A method for preparing magnetocaloric nanoparticles based on piezoelectric catalysis of molybdenum disulfide nanoflowers

Abstract

The present invention belongs to the technical field of antibacterial and anti-inflammatory nanoparticle preparation, specifically relating to a method for preparing magnetocaloric nanoparticles based on piezoelectrically catalytic molybdenum disulfide nanoflowers. The preparation method comprises the following steps: S1: preparing MoS2; in S1, the MoS2 preparation steps are as follows: 1.235g of ammonium molybdate and 1.052g of thioacetamide are dissolved in 20mL of deionized water and stirred uniformly to obtain a colorless, transparent solution; the solution is transferred to a 50mL autoclave and reacted at 220°C for 8h; the solution is repeatedly washed with distilled water and anhydrous ethanol, and the product is vacuum-dried at 60°C for 48h to obtain MoS2. The present invention is a novel antibacterial nanomaterial that integrates MHT, PCT, and enzyme activity, enhancing the synergistic effects of each individual therapeutic approach, achieving multiple antibacterial effects, and improving the treatment efficiency of bacterial pneumonia.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of antibacterial and anti-inflammatory nanoparticles, and specifically relates to a method for preparing magnetocaloric nanoparticles based on piezoelectric catalysis of molybdenum disulfide nanoflowers. Background Art

[0002] Bacteria are ubiquitous in our daily lives. Most, such as those in the intestines, are not only harmless but some can even be beneficial. However, some bacteria, once they enter a host and multiply, can cause infection. Bacterial pneumonia accounts for 80% of all pneumonia cases in adults, making it a common clinical illness and the most common form of pneumonia. Caused by Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, and Haemophilus influenzae, bacterial pneumonia can lead to severe lung damage, impaired respiratory function, and even death. Since the advent of antibiotics, the prognosis of bacterial pneumonia has significantly improved. However, while these antibiotics kill or inhibit bacterial growth, they also place selective pressure on bacterial survival. Due to this selective pressure and the overuse of antibiotics, bacteria have gradually developed resistance, creating difficulties and challenges in the treatment of patients with bacterial pneumonia. To address this situation, researchers have developed numerous novel alternatives to traditional antibiotic treatments, including bacteriophage therapy, combined antibody-antibiotic therapies, and nanomaterials, all of which have been experimentally proven effective. Excitingly, nanomaterial-based therapies have made remarkable progress in the field of antimicrobial therapy, demonstrating enormous potential. Nanomaterials, due to their diverse composition, offer a range of unique advantages. Nanomaterials with specific physicochemical properties can respond to stimuli such as light, ultrasound, and alternating magnetic fields, exhibiting enhanced antimicrobial properties in specific environments. These include novel treatment modalities such as photodynamic therapy (PDT), sonodynamic therapy (SDT), piezoelectric catalytic therapy (PCT), and magnetic hyperthermia therapy (MHT). Compared to light, ultrasound can be focused into a high three-dimensional scattering point within deep tissues. However, light diffuses significantly as it passes through tissue, resulting in a very limited penetration depth. Consequently, SDT is far more effective than PDT for treating deep-seated lesions.

[0003] Piezocatalytic therapy (PCT) has emerged as a novel therapeutic modality for ultrasound-mediated generation of reactive oxygen species (ROS), such as hydroxyl radicals (·OH), superoxide anions (·O2 - ) and singlet oxygen ( 1O2) is similar to the principle of SDT. The sonosensitizer of piezoelectric materials can be excited by low-frequency ultrasound to generate activated electrons and holes. These electrons and holes can participate in the redox reaction of O2 or H2O to produce ROS. With their unique piezoelectric effect, piezoelectric materials such as barium titanate (BaTiO3), MoS2 nanosheets and black phosphorus can immediately generate a built-in electric field when subjected to external mechanical stress. Under ultrasonic excitation, the piezoelectric material is polarized, and the electrons gather on one side, but quickly neutralize the surrounding charges, which greatly limits their catalytic performance. To this end, we constructed a piezoelectric nanoheterojunction. When the heterojunction is formed, the polarized electrons excited by ultrasound are effectively migrated at the interface; therefore, the electron yield can be increased to generate more ROS. However, it is difficult for a single PCT to achieve the ideal antibacterial effect. It can be combined with other highly penetrating treatments to effectively and synergistically fight against lung infection lesions.

[0004] MHT is an emerging physical hyperthermia therapy technique. It utilizes magnetic nanomaterials in an external alternating magnetic field to generate heat through hysteresis or relaxation losses, rapidly raising the temperature of the lesion to 45°C–50°C, thereby killing bacteria. Compared with other hyperthermia treatment modalities, MHT offers significant advantages in overcoming common limitations, such as the poor conformality of microwave and radiofrequency ablation, the inability of ultrasound to penetrate gas-filled cavities, and the limited penetration of light into the human body. Therefore, magnetic hyperthermia is considered one of the most promising physical therapies for treating deep-seated lesions, offering a safer and more effective new antimicrobial treatment option. However, MHT has a significant limitation, namely the requirement for high doses of MNPs. To address this, researchers have developed numerous combination therapies to overcome the inability of single-modality treatments to completely cure tissue infections and achieve optimal therapeutic outcomes. Combining PCT with MHT enhances their synergistic effects, improving antimicrobial efficacy while minimizing adverse reactions.

