A hybrid membrane-wrapped BCZT nanomaterial, its preparation method and application

Abstract

The present invention discloses a hybrid film-coated BCZT nanomaterial, its preparation method and application, belonging to the field of biomedical technology. The preparation method of the hybrid film-coated BCZT nanomaterial comprises the following steps: preparing BCZT piezoelectric nanomaterials; preparing hybrid film vesicles; using a liposome extruder and an ultrasonic disperser to uniformly coat the hybrid film vesicles on the surface of the BCZT piezoelectric nanomaterials to obtain the hybrid film-coated BCZT nanomaterial. The BCZT nanomaterial disclosed in the present invention can efficiently generate reactive oxygen species (ROS) under ultrasonic catalysis, have a killing effect on bacteria, have strong tissue penetration, can penetrate deep into the infected site, and achieve an effective antibacterial effect. Finally, by using the hybrid film coating technology to coat the BCZT material, the stability and biocompatibility of the nanomaterial can be improved, making it more stable in the in-vivo environment and reducing possible side effects.

Description

Technical Field

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

[0002] With the widespread use of antibiotics, the emergence and spread of multi-drug resistant bacteria (MDR) have become a major challenge facing global public health. These drug-resistant bacteria are resistant to a variety of commonly used antibiotics, resulting in a significant reduction in the effectiveness of traditional antibacterial treatment methods. In particular, methicillin-resistant Staphylococcus aureus (MRSA), most of its infections occur in hospitals or other medical institutions, and it is a clinically common bacterium with relatively strong toxicity. Currently, the treatment means for drug-resistant bacteria are relatively limited, so it is urgent to develop new and efficient antibacterial strategies.

[0003] In recent years, the use of nanomaterials for sonodynamic therapy (SDT) as a potential antibacterial technology has attracted wide attention. SDT involves exposing target tissues to non-toxic sensitizing chemicals and low-intensity ultrasound, and generating reactive oxygen species (ROS) in response to ultrasound to achieve site-specific cytotoxicity, which can be used to treat bacterial infections. This method can potentially be used for superficial and deep microbial infections. However, piezoelectric materials have problems such as poor targeting and short in vivo residence time in clinical antibacterial applications, which limit their clinical antibacterial effects. Therefore, constructing a treatment system that can both target pathogenic bacteria, extend the in vivo residence time, and effectively treat drug-resistant bacteria is an urgent problem to be solved for nanomaterial sterilization currently.

[0004] To solve these problems, researchers are exploring various strategies. For example, targeted delivery agents such as nanoparticle composites, liposomes, and microbubbles are used to improve the bioavailability of sonosensitizers and reduce the in vivo clearance rate. 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 bilayer polyethylene glycol polymer chain with a dynamic outer layer structure on the nanoparticle surface, their circulation time in the blood can be greatly extended, thereby increasing their effective accumulation at the infection site. In summary, nanomaterials have broad application prospects in the field of antibacterial treatment, but still face many challenges, such as improving targeting, extending the in vivo circulation time, and how to effectively integrate into clinical treatment. Summary of the Invention

[0005] The present invention aims to provide a hybrid membrane-coated BCZT nanomaterial, its preparation method and application. By synthesizing BCZT nanomaterials with good piezoelectric properties and combining hybrid membrane coating technology, a treatment system capable of precisely targeting pathogenic bacteria in vivo and reprogramming immune cells is constructed. This system not only improves the stability and biocompatibility of the 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] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A preparation method of a hybrid membrane-coated BCZT nanomaterial, comprising the following steps:

[0008] S1. Prepare BCZT piezoelectric nanomaterials;

[0009] S2. Prepare hybrid membrane vesicles;

[0010] S3. Using a liposome extruder and an ultrasonic disperser, uniformly coat the hybrid membrane vesicles obtained in S2 on the surface of the BCZT piezoelectric nanomaterials obtained in step S1 to obtain a hybrid membrane-coated BCZT nanomaterial.

