Magnetic multifunctional nano-drug for targeting bacterial pneumonia as well as preparation method and application of magnetic multifunctional nano-drug

Abstract

According to the magnetic multifunctional nano-drug, on the basis of traditional ZIF-8, iron ions and defect structures are introduced, a synergistic antibacterial mode of magnetocaloric therapy-sonodynamic therapy-chemodynamic therapy is established for the first time, heat is generated under an alternating magnetic field, and the magnetic multifunctional nano-drug is used for treating bacterial pneumonia. Active oxygen is rapidly generated under ultrasound, existence of iron ions causes Fenton reaction to achieve chemical power therapy and induce ferroptosis to achieve synergistic antibiosis, release of zinc ions promotes polarization of macrophages to reduce inflammation of disease sites, the problems that focus depth is deep, drug resistance is generated, single treatment effect is poor and the like can be solved, and the treatment effect is good. A new thought is provided for treating the bacterial pneumonia.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-drugs, and particularly relates to a magnetic multifunctional nano-drug targeting bacterial pneumonia, a preparation method thereof, and an application thereof Background Art

[0002] In recent years, microbial infections have seriously endangered public health and are considered to be one of the most serious public health events in the 21st century. Diseases caused by bacterial infections, such as bacterial pneumonia and chronic diabetic wound infections, have become common and highly prevalent diseases. Among them, bacterial pneumonia is more difficult to treat due to its deep lesion location, presence of complications, and easy induction of organ failure, and has a higher mortality rate in children, the elderly, immunocompromised individuals, and patients with chronic respiratory diseases. In existing treatment methods, antibiotic treatment dominates, such as the use of amoxicillin, doxycycline, macrolide drugs, fluoroquinolone drugs, etc., and has achieved good results in the initial stage of treatment. However, due to overuse, multi-drug resistant bacteria (MDR) have now emerged, including carbapenem-resistant Klebsiella pneumoniae (CRKP), methicillin-resistant Staphylococcus aureus (MRSA), and penicillin-resistant Streptococcus pneumoniae (PRSP), etc. This has greatly reduced the effectiveness of traditional antibiotic treatment, increased the treatment difficulty, and brought a greater economic burden to patients. At the same time, the research and development speed of new antibiotics far lags behind the emergence speed of multi-drug resistant bacteria and is insufficient to cope with the antibiotic resistance crisis that has occurred globally. Therefore, there is an urgent need to develop an effective new antibacterial treatment method

[0003] Multifunctional nanomedicine has attracted wide attention due to its non-drug resistance, high biosafety, and high treatment efficiency. The emergence of new treatment methods developed based on it, such as Photothermal therapy (PTT), Photodynamic therapy (PDT), Sonodynamic therapy (SDT), Magnetic hyperthermia (MHT), etc., brings hope to patients. Phototherapy can convert light energy into ROS (Reactive oxygen species, ROS) or local heat through the use of photosensitizers to kill bacteria and achieve the treatment effect. However, the penetration of light is limited and insufficient to treat deep tissue infections, and its disadvantages such as phototoxicity and easy quenching further limit its application. While ultrasound (US)-induced sonodynamic therapy and alternating magnetic field (AMF)-induced magnetic hyperthermia have strong tissue penetration and are two promising strategies for treating deep tissue diseases.

[0004] In recent years, ferroptosis, as a new type of induced cell death, has attracted attention. Specifically, lipoxygenase and intracellular iron mediate the oxidation of polyunsaturated fatty acids. Unstable iron free radicals generate free radicals through the Fenton reaction, ultimately causing lipid peroxidation of the cell membrane and membrane damage, and finally leading to cell death. This type of cell death was first discovered in eukaryotic cells and provides a new treatment method for diseases such as tumors, organ damage, and ischemia, such as inducing the death of tumor cells in combination with sonodynamic, photothermal, and immunotherapy. Recently, it has been reported that this method also exists in prokaryotic cells, which can provide new ideas for bacterial infections.