[0005] Currently, emerging exogenous stimulation therapies (such as magnetic fields, ultrasound, heat, and light) have been used to treat bacterial infection sites. Under the influence of exogenous stimuli, stimuli-responsive biomaterials can achieve on-demand antimicrobial effects by changing their own physicochemical properties, and therefore show great application prospects in the future. However, the bioavailability of these methods is relatively low. In addition, during bacterial invasion, the microenvironment of bacterial infection is often accompanied by changes in multiple factors, such as neutrophil pleurosis, high proteolytic enzyme content, high local temperature, weak acidity, and low oxygen concentration. This is also accompanied by overexpression of hydrogen peroxide (H2O2) and ROS in the inflammatory area. Compared with exogenous stimuli, endogenous stimuli in the disease microenvironment (e.g., pH, enzymes, redox, or glucose) are region-specific and can respond to changes in the wound microenvironment in a timely manner and minimize infection. Nanomaterials with enzyme-like properties, namely nanozymes, have microenvironment-responsive catalytic activity. In particular, nanozymes including peroxidase (POD), oxidase (OXD), and catalase (CAT) can simultaneously produce ROS or eliminate ROS to maintain the balance of ROS in cells, effectively eradicating bacteria and even biofilms in the infected area while producing O2 to alleviate local inflammation in the bacterial infection microenvironment. However, both exogenous and endogenous stimulus-responsive platforms can affect the activity of bacteria, but strategies based on a single stimulus often show insufficient antibacterial performance, especially for the elimination of biofilms. Therefore, the development of an antibacterial platform that responds to both endogenous and exogenous stimuli is of great significance for the efficient sterilization of infected lesions. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing magnetocaloric nanoparticles based on piezoelectric catalytic molybdenum disulfide nanoflowers, which can integrate MHT, PCT and enzyme activity into a new type of antibacterial nanomaterial, enhance the synergistic effect of each single treatment method, achieve multiple antibacterial effects, and improve the treatment efficiency of bacterial pneumonia.

[0007] The technical solutions adopted by the present invention are as follows:

[0008] A method for preparing magnetocaloric nanoparticles based on piezoelectric catalytic molybdenum disulfide nanoflowers, the preparation method comprising the following steps:

[0009] S1: Preparation of MoS2;

[0010] In the S1, the preparation steps of MoS2 are as follows:

[0011] 1.235 g of ammonium molybdate and 1.052 g of thioacetamide were weighed and dissolved in 20 mL of deionized water. After stirring evenly, a colorless transparent solution was obtained. The solution was transferred to a 50 mL high-pressure reactor and reacted at 220 ° C for 8 h. The solution was repeatedly washed with distilled water and anhydrous ethanol several times. The product was vacuum dried at 60 ° C for 48 h to obtain MoS2.

[0012] S2: preparing MnFe2O4;

[0013] In the S2, the preparation steps of MnFe2O4 are as follows:

[0014] S21: MnCl2·4H2O, FeCl3·6H2O and NaOH are the main co-precipitates, with M Mn :M Fe =1:4 ratio, dissolve MnCl2·4H2O and FeCl3·6H2O in a certain amount of distilled water to obtain a salt solution;

[0015] S22: Under magnetic stirring, NaOH solution was added to the salt solution to maintain the pH at 9 and stirred vigorously at room temperature for 30 min to obtain a brown-black colloidal mixture;

[0016] S23: The brown-black colloidal mixture is then placed into a polytetrafluoroethylene liner, which is then placed into a high-pressure reactor for a constant temperature reaction at 200°C for 16 hours.

[0017] S24: Wash with distilled water and anhydrous ethanol several times, dry the product in vacuum at 60°C for 48 hours, and grind to obtain MnFe2O4.

[0018] S3: Preparation of MoS2@MnFe2O4 nanocomposite materials;

[0019] In the S3, the preparation steps of the MoS2@MnFe2O4 nanocomposite material are as follows:

[0020] S31: Weigh 0.462 g of MnFe2O4 and add it to 10 ml of deionized water. Ultrasonicate for 30 minutes to obtain solution A.

[0021] S32: Dissolve 1.235 g of ammonium molybdate and 1.052 g of thioacetamide in 10 mL of deionized water and stir to obtain solution B;

[0022] S33: Solution A and solution B were mixed and placed in a shaker for 30 min, and then transferred to a high-pressure reactor for reaction at 220° C. for 8 h;

[0023] S34: Wash with distilled water and anhydrous ethanol several times, and dry the product in vacuum at 60°C for 48h.