[0011] Preferably, in step S1, the preparation method of the BCZT piezoelectric nanomaterials comprises the following steps:

[0012] A1. Dissolve TiCl4 in absolute ethanol to obtain solution A;

[0013] A2. Dissolve BaCl2·2H2O, CaCl2·2H2O and ZrCl4 in deionized water to obtain solution B;

[0014] A3. Mix solution A and solution B evenly, add NaOH and PVP, mix well and transfer to a stainless steel autoclave lined with polytetrafluoroethylene for reaction. After the reaction, repeatedly wash the obtained precipitate with deionized water and absolute ethanol, and dry to obtain BCZT piezoelectric nanomaterials.

[0015] Preferably, in step S2, the preparation method of the hybrid membrane vesicles comprises the following steps:

[0016] B1. Co-incubate macrophages with pathogenic bacteria to activate the surface targeting proteins, and extract the pre-activated macrophage membranes;

[0017] B2. Take blood to separate platelets and extract platelet membranes;

[0018] B3. Extract M1 macrophage exosome membranes;

[0019] B4. Ultrasonically fuse the above-mentioned macrophage membranes, platelet membranes, and M1 macrophage exosome membranes to form hybrid membrane vesicles.

[0020] The present invention also discloses a BCZT nanomaterial wrapped with the hybrid membrane prepared by the above-mentioned preparation method.

[0021] The present invention also discloses the application of the BCZT nanomaterial wrapped with the hybrid membrane prepared by the above-mentioned preparation method or the BCZT nanomaterial wrapped with the hybrid membrane in the treatment of drug-resistant bacterial infections.

[0022] The present invention also discloses a method for treating drug-resistant bacteria using a BCZT nanomaterial system wrapped with a hybrid membrane, which includes the following steps:

[0023] T1. Target TLR2 and TLR6 on the surface of pre-activated macrophage membranes to Gram-positive pathogenic bacteria or target TLR4 to Gram-negative pathogenic bacteria;

[0024] T2. Reprogram macrophages using M1 macrophage exosome membranes to convert M2-type immunosuppressive macrophages into anti-pathogenic M1-type macrophages;

[0025] T3. Use an ultrasonic physiotherapy instrument to ultrasonically activate the BCZT nanomaterial wrapped with the hybrid membrane to generate ROS, effectively killing drug-resistant bacteria.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] The present invention discloses a nanomaterial BCZT with excellent piezoelectric properties, and in combination with the hybrid membrane wrapping technology, provides a brand-new strategy for the treatment of multi-drug resistant bacteria. Firstly, by utilizing pre-activated macrophage membranes, the BCZT nanomaterial can accurately target the inflammatory sites where pathogenic bacteria are located, significantly improving the targeting of treatment. Secondly, through the CD47 "don't eat me" protein on the platelet membrane surface, the BCZT nanomaterial can effectively escape the phagocytosis and clearance of immune cells in the body, thus having a longer existence time in the body and improving the treatment effect. In addition, the platelet membrane can bind to the α-hemolysin on the surface of pathogenic bacteria, protecting other immune cells at the inflammatory site and enhancing the immune effect. At the same time, the M1 macrophage exosome membrane can transform immunosuppressive M2 macrophages into anti-pathogenic M1 macrophages, further enhancing the body's immune response to drug-resistant bacteria. The sonodynamic therapy (SDT) using the BCZT nanomaterial has better tissue penetration, spatiotemporal controllability and non-invasiveness. These characteristics make SDT have significant advantages in the treatment of bacterial disease infections. The BCZT nanomaterial can efficiently generate reactive oxygen species (ROS) under ultrasonic catalysis, exerting a killing effect on bacteria. It has strong tissue penetration and can penetrate deep into the infected site to achieve effective antibacterial effects. Finally, by wrapping the BCZT material through the hybrid membrane wrapping technology, the stability and biocompatibility of the nanomaterial can be improved, making it more stable in the in-vivo environment and reducing possible side effects. In summary, the present invention provides an efficient, targeted and immune-enhancing new method for the treatment of multi-drug resistant bacteria, and is expected to play an important role in clinical treatment.

