaterial prepared by the preparation method according to any one of claims 1 to 3 or the hybrid membrane-encapsulated BCZT nanomaterial according to claim 4 in treating drug-resistant bacterial infections.

6. A method for treating drug-resistant bacteria using a hybrid membrane-wrapped BCZT nanomaterial system, characterized in that: The steps include: T1, targeting TLR2 and TLR6 on the membrane surface of preactivated macrophages to Gram-positive pathogens or targeting TLR4 to Gram-negative pathogens; T2, using M1 macrophage exosome membranes to reprogram macrophages, converting M2 immunosuppressive macrophages into M1 macrophages that resist pathogenic microorganisms; T3. Ultrasonic therapy is used to activate the hybrid membrane wrapped with BCZT nanomaterials to produce ROS, which effectively kills drug-resistant bacteria.

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