[0024] S4: Preparation of MoS2@MnFe2O4@PVP;

[0025] In the S4, the preparation steps of MoS2@MnFe2O4@PVP are as follows:

[0026] S41: 10 mg of MoS2@MnFe2O4 nanoparticles were dissolved in 10 mL of deionized water and ultrasonicated for 30 min to obtain a black suspension A;

[0027] S42: Dissolve 40 mg of polyvinylpyrrolidone in 10 mL of deionized water to obtain a clear and transparent solution B;

[0028] S43: Under ultrasound, solution A was slowly added dropwise to solution B, and ultrasound was continued for 10 min. The solution was then placed in a 37°C constant temperature shaker and shaken at 180 rpm for 12 h to obtain solution C.

[0029] S44: Solution C was repeatedly washed with anhydrous ethanol and deionized water three times, dried under vacuum at 60°C overnight, and ground to obtain MoS2@MnFe2O4@PVP nanoparticles.

[0030] S5: Determine the antibacterial properties of MoS2@MnFe2O4@PVP nanocomposites;

[0031] In S5, the antibacterial properties of the nanocomposite were studied using MRSA and MDR-Kp as model bacteria by using a plate coating method to study the in vitro antibacterial effect of the nanocomposite. There was no significant reduction in the colonies after co-incubation with normal saline and application of stimulation conditions.

[0032] Under the synergistic effect of magnetic heat and ultrasound, the nanocomposite material has an antibacterial efficiency of 99.5% and 99.8% against MRSA and MDR-Kp respectively, and the nanocomposite material has in vitro antibacterial ability.

[0033] S6: Evaluation of the anti-biofilm ability of MoS2@MnFe2O4@PVP nanocomposites in vitro;

[0034] In S6, MRSA and MDR-Kp were used to evaluate the in vitro anti-biofilm activity, and the evaluation steps were as follows:

[0035] S61: Place a 9 mm cell slide into a 24-well plate and add 1 mL of 1×10 7 CFU / mL of MRSA or MDR-Kp suspension was inoculated into the wells containing cell slides;

[0036] S62: Incubate continuously in a biochemical incubator at 37°C for 96 hours, changing the culture medium every 24 hours, to allow biofilm to form on the surface; after the biofilm is formed, wash repeatedly with sterile saline to remove unbound bacteria;

[0037] S63: After treatment under different conditions, bacteria were labeled with SYTO9 and PI dyes in the dark, incubated for 30 minutes, and rinsed three times with sterile saline. Fluorescence imaging of live and dead bacteria was performed under a laser confocal microscope.

[0038] S64: In the case of MDR-Kp or MRSA, the mortality of the MMP-treated groups was higher than that of the control group, demonstrating that the nanocomposite played a significant role in destroying the biofilm.

[0039] S7: Verify the therapeutic effect of MoS2@MnFe2O4@PVP nanocomposite on bacterial pneumonia in vivo.

[0040] In S7, the steps of verifying the treatment effect are as follows:

[0041] S71: A BALB / C mouse lung infection model induced by MDR-Kp was established, and seven different groups were treated, including PBS, PBS+AMF+US, MMP, MMP+US, MMP+AMF, MMP+AMF+US, and Health;

[0042] S72: Lung tissue homogenate was collected and smeared on day 3 after treatment, and the residual bacteria in the lung tissue were evaluated using the plate smear method;

[0043] S73: Bacteria were evident in the lung tissues of the other treatment groups, but MMP+AMF+US-treated mice showed a significant decrease in bacteria in their lungs.

[0044] S74: In the PBS group, PBS+AMF+US group, and MMP group, CD86 was highly expressed in the lungs, which enhanced the inflammatory response and induced lung damage; in the MMP+AMF+US group, CD86 protein was significantly downregulated, while CD206 expression was at its peak. MMP+AMF+US treatment has antibacterial ability in mice.

[0045] The technical effects achieved by the present invention are:

[0046] The present invention discloses a method for preparing magnetocaloric nanoparticles based on piezoelectric catalytic molybdenum disulfide nanoflowers. The novel antibacterial nanomaterial integrates MHT, PCT and enzyme activity, thereby enhancing the synergistic effect of each single treatment method, achieving multiple antibacterial effects, and improving the treatment efficiency of bacterial pneumonia. The MMP is based on MnFe2O4 as the core, and when an alternating magnetic field is applied, the temperature is raised to kill bacteria and promote the efficiency of PCT in producing ROS.

[0047] The present invention provides a magnetocaloric nanoparticle preparation method based on piezoelectric catalytic molybdenum disulfide nanoflowers. MMP can exert different enzymatic activity effects under the bacterial infection microenvironment at different stages, and improve the reaction efficiency of enzyme activity through the combined action of magnetocaloric and piezoelectric catalysis, accelerate the production of ROS and O2, kill bacteria and promote the polarization of macrophages, activate the body's immune response and regulate lung inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 1 is a schematic diagram of an embodiment of the present invention;

[0049] Figure 2a This is an SEM image of the MoS2 nanomaterial according to an embodiment of the present invention;

[0050] Figure 2b is the XRD pattern of MoS2 in the embodiment of the present invention;

[0051] Figure 3a This is an SEM image of the MnFe2O4 nanomaterial according to an embodiment of the present invention;