[0005] Currently, the single treatment effect is not ideal: the ROS concentration needs to reach a certain level to completely kill bacteria, and the long-term application of stimuli and high ROS concentration will undoubtedly damage the surrounding normal tissues; compared with eukaryotic cells, prokaryotic cells such as bacteria require a higher temperature to cause their death due to their thicker cell walls. Higher heat will enhance the stress resistance and also cause casualties to the surrounding normal tissues. Therefore, the combined treatment of multiple treatment methods is considered to be the development trend. Currently, there is still a lack of treatment methods and drugs that can effectively solve problems such as deep lesion depth, drug resistance generation, and poor single treatment effect. Summary of the Invention

[0006] To solve the above technical problems, a magnetic metal-organic framework compound was designed and synthesized in this solution, which can be used as an inhalable multifunctional nanomedicine for the treatment of bacterial pneumonia. This compound generates heat under an alternating magnetic field, rapidly produces reactive oxygen species under ultrasound, and the presence of iron ions triggers the Fenton reaction to achieve chemodynamic therapy, inducing ferroptosis for synergistic antibacterial effect. At the same time, the release of zinc ions promotes macrophage polarization to reduce inflammation at the disease site, providing a new idea for the treatment of bacterial pneumonia.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] First aspect, a preparation method of a magnetic multifunctional nanomedicine targeting bacterial pneumonia, comprising the following steps:

[0009] (1) Preparation of Fe@ZIF-8 (FZ) nanoparticles: Through the solution reaction of metal salts and organic ligands, a metal-organic framework (MOF) structure is formed, and after centrifugation, washing and drying, Fe@ZIF-8 nanoparticles are obtained.

[0010] (2) Preparation of Fe@ZIF-8 (500) nanoparticles: Using the thermal reduction method, FZ is calcined at high temperature under an inert atmosphere to obtain carbon-based composite nanoparticles.

[0011] (3) Preparation of Fe@ZIF-8 (500)@PVP nanoparticles (FZP): Combine FZ(500) with polyvinylpyrrolidone (PVP), through ultrasonic and oscillating reactions, and after freeze-drying, nanoparticles with PVP surface modification are obtained.

[0012] Preferably, the metal salt molecule in step (1) is at least one of Zn(NO3)2·6H2O and FeSO4·7H2O; the organic ligand is at least one of 2-methylimidazole and n-butylamine.

[0013] Preferably, the reaction conditions in step (1) are: reaction temperature 40 - 50 °C, stirring speed 300 - 1000 rpm, and reaction time 12 - 36 h.

[0014] Preferably, the centrifugation speed in step (1) is 9000 - 11000 rpm, and the centrifugation time is 10 - 20 min. Under this centrifugation condition, the precipitate can be effectively precipitated.

[0015] Preferably, the drying temperature in step (1) is 55 - 60 °C.

[0016] Preferably, the inert atmosphere in step (2) is argon; the reaction conditions for the high-temperature calcination are: rising to 400 - 600 °C at a rate of 3 - 5 °C / min and calcining for 1 - 3 h.

[0017] Preferably, the reaction conditions described in step (3) are: reaction temperature 30 - 40°C, stirring speed 100 - 200 rpm, and reaction time 15 - 20 h.

[0018] Preferably, in the nano - drug, Zn:Fe = 1:1 - 1.5, and the particle size is 100 - 150 nm.

[0019] In a second aspect, the present invention provides a magnetic multifunctional nano - drug targeting bacterial pneumonia prepared by the above - mentioned preparation method.

[0020] In a third aspect, the present invention provides an application of the above - mentioned magnetic multifunctional nano - drug in the preparation of a drug for treating bacterial pneumonia.

[0021] In a fourth aspect, the present invention provides an application of the above - mentioned magnetic multifunctional nano - drug in the preparation of a sonodynamic therapy treatment reagent.

[0022] In a fifth aspect, the present invention provides an application of the above - mentioned magnetic multifunctional nano - drug in the preparation of a magnetic hyperthermia therapy treatment reagent.