[0028] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Brief Description of the Drawings

[0029] Figure 1 It is the preparation process of the hybrid membrane-wrapped BCZT nanomaterial system of the present invention;

[0030] Figure 2 It is the transmission electron micrograph of the BCZT piezoelectric nanomaterial provided in Example 1 of the present invention;

[0031] Figure 3 It is the lattice structure of the BCZT piezoelectric nanomaterial provided in Example 1 of the present invention;

[0032] Figure 4 It is the statistical chart of the change in the content of macrophage membrane-targeted Gram-positive bacterial membrane protein genes provided in Example 2 of the present invention, where Figure 4 A in it is the statistical chart of the change in the expression level of macrophage TLR2 gene over time, Figure 4 and B in it is the statistical chart of the change in the expression level of macrophage TLR4 gene over time;

[0033] Figure 5 TEM image of the hybrid film - wrapped BCZT nanomaterial provided in Embodiment 3 of the present invention;

[0034] Figure 6 Electron microscope comparison images of the BCZT nanomaterial before and after being wrapped by the hybrid film of the present invention. Among them, Figure 6 A in [reference number] is the TEM image of BCZT, Figure 6 B in [reference number] is the TEM image of BCZT@HM;

[0035] Figure 7 Flow cytometry diagrams of the BCZT nanomaterial before and after being wrapped by the hybrid film of the present invention;

[0036] Figure 8 Statistical chart of Western blot detection of the BCZT nanomaterial before and after being wrapped by the hybrid film of the present invention;

[0037] Figure 9 Relative fluorescence intensity of ROS generated by BCZT under ultrasonic treatment of the BCZT nanomaterial before and after being wrapped by the hybrid film of the present invention;

[0038] Figure 10 Electron microscope image of the system of the BCZT nanomaterial wrapped by the hybrid film of the present invention binding to pathogenic bacteria;

[0039] Figure 11 Colony number diagram of pathogenic bacteria co - treated by the system of the BCZT nanomaterial wrapped by the hybrid film of the present invention and immune cells. Detailed implementation manners

[0040] The technical solutions of the present invention will be further described below with reference to the drawings and embodiments.

[0041] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention pertains.

[0042] In the present invention, unless otherwise specified, other test materials and instrument equipment are all conventional test materials in the art and can be obtained through commercial channels.

[0043] Embodiment 1 This embodiment provides a BCZT piezoelectric nanomaterial, and the preparation method includes the following steps:

[0044] A1. Dissolve 8 mmol of TiCl4 in 10 ml of absolute ethanol to obtain solution A;

[0045] A2. Dissolve 12 mmol of BaCl2·2H2O, 3 mmol of CaCl2·2H2O, and 2 mmol of ZrCl4 in 30 ml of deionized water to obtain solution B;

[0046] A3. Mix solution A and solution B evenly, add 80 mmol of NaOH and 1.0 g of PVP, mix well and transfer to a 50 ml stainless steel autoclave lined with polytetrafluoroethylene for reaction at 180 °C for 24 h. After the reaction, wash the obtained precipitate repeatedly with deionized water and absolute ethanol until the pH is 7, and dry at 60 °C for 24 h to obtain BCZT piezoelectric nanomaterials.

[0047] Example 2. This example provides a hybrid membrane vesicle, and the preparation method includes the following steps:

[0048] 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 100 μg / ml of gentamicin sulfate after culturing for 1 h, incubate for 24 h, then wash the macrophages 3 times with PBS, disrupt the macrophage cell membrane with a hypotonic sucrose solution, grind with a homogenizer at least 20 times, centrifuge at 600 x g for 30 min, take the supernatant, and centrifuge the supernatant at 15000 x g for 60 min to obtain a precipitate, which is the macrophage cell membrane that activates the target protein;