[0052] Figure 3b is the XRD pattern of MnFe2O4 in the embodiment of the present invention;

[0053] Figure 4a 2 is the SEM image of MoS2@MnFe2O4 synthesized in different molar ratios according to the embodiment of the present invention;

[0054] Figure 4b is the DLS particle size distribution of MoS2@MnFe2O4 synthesized at different molar ratios in the examples of the present invention;

[0055] Figure 4c 1 is the XRD pattern of MoS2@MnFe2O4 synthesized in different molar ratios in the examples of the present invention;

[0056] Figure 5a This is the magnetocaloric temperature rise curve of MoS2@MnFe2O4 synthesized in different molar ratios in the embodiment of the present invention in AMF;

[0057] Figure 5b This is a comparison chart of the degradation rates of DPBF of MoS2@MnFe2O4 synthesized at different molar ratios under ultrasonic irradiation in the embodiments of the present invention;

[0058] Figure 5c This is a comparison chart of the degradation rates of DPBF under ultrasonic irradiation of MoS2@MnFe2O4 synthesized at different molar ratios under a heating environment in an embodiment of the present invention;

[0059] Figure 6a is a SEM image of the MMP of an embodiment of the present invention;

[0060] Figure 6b is a TEM image of the MMP of an embodiment of the present invention;

[0061] Figure 6c is an EDS element mapping image of the MMP according to an embodiment of the present invention;

[0062] Figure 6d is the XRD pattern of the MMP of the embodiment of the present invention;

[0063] Figure 6e is the FTIR spectrum of the MMP of the embodiment of the present invention;

[0064] Figure 7a is the UV-visible-diffuse reflectance spectrum of different samples in the examples of the present invention;

[0065] Figure 7b is the photoluminescence spectrum of different samples according to the embodiment of the present invention;

[0066] Figure 7c 1 is the electrochemical impedance spectroscopy diagram of different samples of the embodiment of the present invention;

[0067] Figure 7d is the transient current response in the ultrasonic on / off state of the embodiment of the present invention;

[0068] Figure 7e The ESR test of MMP under different conditions in the embodiment of the present invention 1 O2 production;

[0069] Figure 7f Schematic diagram of the energy band structure of the MMP according to an embodiment of the present invention;

[0070] Figure 7g It is a hysteresis loop test of the MMP of the embodiment of the present invention;

[0071] Figure 7h is the magnetocaloric temperature rise curve of MMP at different concentrations in the embodiment of the present invention;

[0072] Figure 7i is the magnetocaloric cycle curve of the MMP of the embodiment of the present invention;

[0073] Figure 8a is the POD enzyme activity of the MMP of the present invention;

[0074] Figure 8b is the change in absorbance at 652 nm of the POD enzyme activity of different samples in the examples of the present invention over time;

[0075] Figure 8c is an ESR spectrum for detecting OH in an embodiment of the present invention;

[0076] Figure 8d This is the situation in which MoS2, MnFe2O4 and MMP catalyze H2O2 to produce O2 under different pH conditions in the examples of the present invention;

[0077] Figure 8e is the absorbance of MMP at 410 nm under different stimulation conditions in the examples of the present invention;

[0078] Figure 9a These are pictures of bacterial colonies on plates after different treatments in the examples of the present invention;

[0079] Figure 9b The embodiment of the present invention is based on Figure 9a Survival statistics of MRSA and MDR-Kp (n=3);

[0080] Figure 10a 3DCLSM images of biofilms stained under different treatment conditions and using a live / dead double staining scheme according to an embodiment of the present invention;

[0081] Figure 11a This is a photograph of bacterial colonies on a plate of lung tissue homogenate after different treatments of infected mice in the examples of the present invention;

[0082] Figure 11b The quantitative statistics of colonies on lung homogenate plates of infected mice after different treatments in the examples of the present invention (n=3);

[0083] Figure 11c This is the immunohistochemical analysis of CD206 and CD86 in lung tissues of infected mice after different treatments according to the examples of the present invention. DETAILED DESCRIPTION

[0084] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following examples. It should be understood that the following text is only used to describe one or more specific embodiments of the present invention and does not strictly limit the scope of protection of the present invention.

[0085] Diseases caused by bacterial infections, such as skin wound infections, respiratory and lung infections, and soft tissue infections, are often treated with antibiotics. However, with the abuse of antibiotics, a large number of drug-resistant bacteria have emerged, and antibiotic-resistant (AMR) bacterial infections have become a global health crisis. At present, there are many treatment strategies for bacterial infections, such as PTT, PDT, and CDT. Generally speaking, single-mode antibacterial strategies are limited and it is difficult to achieve ideal antibacterial effects. In addition, PTT and PDT cannot penetrate deep tissues and are only effective and applicable in the treatment of superficial infections. The present invention has developed a new nanoformulation MoS2@MnFe2O4@PVP (MMP) for the treatment of deep-seated bacterial pneumonia. In addition to having the effects of MHT and PCT, this nanoformulation can effectively sterilize deep-seated infection lesions. It can also exert multiple enzyme activities in different bacterial infection micro-acidic environments, improve antibacterial efficiency, and enhance the body's immune regulation function for inflammatory sites. In addition, magnetothermal and piezoelectric catalysis can cascade enhance nanoenzyme activity. MMP nanoparticles, as a safe and effective integrated antibacterial and anti-inflammatory nanoformulation, provide a new way to replace traditional antibiotics in the treatment of bacterial diseases.