[0023] Beneficial effects

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention discloses for the first time a magnetic multifunctional nano - drug targeting bacterial pneumonia. Based on the traditional ZIF - 8, iron ions and defect structures are introduced, and a synergistic antibacterial mode of magnetic hyperthermia - sonodynamic therapy - chemodynamic therapy is established for the first time. It generates heat under an alternating magnetic field, rapidly generates reactive oxygen species under ultrasound, the presence of iron ions triggers the Fenton reaction to achieve chemodynamic therapy, induces ferroptosis for synergistic antibacterial, and at the same time, the release of zinc ions promotes macrophage polarization to reduce inflammation at the disease site, which can solve problems such as deep lesion depth, drug resistance generation, and poor single - treatment effect, providing a new idea for the treatment of bacterial pneumonia.

[0026] 1. Magnetic - acoustic - chemical multimodal synergistic therapy: Through the magnetic MOF material (Fe@ZIF - 8) combined with hyperthermia (generating heat by alternating magnetic field), sonodynamic (generating reactive oxygen species by ultrasound), chemodynamic (Fe2+ / Fe3+ Fenton reaction) and ferroptosis mechanism, multi - pathway synergistic bactericidal is achieved, breaking through the limitations of the single - action mode of traditional antibiotics.

[0027] 2. For the first time, a magnetic / acoustic / acid - responsive MOF is synthesized by introducing metal element doping and defect structures, improving the magnetic hyperthermia effect and sonodynamic effect for the treatment of deep - layer lung bacterial infections.

[0028] 3. Optimization of PVP surface modification: Coating with PVP enhances the hydrophilicity, dispersibility and pulmonary mucus penetration ability of nanoparticles, adapts to nebulized inhalation administration, directly targets the lung infection site, and increases the local drug concentration.

[0029] 4. Precise release of zinc ions: Zn2+ in the ZIF-8 framework is slowly released in the lesion microenvironment, promoting the polarization of macrophages towards an anti-inflammatory phenotype (M2 type), simultaneously inhibiting the cytokine storm, and achieving the dual synergistic effects of "bactericidal - anti-inflammatory".

[0030] In summary, through the three-level structural design of "magnetic MOF synthesis - carbonization modification - surface functionalization", the present invention innovatively integrates magnetothermal, sonodynamic, chemodynamic, ferroptosis and immunomodulatory functions on a single nanoplatform, with the advantages of efficient sterilization, precise anti-inflammatory and inhalable delivery, providing a new strategy for the treatment of bacterial pneumonia. Brief Description of the Drawings

[0031] Figure 1 It is a schematic diagram of the preparation process and mechanism of action of the present invention.

[0032] Figure 2 It is the characterization result of Example 1: Figure 2 Figure A is the SEM image of the magnetic multifunctional nanodrug (FZP) prepared in Example 1; Figure 2 Figure B is the DLS image of FZP; Figure 2 Figure C is the XRD image of FZP; Figure 2 Figure D is the FT-IR image of FZP; Figure 2 Figure E is the Zeta potential image of FZP.

[0033] Figure 3 It is the characterization result of the magnetothermal effect of FZP prepared in Example 1: Figure 3 Figure A is the real-time infrared thermal imaging image of FZP; Figure 3 Figure B is the Raman spectrum image of FZP; Figure 3 Figure C is the magnetothermal heating curve of FZP; Figure 3 Figure D is the ROS production curves of water, ZIF-8, Fe@ZIF-8, FZP under ultrasonic stimulation; Figure 3 Figure E is the ESR test image of ROS production of water, ZIF-8, Fe@ZIF-8, FZP under ultrasonic stimulation; Figure 3 Figure F is the ESR test image of ROS production of FZP at different temperatures; Figure 3 Figure G is the UV-Vis-DRS image of ZIF-8, Fe@ZIF-8, FZP.

[0034] Figure 4 It is the characterization result of the CDT effect of FZP prepared in Example 1:Figure 4 Figure A shows the increased absorbance value of TMB after incubation of FZP in different pH buffers; Figure 4 Figure B shows the absorbance of TMB at 632 nm after incubation of FZP at different temperatures; Figure 4 Figure C shows the oxygen production of FZP reacting with H2O2 in different pH buffers; Figure 3 Figure D shows the oxygen production of FZP reacting with H2O2 at different temperatures; Figure 4 Figure E shows the decreased absorbance value after the reaction of DNTB with GSH at different pH buffers and times after incubation of FZP; Figure 4 Figure F shows the absorbance curve of the reaction of DNTB with GSH at different pH buffers and temperatures after incubation of FZP.