[0049] B2. After adding an anticoagulant to whole blood, centrifuge at 100 x g for 20 min to take the supernatant, centrifuge the supernatant at 800 x g for 20 min, resuspend the obtained precipitate in PBS, then freeze-thaw repeatedly to obtain lysed platelets, and centrifuge at 12000 x g for 15 min at 4 °C to obtain platelet membranes;

[0050] B3. After treating macrophages with LPS, obtain M1 macrophages, culture M1 macrophages in exosome-free serum medium for 24 h, centrifuge the supernatant medium at 200 x g for 20 min, take the supernatant and centrifuge at 10000 x g for 30 min, filter the supernatant through a 0.22 μm filter membrane, and centrifuge at 100000 x g for 70 min. Freeze-thaw the obtained precipitate repeatedly to obtain M1 macrophage exosome membranes;

[0051] B4. Ultrasonically fuse the above macrophage cell membranes, platelet membranes and M1 macrophage exosome membranes at 42 kHz and 100 W for 5 min to form hybrid membrane vesicles.

[0052] Example 3. This example provides a BCZT nanomaterial wrapped with a hybrid membrane, and the preparation method includes the following steps:

[0053] Mix the BCZT piezoelectric nanomaterials prepared in Example 1 and the hybrid membrane vesicles prepared in Example 2 in a ratio of 1:1, and ultrasonically treat and mix them at 42 kHz and 100 W for 5 min. Then, use a liposome extruder to extrude the mixed solution through polycarbonate membranes with pore sizes of 400 nm and 200 nm for 20 times. The resulting solution after extrusion contains BCZT nanomaterials encapsulated by hybrid membranes, BCZT@HM.

[0054] The physicochemical properties of Examples 1 - 3 were determined through the following tests.

[0055] 1. Conduct a transmission electron microscopy (TEM) test on the BCZT piezoelectric nanomaterials provided in Example 1. The results are as shown in Figure 2 and Figure 3 .

[0056] From Figure 2 and Figure 3 , it can be seen that through TEM imaging, the morphology of the BCZT nanomaterials was characterized, and the lattice of the nanomaterials was observed using high resolution to infer their crystal forms.

[0057] 2. Co - incubate the macrophages in Example 2 with methicillin - resistant Staphylococcus aureus, and detect the RNA content in the macrophages. The results are as shown in Figure 4 .

[0058] From Figure 4 , it was found that as time increased, the expression levels of the TLR2 and TLR6 genes targeting Gram - positive bacteria increased.

[0059] 3. Conduct a scanning electron microscopy test on the BCZT nanomaterials encapsulated by hybrid membranes provided in Example 3. The results are as shown in Figures 5-6 .

[0060] From Figure 5 , it can be seen that a membrane structure covers the surface of the nanomaterials.

[0061] From Figure 6 , it can be seen that after physical extrusion, a membrane successfully covers the nanomaterials.

[0062] 4. Conduct a fluorescence detection on the materials provided in Example 1 and Example 3. The test method is as follows: Add 10 μM of DIO dye to the hybrid membrane vesicles obtained in Example 2, incubate at room temperature for 10 min, then centrifuge at 12000 xg for 10 min at 4 °C, discard the supernatant, add 0.5 ml of PBS, repeat the centrifugation and washing 3 times. Subsequently, obtain fluorescent BCZT@HM through the method of Example 3. Detect the fluorescence of the obtained BCZT@HM and BCZT using a flow cytometer. The results are as shown in Figure 7 .

[0063] From Figure 7It can be seen that the hybrid membrane in Example 3 is successfully combined with the BCZT nanomaterial.

[0064] 5. Western blot detection was performed on the materials provided in Example 1 and Example 3. The test method was as follows: BCZT and BCZT@HM obtained in Example 1 and Example 3 were centrifuged at 3000 x g, the supernatant was discarded, 200 μl of cell lysate (containing protease inhibitor) was added, and lysed on ice bath for 30 min. Then centrifuged at 10000 x g for 10 min at 4 °C, the supernatant was collected, 50 μl of 5x loading buffer was added, boiled in a metal bath at 95 °C for 10 min, and then protein immunoblotting was carried out. The results are as Figure 8 .