[0086] like Figure 1 As shown, a method for preparing magnetocaloric nanoparticles based on piezoelectric catalytic molybdenum disulfide nanoflowers comprises the following steps:

[0087] S1: Preparation of MoS2;

[0088] In the S1, the preparation steps of MoS2 are as follows:

[0089] 1.235 g of ammonium molybdate and 1.052 g of thioacetamide were weighed and dissolved in 20 mL of deionized water. After stirring evenly, a colorless transparent solution was obtained. The solution was transferred to a 50 mL high-pressure reactor and reacted at 220 ° C for 8 h. The solution was repeatedly washed with distilled water and anhydrous ethanol several times. The product was vacuum dried at 60 ° C for 48 h to obtain MoS2.

[0090] Characterization of MoS2

[0091] The basic morphology of MoS2 was observed by scanning electron microscopy (SEM). Figure 2a As shown in Figure 2, the MoS2 synthesized by the hydrothermal method is composed of multi-layer two-dimensional nanosheets with a particle size of about 400nm. The crystal structure was verified by X-ray powder diffractometer (XRD) analysis. The results are shown in Figure 2. Figure 2b As shown, the XRD pattern of the obtained MoS2 corresponds to the standard card (PDF#04-008-2233), indicating that we have successfully prepared MoS2.

[0092] S2: preparing MnFe2O4;

[0093] In the S2, the preparation steps of MnFe2O4 are as follows:

[0094] S21: In this experiment, MnCl2·4H2O, FeCl3·6H2O and NaOH were the main co-precipitates, and M Mn :M Fe =1:4 ratio, dissolve MnCl2·4H2O and FeCl3·6H2O in a certain amount of distilled water to obtain a salt solution;

[0095] S22: Under magnetic stirring, NaOH solution was added to the salt solution to maintain the pH at 9 and stirred vigorously at room temperature for 30 min to obtain a brown-black colloidal mixture;

[0096] S23: The brown-black colloidal mixture is then placed into a polytetrafluoroethylene liner, which is then placed into a high-pressure reactor for a constant temperature reaction at 200°C for 16 hours.

[0097] S24: Wash with distilled water and anhydrous ethanol several times, dry the product in vacuum at 60°C for 48 hours, and grind to obtain MnFe2O4.

[0098] Structural characterization of MnFe2O4

[0099] SEM images of MnFe2O4 nanomaterials Figure 3a As shown in Figure 2, the morphology of MnFe2O4 is irregular nanocubic shape, such as Figure 3b As shown in the figure, by analyzing the XRD pattern, it is shown that we have successfully synthesized MnFe2O4 nanomaterials.

[0100] S3: Preparation of MoS2@MnFe2O4 nanocomposite materials;

[0101] In the S3, the preparation steps of the MoS2@MnFe2O4 nanocomposite material are as follows:

[0102] S31: Weigh 0.462 g of MnFe2O4 and add it to 10 ml of deionized water. Ultrasonicate for 30 minutes to obtain solution A.

[0103] S32: Dissolve 1.235 g of ammonium molybdate and 1.052 g of thioacetamide in 10 mL of deionized water and stir to obtain solution B;

[0104] S33: Solution A and solution B were mixed and placed in a shaker for 30 min, and then transferred to a high-pressure reactor for reaction at 220° C. for 8 h;

[0105] S34: Wash with distilled water and anhydrous ethanol several times, and dry the product in vacuum at 60°C for 48h.

[0106] Structural characterization of MoS2@MnFe2O4 with different synthesis ratios

[0107] During the synthesis of MoS2@MnFe2O4, we also explored the effects of different molar ratios of MnFe2O4 and MoS2 on the structure of the composite material. Figure 4a As shown in Figure 2, the SEM analysis results show that the morphology of the composite material still maintains the nanoflower shape under different molar ratios of MoS2 and MnFe2O4. Figure 4b The DLS analysis results showed that the sizes of composite materials with different molar ratios did not differ significantly, and all remained around 500 nm, which was consistent with the SEM results. Figure 4c ,XRD patterns show that the intensities of some characteristic peaks of MnFe2O4 are significantly enhanced as the proportion of MnFe2O4 increases.

[0108] Performance characterization of MoS2@MnFe2O4 with different synthesis ratios

[0109] The magnetocaloric and piezoelectric properties of MoS2@MnFe2O4 synthesized with different molar ratios are very different. Figure 5a As shown in the figure, the magnetocaloric heating curve in the alternating magnetic field (AMF) shows that the magnetocaloric properties of the MoS2@MnFe2O4 nanocomposite are proportional to the proportion of MnFe2O4. Figure 5b As shown in Figure 2, the ability of MoS2@MnFe2O4 to generate ROS by ultrasound is inversely proportional to the proportion of MnFe2O4. Figure 5c It shows that the performance of ROS generated by ultrasonic irradiation of MoS2@MnFe2O4 at 45℃ is improved compared with that at room temperature. Through its performance analysis, we finally selected the composite condition with a molar ratio of MnFe2O4:MoS2=2:1.