[0035] Figure 5 The following are the in vitro antibacterial performance characterization results of FZP prepared in Example 1: Figure 5 Figure A shows the plate-coated colony photos and the quantified bacterial survival rate of Gram-negative multidrug-resistant Klebsiella pneumoniae (MDR-KP) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) after treatment under different stimulation conditions; Figure 5 Figure B shows the live / dead fluorescence microscopy images and the quantified bacterial survival rate of MDR-KP and MRSA after treatment under different stimulation conditions.

[0036] Figure 6 The following are the in vitro antibiofilm performance characterization results of FZP prepared in Example 1: Figure 6 Figure A shows the confocal imaging pictures and the quantified fluorescence intensity of MDR-KP and MRSA biofilms after treatment under different stimulation conditions; Figure 6 Figure B shows the crystal violet staining pictures and the absorbance ratio after dissolution of MDR-KP and MRSA biofilms after treatment under different stimulation conditions.

[0037] Figure 7 The following are the characterization results of FZP-induced bacterial ferroptosis prepared in Example 1: Figure 7 Figure A shows the DCFH-DA fluorescence microscopy images of MDR-KP after treatment under different conditions; Figure 7 Figure B shows the DCFH-DA fluorescence microscopy images of MRSA after treatment under different conditions; Figure 7 Figure C shows the BODIPY 581 / 591C11 fluorescence microscopy images of MDR-KP after treatment under different conditions; Figure 7 Figure D shows the BODIPY 581 / 591C11 fluorescence microscopy images of MRSA after treatment under different conditions.

[0038] Figure 8Characterization results of the in vivo antibacterial and anti-inflammatory abilities of the FZP prepared in Example 1: Figure 8 Figure A in Figure 8 is a photo of the colonies spread on the plate of the lung homogenate of the infected mice after different treatments; Figure 8 Figure B in Figure 8 is a quantitative statistical chart of the colonies spread on the plate of the lung homogenate of the infected mice after different treatments; Figure 8 Figure C in Figure 8 is a photo of the H&E staining of the lungs of the infected mice after different treatments; Figure 8 Figure D in Figure 8 is an immunofluorescence staining image of the lungs of the infected mice after different treatments; Figure 8 Figure E in Figure 8 is an H&E staining image of the heart, liver, spleen, and kidneys of the infected mice after different treatments. Detailed implementation mode

[0039] Next, the technical solutions in the present invention will be clearly and completely described in combination with the embodiments in the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0040] Example 1

[0041] A preparation method of a magnetic multifunctional nano-drug targeting bacterial pneumonia, comprising the following steps:

[0042] (1) Preparation of Fe@ZIF-8:

[0043] Weigh 0.3125 g of Zn(NO3)2·6H2O and 0.6881 g of FeSO4·7H2O into a 50 mL single-necked flask, add 5 mL of ultrapure water, stir until dissolved to obtain solution A; weigh 0.9483 g of 2-methylimidazole into a 50 mL beaker, add 5 mL of ultrapure water, stir until dissolved, add 8.175 mL of n-butylamine and mix evenly to obtain solution B. Under the condition of vigorous stirring, slowly drop solution B into solution A, stir evenly and then transfer it to an oil bath, and react at 45 °C and 500 rpm for 24 h. After the reaction, collect the sample, leave the waste liquid at 10000 rpm for 15 min, and wash the sample three times with ultrapure water and methanol respectively under the same centrifugation conditions, and transfer it to a vacuum oven and dry it overnight at 60 °C to obtain dry nanoparticles of Fe@ZIF-8 (abbreviated as FZ).

[0044] (2) Preparation of Fe@ZIF-8(500):

[0045] Weigh 100 mg of Fe@ZIF-8, grind it into powder and transfer it to a quartz boat, use a tube furnace to rise to 500 °C at a rate of 5 °C / min under an argon atmosphere, and calcine for 2 h to obtain a black powder, which is Fe@ZIF-8(500).