[0065] From Figure 8 it can be seen that the proteins on the membrane surface still exist after being wrapped by the hybrid membrane.

[0066] 6. Ultrasonic detection was performed on the materials provided in Example 1 and Example 3. The test method was as follows: The materials obtained in Example 1 and Example 3 were added with 0.15 mM of 9,10-anthrylene-bis(methylene), and ultrasonic treatment was carried out with an ultrasonic physiotherapy instrument at 1.5 W. The fluorescence change diagram was obtained using a UV spectrophotometer. The results are as Figure 9 .

[0067] From Figure 9 it can be seen that the amount of ROS generated by ultrasound before and after wrapping with the BCZT hybrid membrane is not affected by the membrane.

[0068] 7. The hybrid membrane-wrapped BCZT nanomaterial provided in Example 3 was tested for drug-resistant bacteria, including the following steps:

[0069] T1. Target TLR2 and TLR6 on the surface of pre-activated macrophage membranes in Example 2 to Gram-positive pathogenic bacteria;

[0070] T2. Achieve immune escape through the CD47 protein on the surface of platelets, and bind to α-hemolysin on the surface of pathogenic bacteria to protect immune cells;

[0071] T3. Reprogram macrophages using the membrane of M1 macrophage exosomes to transform M2-type immunosuppressive macrophages into anti-pathogenic microorganism M1-type macrophages;

[0072] T4. Start the hybrid membrane-wrapped BCZT nanomaterial provided in Example 3 to generate ROS through ultrasonic physiotherapy instrument ultrasound to effectively kill drug-resistant bacteria. The results are as Figures 10-11 .

[0073] From Figure 10 it can be seen that Figure 10 is the electron microscopy binding diagram 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.

[0074] Figure 11 The piezoelectric material and the BCZT nanomaterial wrapped with a hybrid film are co-incubated with pathogenic bacteria in the presence of macrophages, and the number of pathogenic bacteria is detected by ultrasonic treatment, demonstrating that the hybrid film 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.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a hybrid membrane-wrapped BCZT nanomaterial, characterized in that It includes the following steps: S1. Prepare BCZT piezoelectric nanomaterials; S2. Prepare hybrid membrane vesicles; S3. Using a liposome extruder and an ultrasonic disperser, uniformly wrap the hybrid membrane vesicles obtained in S2 on the surface of the BCZT piezoelectric nanomaterials obtained in step S1 to obtain BCZT nanomaterials wrapped with a hybrid membrane. In step S1, the preparation method of the BCZT piezoelectric nanomaterials includes the following steps: A1. Dissolve TiCl4 in absolute ethanol to obtain solution A; A2. Dissolve 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, transfer the mixture to a stainless steel autoclave lined with polytetrafluoroethylene for reaction after mixing evenly. After the reaction is completed, repeatedly wash the obtained precipitate with deionized water and absolute ethanol, and dry it to obtain BCZT piezoelectric nanomaterials; In step S2, the preparation method of the hybrid membrane vesicles includes the following steps: B1. Co-incubate macrophages with pathogenic bacteria to activate the surface targeting proteins, and extract the pre-activated macrophage membranes; B2. Separate platelets from blood and extract platelet membranes; B3. Extract M1 macrophage exosome membranes; B4. Ultrasonically fuse the above macrophage membranes, platelet membranes and M1 macrophage exosome membranes to form hybrid membrane vesicles.

2. The BCZT nanomaterials wrapped with a hybrid membrane prepared by the preparation method according to claim 1.

3. The application of the BCZT nanomaterials wrapped with a hybrid membrane prepared by the preparation method according to claim 1 or the BCZT nanomaterials wrapped with a hybrid membrane according to claim 2 in the preparation of drugs for treating drug-resistant bacterial infections.

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