[0110] S4: Preparation of MoS2@MnFe2O4@PVP (MMP for short);

[0111] In the S4, the preparation steps of MoS2@MnFe2O4@PVP are as follows:

[0112] S41: 10 mg of MoS2@MnFe2O4 nanoparticles were dissolved in 10 mL of deionized water and ultrasonicated for 30 min to obtain a black suspension A;

[0113] S42: Dissolve 40 mg of polyvinylpyrrolidone in 10 mL of deionized water to obtain a clear and transparent solution B;

[0114] S43: Under ultrasound, solution A was slowly added dropwise to solution B, and ultrasound was continued for 10 min. The solution was then placed in a 37°C constant temperature shaker and shaken at 180 rpm for 12 h to obtain solution C.

[0115] S44: Solution C was repeatedly washed with anhydrous ethanol and deionized water three times, dried under vacuum at 60°C overnight, and ground to obtain MoS2@MnFe2O4@PVP nanoparticles.

[0116] Structural characterization of MMPs

[0117] The surface morphology and microstructure of the material were observed using SEM. Figure 6a As shown in Figure 2, the MMP synthesized by hydrothermal method has a nano-flower structure with a rough surface and an average particle size of 490nm. The basic morphology of the material was observed using a transmission electron microscope (TEM). Figure 6b As shown in the figure, the TME image clearly shows that MoS2 nanosheets grow in situ on the surface of MnFe2O4, and multilayer nanosheets wrap the nanocubes to form a core-shell structure with abundant wrinkles on the surface. Figure 6c As shown in the figure, Mn, Fe, and O elements are mainly distributed in the nucleus, while Mo and S elements are mainly distributed in the shell. The results further indicate that the two-dimensional MoS2 nanosheets are evenly distributed on the surface of MnFe2O4. Figure 6d As shown in the XRD pattern analysis, compared with the standard cards of MnFe2O4 (PDF#04-004-5458) and MoS2 (PDF#04-008-2233), the results show that the diffraction peaks match, and the characteristic peaks of MnFe2O4 and MoS2 are present in MMP, proving that the nanoparticles are successfully composited. Figure 6e As shown, the peak is located at 1279 cm -1 The 1660 cm-1 -1 The strong peak at indicates the presence of C=O stretching vibration absorption from the pyrrolidone ring.

[0118] Magnetocaloric and piezoelectric properties of MMP

[0119] like Figure 7a As shown, UV-Vis diffuse reflectance spectra (UV-Vis-DRS) were obtained for the different samples to measure the changes in their optical properties. The larger absorbance of MMP compared to MnFe2O4 indicates a higher tendency for MMP to be activated by exogenous stimuli with lower energy. In addition, photoluminescence spectroscopy (PL) was used to measure the electron-hole combination efficiency of various samples. Figure 7bAs shown in Figure 2, MMP exhibits lower photoluminescence intensity compared to MoS2, which may be due to the formation of heterojunction that accelerates charge separation. In addition, EIS tests were performed to evaluate the charge transfer efficiency, as shown in Figure 2. Figure 7c As shown in the figure, compared with MnFe2O4 and MoS2, MMP exhibits the smallest electrical resistance, indicating that the composite of MnFe2O4 and MoS2 reduces the electron transfer resistance, and the interface of MMP can promote charge transfer, thanks to the successful construction of the heterojunction. Figure 7d As shown in Figure 2, under 5 cycles of ultrasound irradiation, MMP showed good current responsiveness. Secondly, we further explored the types of ROS produced by MMP nanomaterials and the ROS production capacity under different stimulation conditions by electron spin resonance spectroscopy (ESR), such as Figure 7e As shown, MMP can generate singlet oxygen ( 1 O2); and the MMP+US+45℃ group had a more distinct 1:1:1 1 The characteristic peak of O2 is due to the reduction of the band gap through the composite of MnFe2O4 and MoS2, thereby enhancing its ultrasonic catalytic effect; at the same time, the higher temperature accelerates the electron transfer rate by increasing the entropy of the chemical reaction, and promotes the effective separation of electrons and holes; and the ROS intensity generated by the composite material is stronger than that of the single material, further verifying the successful construction of the heterojunction and the improvement of the sonodynamic performance.

[0120] In order to clarify the generation mechanism of ROS, the band gap and valence band of MnFe2O4 and MoS2 were tested respectively. First, the band gap values ​​(E g ) are 1.27eV and 1.56eV respectively; the valence band values ​​(E VB ) are 1.05V and 0.79V respectively. According to formula E CB =E VB -E g The conduction band positions of MnFe2O4 and MoS2 are -0.22V and -0.77V respectively. Therefore, the energy band structure diagram of MMP is drawn ( Figure 7f ) It can be seen that MnFe2O4 and MoS2 can form a Z-type heterojunction.

