Preparation method and application of multi-field response type nano diagnosis and treatment agent

Abstract

The invention belongs to the technical field of nano diagnosis and treatment agents, and particularly relates to a preparation method and application of a multi-field response type nano diagnosis and treatment agent, the preparation method of the multi-field response type nano diagnosis and treatment agent comprises a core layer, a middle shell layer and an outer wrapping layer, the core layer is DCNPs, the middle shell layer is Nd0. 03Fe0. 97Se2 wrapping the surface of the DCNPs, and the outer wrapping layer is an Nd0. 03Fe0. 97Se2 wrapping the surface of the DCNPs. And the outer coating layer is HA which is coated on the outer side of the Nd < 0.03 > Fe < 0.97 > Se < 2 >. According to the invention, a multi-mode therapy of membrane structure defect induction-oxidative stress-metabolism interference-energy blocking can be realized, the mucous barrier penetrating ability is good, the retention ability of DNH in a pulmonary infection focus is improved, efficient drug delivery is realized, real-time NIR-II diagnosis can be assisted, and the application prospect is wide. The efficient treatment of lung bacterial infection diseases is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanodiagnosis and treatment agents, and particularly relates to a preparation method of a multi-field response type nanodiagnosis and treatment agent and application thereof. BACKGROUND

[0002] Healthcare-associated infections (HAIs) caused by multi-drug resistant (MDR) bacteria are a major threat in the field of global public health, among which bacterial pneumonia caused by K. pneumoniae (K.p) and the like has high mortality and is difficult to treat - the abuse of broad-spectrum antibiotics has intensified bacterial drug resistance, and traditional antibacterial therapy often fails to control infection due to treatment failure or recurrence.

[0003] There are two core treatment bottlenecks for lung infections: first, the lung mucus layer has high viscosity after infection due to excessive secretion of mucin, and traditional drugs are difficult to penetrate the barrier to reach the infection site, resulting in low delivery efficiency; second, existing alternative antibacterial therapies (such as photothermal therapy PTT, sonodynamic therapy SDT, and chemical dynamic therapy CDT) have significant limitations: PTT requires a high temperature of 65℃ or above to kill bacteria, but a temperature of 45℃ or above damages normal human cells; SDT / PDT produces reactive oxygen species (ROS) that easily cause oxidative damage to normal tissues; CDT relies on H2O2 and an acidic microenvironment at the infection site, but the lung infection microenvironment often cannot provide sufficient reaction conditions, resulting in insufficient ·OH generation.

[0004] In summary, there is an urgent need for an integrated nanoplatform that has the functions of "efficient penetration of lung mucus, multi-modal synergistic bactericidal activity (without drug resistance), and real-time NIR-II diagnosis" to solve the diagnosis and treatment problems of MDR bacterial lung infections. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a multi-field response type nanodiagnosis and treatment agent and application thereof, which can realize multi-modal therapy of membrane structure defect induction-oxidative stress-metabolic interference-energy blockage, has good mucus barrier penetration ability, improves the retention ability of DNH in lung infection foci, realizes efficient drug delivery, and can assist in real-time NIR-II diagnosis, thereby realizing efficient treatment of lung bacterial infection diseases.

[0006] The technical solutions adopted by the present application are as follows: A multi-field response type nanodiagnosis and treatment agent comprises a core layer, an intermediate shell layer, and an outer coating layer. The core layer is DCNPs. The intermediate shell layer is Nd 0.03 Fe 0.97 Se2 wrapped on the surface of the DCNPs. The outer coating layer is Nd 0.03 Fe 0.97HA outside Se2.

[0007] Further, the Nd 0.03 Fe 0.97 The doping amount of Nd in Se2 is 3%.