[0046] (3) Preparation of Fe@ZIF-8(500)@PVP:

[0047] Dissolve 40 mg of Fe@ZIF-8(500) in 20 mL of ultrapure water and mix well by ultrasonic treatment to obtain solution A; weigh 40 mg of polyvinylpyrrolidone (PVP) and dissolve it in 20 mL of ultrapure water to obtain solution B. Slowly drop solution A into solution B under ultrasonic conditions, then transfer the solution to a shaker and react at 37 °C and 180 rpm for 18 h. Discard the waste liquid at 12000 rpm for 20 min, wash it three times with ultrapure water, and place it in a freeze dryer for freeze-drying to obtain Fe@ZIF-8(500)@PVP nanoparticles.

[0048] The present invention will hereinafter analyze and explain the structure and performance of Example 1.

[0049] 1. Physical characterization of FZP

[0050] The present invention characterized the FZP prepared in Example 1 by SEM, DLS, XRD, FT-IR spectroscopy and Zeta potential, and the results are as Figure 2 shown, Figure 2 A is the SEM image of FZP, Figure 2 B is the DLS image of FZP, Figure 2 C is the XRD pattern of FZP, Figure 2 D is the FT-IR image of FZP, Figure 2 E is the Zeta potential image of FZP.

[0051] Through Figure 2 A, it can be found that FZP is spherical nanoparticles with a size of about 100 - 150 nm, which is consistent with Figure 2 B. In the XRD pattern shown in Figure 2 C, the ZIF-8 crystal form of FZP disappears, and it is basically consistent with the peak shape and peak intensity of calcined FZ(500), proving that the crystal form is not affected after surfactant coating. In the Fourier transform infrared (FT-IR) spectrum shown in Figure 2 D, the absorption peaks characterizing the imidazole ring of FZ(500) and FZP weaken at 1000 - 1600 cm -1 , proving the destruction of the MOF crystal form, which is consistent with XRD; FZP has corresponding C-N and C=O stretching vibrations of PVP at 1279 cm -1 and 1660 cm -1 . Since PVP is electrically neutral, Figure 2 the Zeta potential of FZP shown in E decreases compared with positively charged FZ(500), but still remains positive. In summary, FZP has been successfully prepared.

[0052] 2. Magnetothermal effect and sonodynamic effect of FZP

[0053] The present invention disperses FZP in ultrapure water to 1 mg / mL and detects its magnetic thermal heating condition under an alternating magnetic field of 20.0 A; the sonodynamic effect of FZP is detected, and the oxidation of DPBF is detected under the conditions of 1.0 MHz, 3.0 W / cm 2 ², and 30 min, and an electron spin resonance spectrometer (ESR) is used to detect the ability of FZP nanomaterials to generate ROS. Further, constant water bath heat simulation is adopted, and ultrasonic stimulation is carried out at temperatures of 25 °C, 37 °C, and 50 °C.

[0054] Figure 3 For the characterization results of the magnetic thermal effect and sonodynamic effect of FZP, it can be found from Figure 3 Figures A and C that at 10 min, the temperature can rise to 55.2 °C, slightly higher than that of Fe3O4, which can meet the sterilization temperature and has a good magnetic thermal heating effect. It can be found from Figure 3 Figures D, E, and F that the maximum decrease in absorbance of FZP at 410 nm after incubation with DPBF is the largest, and the ROS production is the highest. It shows a peak pattern of 1:1:1 in ESR and the highest intensity, proving that the sonodynamic effect of FZP is the strongest, and the ROS type is 1 O2. As the temperature increases, the ROS production also increases. The reason is that the increase in temperature accelerates the electron transfer rate, promotes the effective separation of electron holes, and improves the sonodynamic effect, which also provides a basis for magnetic thermal synergistic therapy.

[0055] In order to explore the sonodynamic enhancement mechanism, the present invention conducts ultraviolet diffuse reflection (UV-Vis-DRS) and Raman spectroscopy detections. The results are as shown in Figure 3 Figures B and G. FZP shows obvious D peaks and G peaks, with obvious defects; from ZIF-8 to Fe@ZIF-8 and then to Fe@ZIF-8(500)@PVP, DRS detects that the band gap decreases from 3.68 eV to 2.15 eV and then to 1.85 eV. It is speculated that the reason for the sonodynamic enhancement is that after doping with metal ions, the energy band structure of MOF is adjusted, introducing a new metal-metal charge transfer mode. At the same time, after calcination, the crystal form of MOF changes, the micro-mesoporous framework enhances the cavitation effect, the defect structure increases, the energy band gap decreases, the electrons induced by ultrasound are rapidly transmitted, and electrons can also be captured to prevent electron-hole recombination, thus improving the sonodynamic performance.