[0121] In order to explore the magnetic effect of MMP, Figure 7gAs shown in the figure, we used a vibrating sample magnetometer to measure the hysteresis loop of MMP. At room temperature, the coercivity of MMP was 62.123 Oe and the saturation magnetization reached 11.42068 emu / g. This result shows that MMP has ferromagnetism and can generate heat through the hysteresis loss mechanism under the action of AMF. We also tested the magnetothermal heating effect of different concentrations of MMP in AMF, as shown in the figure. Figure 7h As shown in Figure 2, the magnetothermal effect of MMP is positively correlated with its concentration; 0.5 mg / mL of MMP can be elevated to 47°C in AMF within 10 minutes, a temperature that can inhibit bacterial growth. Figure 7i As shown in the figure, the magnetocaloric temperature rise curves showed almost no change during the 5 cycles of applying AMF, indicating that MMP has good magnetocaloric stability. MMP has good magnetocaloric ability in vitro and can be used in antibacterial applications.

[0122] Characterization of MMP dual enzyme activity

[0123] Because it contains a variety of variable valence metals, MMP is likely to have nanozyme activity. Therefore, TMB was selected as a substrate to verify whether it has POD-like properties. Subsequently, the POD-like enzyme activity of MMP under different treatment conditions was evaluated. H2O2 was added to the colorimetric reaction mixture to produce ·OH when POD-like nanozymes catalyzed H2O2, which could oxidize colorless TMB into blue oxidized TMB (oxTMB). Figure 8a As shown in Figure 2, MMP+TMB+H2O2 has a significant absorption peak at 652nm, indicating that MMP has good POD enzyme activity. MMP+TMB has no absorption peak at 652nm, thus excluding oxidase activity. Figure 8b As shown in the figure, under the same concentration of H2O2 and TMB, the absorption value of MMP+AMF+US at 652nm is the highest. Subsequently, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin probe to detect the content of ·OH by ESR. Figure 8c As shown, MMP+AMF+US showed the best efficiency, which further clarified that magnetothermal synergistic piezoelectricity plays an important role in enhancing POD-like. It is proportional to the substrate concentration. In contrast, the POD-like activity of MMP+AMF+US is better than that of other groups. Since MMP can catalyze H2O2 to produce O2, the enzyme-like activity of MMP is evaluated by measuring the content of O2 produced using a dissolved oxygen meter. Figure 8dAs shown in the figure, when in a slightly acidic medium (pH = 5.4, 6.5), MMP exhibits poor CAT-like activity. When in a neutral medium (pH = 7.4), the O2 production of MMP is very significant, which indicates that MMP has good CAT-like activity and can produce oxygen in the bacterial infection microenvironment. In addition, the titanium peroxide complex generated by the reaction of H2O2 and titanium sulfate is a yellow precipitate, which dissolves in strong acid. The depth of its yellow color is inversely proportional to the concentration of hydrogen peroxide. The absorbance at 410nm is detected by a spectrophotometer. Figure 8e It can be seen that the absorbance of MPP+AFM+US is the lowest at 412 nm, indicating that the CAT-like activity of MMP is best under the stimulation of magnetothermal and ultrasound.

[0124] S5: Determine the antibacterial properties of MoS2@MnFe2O4@PVP nanocomposites;

[0125] In S5, acquired pneumonia is usually caused by two common multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Klebsiella pneumoniae (MDR-Kp). The antibacterial properties of the nanocomposite were studied using MRSA and MDR-Kp as model bacteria. The plate coating method was used to study the in vitro antibacterial effect of the nanocomposite. The plate coating method can intuitively demonstrate the inactivation ability of MMP against MRSA and MDR-Kp. Figure 9a As shown, there was no significant reduction in the colonies after co-incubation with saline and application of stimulation conditions;

[0126] like Figure 9b As shown in the figure, the nanocomposite material, under the synergistic effects of magnetic heat and ultrasound, achieved an antibacterial efficiency of 99.5% and 99.8% against MRSA and MDR-Kp, respectively, demonstrating in vitro antibacterial capabilities. This is because the mechanical force and short-lived ROS produced by ultrasound alone cannot completely destroy the thick cell wall structure of bacteria, and the limited temperature can only inhibit their growth without achieving a significant sterilization effect. In contrast, the sterilization effect is particularly pronounced under the synergistic effects of magnetic heat and ultrasound.

[0127] S6: Evaluation of the anti-biofilm ability of MoS2@MnFe2O4@PVP nanocomposites in vitro;

[0128] In S6, MRSA and MDR-Kp were used to evaluate the in vitro anti-biofilm activity, and the evaluation steps were as follows:

[0129] S61: Place a 9 mm cell slide into a 24-well plate and add 1 mL of 1×10 7 CFU / mL of MRSA or MDR-Kp suspension was inoculated into the wells containing cell slides;

[0130] S62: Incubate continuously in a biochemical incubator at 37°C for 96 hours, changing the culture medium every 24 hours, to allow biofilm to form on the surface; after the biofilm is formed, wash repeatedly with sterile saline to remove unbound bacteria;

[0131] S63: If Figure 10a As shown, after treatment under different conditions, bacteria were labeled with SYTO9 and PI dyes in the dark, incubated for 30 minutes, and rinsed three times with sterile saline. Fluorescence imaging of live and dead bacteria was performed under a laser confocal microscope.

[0132] S64: In the case of MDR-Kp or MRSA, the MMP-treated groups showed a higher mortality rate than the control group, demonstrating that the nanocomposite played a significant role in disrupting biofilms. Furthermore, in the final group, MMP exhibited a superior biofilm disruption effect under the synergistic stimulation of magnetic heat and ultrasound, maximizing the antimicrobial potential of MMP.

[0133] S7: Verify the therapeutic effect of MoS2@MnFe2O4@PVP nanocomposite on bacterial pneumonia in vivo.

[0134] In S7, the steps of verifying the treatment effect are as follows:

[0135] S71: To further verify the potential of MMP for the treatment of bacterial pneumonia, a BALB / C mouse lung infection model induced by MDR-Kp was established, and seven different groups were treated, including PBS, PBS+AMF+US, MMP, MMP+US, MMP+AMF, MMP+AMF+US, and Health;

[0136] S72: Lung tissue homogenate was collected and smeared on day 3 after treatment, and the residual bacteria in the lung tissue were evaluated using the plate smear method;

[0137] S73: Figure 11a -b shows that there were obvious bacteria in the lung tissues of other treatment groups, and the bacteria in the lungs of mice treated with MMP+AMF+US were significantly reduced; the expression of co-stimulatory molecules CD206 / CD86 was analyzed by immunohistochemistry (IHC) staining of dissected lung tissues.

[0138] S74: The results are as follows Figure 11cAs shown, CD86 was highly expressed in the lungs of the PBS group, PBS+AMF+US group, and MMP group, which enhanced the inflammatory response and induced lung damage; in the MMP+AMF+US group, CD86 protein was significantly downregulated, while CD206 expression was at its peak. MMP+AMF+US treatment has antibacterial ability in mice, and helps polarize M1 macrophages to M2 phenotype, inhibiting the development of inflammation.

[0139] The foregoing is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained herein shall, unless otherwise specified or limited, be implemented in accordance with conventional means in the art.

Claims

1. Application of magnetocaloric nanoparticles based on piezoelectrically catalytic molybdenum disulfide nanoflowers in the preparation of a drug for treating bacterial pneumonia, characterized in that: The preparation method of the nanoparticles is: S1: The preparation steps of MnFe2O4 are as follows: S11: MnCl2·4H2O, FeCl3·6H2O and NaOH are the main co-precipitates, with M Mn :M Fe =1:4 ratio, dissolve MnCl2·4H2O and FeCl3·6H2O in a certain amount of distilled water to obtain a salt solution; S12: Under magnetic stirring, NaOH solution was added to the salt solution to maintain the pH value at 9, and the mixture was vigorously stirred at room temperature for 30 min to obtain a brown-black colloidal mixture; S13: The brown-black colloidal mixture is then placed into a polytetrafluoroethylene liner, which is then placed into a high-pressure reactor for a constant temperature reaction at 200°C for 16 hours. S14: washing with distilled water and anhydrous ethanol several times, drying the product in a vacuum at 60°C for 48 h, and grinding to obtain MnFe2O4; The preparation steps of S2:MoS2@MnFe2O4 nanocomposite are as follows: S21: Weigh 0.462 g of MnFe2O4 and add it to 10 ml of deionized water. Ultrasonicate for 30 min to obtain solution A. S22: Dissolve 1.235 g of ammonium molybdate and 1.052 g of thioacetamide in 10 mL of deionized water and stir to obtain solution B. S23: Solution A and solution B were mixed and placed in a shaker for 30 min, and then transferred to a high-pressure reactor for reaction at 220°C for 8 h; S24: Wash with distilled water and anhydrous ethanol several times, and dry the product in vacuum at 60 ° C for 48 h to obtain MoS2@MnFe2O4 nanoparticles. S3: The preparation steps of MoS2@MnFe2O4@PVP are as follows: S31: 10 mg of MoS2@MnFe2O4 nanoparticles were dissolved in 10 mL of deionized water and ultrasonicated for 30 min to obtain a black suspension A; S32: Dissolve 40 mg of polyvinylpyrrolidone in 10 mL of deionized water to obtain a clear and transparent solution B; S33: Under ultrasound, solution A was slowly added dropwise to solution B, and ultrasound was continued for 10 min. The solution was then placed in a 37°C constant temperature shaker and shaken at 180 rpm for 12 h to obtain solution C. S34: Solution C was repeatedly washed with anhydrous ethanol and deionized water three times, dried under vacuum at 60°C overnight, and ground to obtain MoS2@MnFe2O4@PVP nanoparticles.

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