[0056] 3. CDT effect of FZP

[0057] Fe in FZP 2+It can react with H2O2 to generate ·OH, increase the production of ROS, further increase bacterial lipid peroxidation, and promote bacterial ferroptosis. TMB can react with ·OH to show blue, and the amount of ·OH production, that is, peroxidase-like (POD-like) activity, is characterized by the absorbance at 632 nm.

[0058] Figure 4 The characterization results of the CDT effect of FZP are as follows: As Figure 4 shown in Figures A and B, as the material concentration increases, the pH decreases, and the temperature increases, the absorbance value is larger and the POD enzyme activity is stronger, which proves its good enzyme activity performance and provides the possibility for antibacterial in the slightly acidic pulmonary inflammation area.

[0059] The present invention explored the catalase-like (CAT-like) activity of FZP. Through the incubation reaction of FZP with H2O2, the oxygen production amount was measured by a dissolved oxygen meter to characterize its CAT enzyme activity. The results are as Figure 4 shown in Figures C and D. The calcined FZ has stronger CAT enzyme activity, and as the concentration increases, the pH increases, and the temperature increases, the oxygen production amount increases, which can solve the problem of deep tissue hypoxia and provide an oxygen source for the generation of singlet oxygen by sonodynamic electron transfer.

[0060] The present invention detected the glutathione peroxidase-like (GSHOx-like) activity of FZP. After incubating FZP with GSH, the Fe 3+ oxidizes GSH to GSSG, and DNTB is added to react with GSH to show color. The decrease in absorbance at 410 nm characterizes the consumption of GSH. The results are as Figure 4 shown in Figures E and F. As the temperature rises and the pH decreases, the absorbance at 410 nm decreases significantly, and GSH is consumed. In summary, FZP has peroxidase-like activity, catalase-like activity, and glutathione oxidase-like activity. Through the Fe 2+ / Fe 3+ cycle, it solves the endogenous GSH antioxidant mechanism, reduces the hindrance of ROS accumulation, provides oxygen for sonodynamic, and increases the ROS content in the lungs.

[0061] 4. In vitro antibacterial performance of FZP

[0062] The present invention evaluated the in vitro antibacterial performance of the FZP prepared in Example 1 using the Gram-negative multi-drug resistant Klebsiella pneumoniae (MDR-KP.) and the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA). After dispersing FZP and co-incubating it with the bacterial solution, after applying the corresponding stimulus, dilution plate coating and counting were carried out, and its antibacterial effect was characterized by statistically calculating its survival rate.

[0063] Figure 5 The characterization results of the in vitro antibacterial performance of FZP are shown byFigure 5 As can be seen from Figure A, under the stimulation of an alternating magnetic field (20.0 A, 8 min) and ultrasound (1 MHz, 3.0 W / cm 2 , 30 min), there is no killing effect on the two types of bacteria; after adding FZP, the mortality rate of MDR-KP can reach 19.9 ± 9.2%; under the single stimulation of an alternating magnetic field, the mortality rate of bacteria can reach 48.5 ± 4.8% under the thermal factor; under the single stimulation of ultrasound, the survival rate of bacteria under the ROS factor reaches 27.3 ± 6.5%, slightly lower than that of the single magnetic hyperthermia group; under the combined treatment of dual stimulation, the killing rate can reach more than 99.4 ± 0.4%; FZP has the same bactericidal effect on MRSA, and the mortality rates under FZP, FZP + AMF, FZP + US, and FZP + AMF + US are 18.3 ± 3.5%, 48.9 ± 5.0%, 29.9 ± 6.6%, and 96.2 ± 2.4% respectively, showing good bactericidal effects, providing a possibility for clinical application.

[0064] The present invention further uses the bacterial live / dead staining method to verify the antibacterial performance of the FZP prepared in Example 1. The SYTO9 dye stains live bacteria and shows green fluorescence under the excitation light; the PI dye stains dead bacteria and shows red fluorescence under the excitation light. From Figure 5 Figure B, it can be seen that in the group incubated with PBS, whether the stimulation is applied or not, it basically shows green fluorescence, and the bacterial mortality rate is low; with the application of stimulation, the red fluorescence gradually strengthens. Under the dual stimulation of an alternating magnetic field and ultrasound, it basically shows red fluorescence, and the intensity reaches the highest, indicating that the bacteria are basically all dead, further verifying the excellent antibacterial performance of FZP.

[0065] 5. Antibiofilm performance of FZP in vitro

[0066] The present invention uses the bacterial live / dead staining method and the crystal violet staining method to evaluate the ability of the FZP prepared in Example 1 to disrupt biofilms. From Figure 6 Figure A, it can be seen that in the control group treated with PBS, whether a single stimulation or a dual stimulation is applied, the biofilm basically shows green fluorescence; while in the group treated with FZP, the intensity of red fluorescence increases. In the group of combined treatment with FZP + AMF + US, the biofilm basically shows red fluorescence, and the bacterial mortality rate is as high as 95%; from Figure 6 Figure B, it can be seen that in the group of combined treatment with FZP + AMF + US, the coloring in the crystal violet staining is lighter, indicating that a large number of bacteria have died, the connection between bacteria has been interrupted, and they have fallen off from the biofilm structure, and the biofilm structure has been damaged. This shows that FZP has excellent biofilm clearance performance and shows a better combined treatment effect under dual stimulation.

[0067] 6. Ability of FZP to induce ferroptosis in bacteria

[0068] The present invention uses the fluorescence probe method to verify the ability of the FZP prepared in Example 1 to induce bacterial ferroptosis. The DCFH-DA probe is used to detect the ROS content in bacteria. When the bacteria are incubated with FZP, the content of ferrous ions increases. Its Fenton reaction and ultrasonic stimulation increase the ROS content, the accumulation of LPO increases, and at the same time, the ratio of GSH / GSSG in the body is imbalanced, ultimately leading to the destruction of the redox balance in the bacteria and resulting in death. From Figure 7 Figures A and B of show that there is basically no green fluorescence in the control group, indicating a low ROS content; after the material is incubated with the bacteria, the Fenton reaction triggered by divalent iron ions causes the ROS level to rise. After applying magnetic hyperthermia stimulation, the temperature rises, the activity of POD enzyme increases, and the ROS production increases. In the ultrasonic group, the sonodynamic performance of the material itself further increases the ROS production, and the green fluorescence is further enhanced. In the synergistic effect of the final group, the ROS content reaches the highest and the green fluorescence is the strongest.

[0069] The present invention uses the BODIPY 581 / 591C11 probe to characterize the accumulation of LPO in bacteria. From Figure 7 Figures C and D of show that in the control group, BODIPY 581 / 591C11 remains in the reduced state and is observed as red fluorescence; with the application of the material and stimulation, BODIPY 581 / 591C11 is oxidized by paper hydrogen peroxide, and the oxidized state shows green fluorescence, indicating an increase in the LPO level and inducing bacterial ferroptosis.

[0070] 7. Antibacterial and anti-inflammatory ability of FZP in vivo

[0071] The present invention uses a BALB / c mouse model to test the antibacterial and anti-inflammatory ability of the FZP prepared in Example 1 in vivo. The mice are divided into seven groups: Health, NS, NS+AMF+US, FZP, FZP+AMF, FZP+US, and FZP+AMF+US. Except for the Health group, the others are used to infect the lungs of the mice with MRSA to establish a bacterial pneumonia model, and corresponding treatments are carried out 24 hours after successful infection. After 4 days, experiments such as lung homogenate plate coating counting, HE staining of main organs, and lung immunofluorescence section are carried out for treatment evaluation.

[0072] From Figure 8 Figures A and B of show that the NS group still maintains a high lung bacterial load on the fourth day, and the bacterial survival rate of the FZP+AMF+US group is only 0.62%, showing excellent antibacterial performance in vivo. From Figure 8As can be seen from Figure C, after the mice were infected with bacterial pneumonia, the alveolar structure was significantly damaged, pulmonary fibrosis was severe, and there was a large amount of inflammatory cell infiltration. After treatment, the pulmonary structures of the FZP+AMF+US group and the Health group were basically the same, the alveolar structure was obvious, and the inflammation was reduced, indicating excellent therapeutic effects. From Figure 8 As can be seen from Figure D, with the application of FZP and the stimulus, the green fluorescence intensity of CD86 gradually decreased, and the yellow fluorescence intensity of CD206 increased correspondingly. Macrophages were transformed from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and the inflammation was alleviated. From Figure 8 As can be seen from Figure E of other major organs, the H&E staining sections were all consistent with the healthy group, indicating that FZP has high biosafety. The above shows that FZP has good anti-inflammatory and antibacterial abilities at the level of mice in vivo and has good biosafety, providing a new idea for the treatment of bacterial pneumonia.

[0073] In summary, the FZP prepared by the present invention generates heat under an alternating magnetic field and rapidly generates reactive oxygen species under ultrasound. The presence of iron ions triggers the Fenton reaction to achieve chemodynamic therapy, induces ferroptosis for synergistic antibacterial, and at the same time the release of zinc ions promotes macrophage polarization to reduce inflammation at the disease site, with the advantages of high-efficiency sterilization, precise anti-inflammation and inhalable delivery, providing a brand-new strategy for the treatment of bacterial pneumonia.

[0074] The above is only the best implementation mode of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications or equivalent replacements can be made to the technical solution of the present invention, and the technical effects of the present invention can also be achieved, which should also be regarded as belonging to the protection scope of the present invention.

Claims

1. A preparation method of a magnetic multifunctional nano-drug targeting bacterial pneumonia, characterized in that, It includes the following steps: (1) Preparation of Fe@ZIF-8 (FZ) nanoparticles: Through the solution reaction of metal salts and organic ligands, a metal-organic framework (MOF) structure is formed, followed by centrifugal washing and drying to obtain Fe@ZIF-8 nanoparticles. (2) Preparation of Fe@ZIF-8(500) nanoparticles: Using the thermal reduction method, FZ is calcined at high temperature in an inert atmosphere to obtain carbon-based composite nanoparticles. (3) Preparation of Fe@ZIF-8(500)@PVP nanoparticles (FZP): Combine FZ(500) with polyvinylpyrrolidone (PVP), through ultrasonic and oscillating reactions, and after freeze-drying, obtain nanoparticles with PVP surface modification.

2. The preparation method according to claim 1, characterized in that: The metal salt molecules described in step (1) are at least one of Zn(NO3)2·6H2O and FeSO4·7H2O; the organic ligands are at least one of 2-methylimidazole and n-butylamine.

3. The preparation method according to claim 1, characterized in that: The reaction conditions described in step (1) are: reaction temperature 40 - 50°C, stirring speed 300 - 1000 rpm, and reaction time 12 - 36 h.

4. The preparation method according to claim 1, wherein: The inert atmosphere described in step (2) is argon; the reaction conditions for the high-temperature calcination are: rising to 400 - 600°C at a rate of 3 - 5°C / min and calcining for 1 - 3 h.

5. The preparation method according to claim 1, characterized in that: Reaction temperature 30 - 40°C, stirring speed 100 - 200 rpm, and reaction time 15 - 20 h.

6. The preparation method according to claim 1, wherein: In the nano-drug, Zn:Fe = 1:1 - 1.5, and the particle size is 100 - 150 nm.

7. A nano-drug prepared by the preparation method of the magnetic multifunctional nano-drug for targeting bacterial pneumonia according to any one of claims 1 - 6.

8. An application of the magnetic multifunctional nano-drug according to claim 7 in the preparation of a drug for treating bacterial pneumonia.

9. An application of the magnetic multifunctional nano-drug according to claim 7 in the preparation of a therapeutic reagent for sonodynamic therapy.

10. An application of the magnetic multifunctional nano-drug according to claim 7 in the preparation of a therapeutic reagent for magnetic hyperthermia therapy.

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