[0008] A preparation method of a multi-field response type nano diagnosis and treatment agent, comprising the following steps: Step 1, preparation of DCNPs: YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O, oleic acid and octadecene are mixed, heated under argon protection, then NH4F and NaOH dissolved in anhydrous methanol are added, high-temperature reaction, centrifugal washing, and the obtained white nanoparticles are dispersed in a cyclohexane solution; YCl3·6H2O, oleic acid and octadecene solution are mixed, heated under argon protection, then NH4F and NaOH dissolved in anhydrous methanol are added, high-speed stirring is carried out, then the white nanoparticles dispersed in the cyclohexane solution are added, high-temperature reaction, centrifugal washing are carried out, and the obtained white nanoparticles are dispersed in a cyclohexane solution, thereby obtaining DCNPs; Step 2, preparation of DCNPs@Nd 0.03 Fe 0.97 Se2: NdCl3·6H2O, FeCl2·4H2O, octadecene and oleylamine are mixed, vacuum dehydration is carried out, then the white nanoparticles dispersed in the cyclohexane solution are added, again, vacuum dehydration is carried out, then selenium powder dissolved in oleylamine is added, heating reaction, centrifugal drying are carried out, and DCNPs@Nd 0.03 Fe 0.97 Se2 are obtained. Step 3, preparation of DCNPs@Nd 0.03 Fe 0.97 Se2 / HA: the DCNPs@Nd 0.03 Fe 0.97 Se2 dispersed in DMSO is mixed with an HA aqueous solution for reaction, centrifugal washing, freeze-drying are carried out, and DCNPs@Nd 0.03 Fe 0.97 Se2 / HA is obtained.

[0009] The application of the multi-field response type nano diagnosis and treatment agent in the preparation of a bacterial infection treatment drug.

[0010] Further, the bacterial infection is bacterial pneumonia caused by multi-drug resistant bacteria.

[0011] Further, the multi-drug resistant bacteria include methicillin-resistant Staphylococcus aureus and multi-drug resistant Klebsiella pneumoniae.

[0012] The technical effects obtained by the application are as follows: The application discloses a preparation method of a multi-field response type nanodiagnosis and treatment agent and application of the multi-field response type nanodiagnosis and treatment agent 0.03 Fe 0.97 The DNH nanocomposite material is doped with a rare earth element neodymium (Nd), thereby improving the sonodynamic effect of the nanomaterial, improving the treatment effect on deep bacterial infection, realizing real-time imaging of an infection site through DCNPs, and simultaneously, the technical scheme proposes that the DNH targets bacteria and interacts with the bacteria, captures bacterial cell membrane protons / electrons, destroys the bacterial respiratory chain, blocks energy metabolism, cooperates with heat, ROS and H2Se, realizes multi-modal therapy of membrane structure defect-oxidative stress-metabolic interference-energy block, realizes efficient treatment of bacterial infection diseases, and the DNH coated with HA has good mucus barrier penetration capacity, improves the retention capacity of the DNH in a pulmonary infection lesion, realizes efficient drug delivery, can be applied to preparation of a bacterial infection treatment drug, and more further, can be better applied to preparation of a bacterial pneumonia treatment drug caused by multi-drug resistant (MDR) bacteria, and the drug can be a solid drug or a medicament. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is an XRD diagram of the DCNPs nanomaterial of the application; Figure 2 is a fluorescence emission spectrum diagram of the DCNPs nanomaterial of the application; Figure 3 is an XRD diagram of Nd X Fe 1-X Se2 of different doping amounts in the application; Figure 4 is an SEM diagram of Nd X Fe 1-X Se2 of different doping amounts in the application; Figure 5 is a magnetocaloric effect diagram of FeSe2 under different Nd doping amounts in the application; Figure 6 is a sonodynamic effect diagram of FeSe2 under different Nd doping amounts in the application; Figure 7 is an XRD diagram of the DCNPs@Nd 0.03 Fe 0.97 Se2 nanocomposite material of the application; Figure 8 is a fluorescence spectrum of the synthesized composite material under different proportions in the application; Figure 9 is a fluorescence spectrum of the synthesized composite material under different proportions in the application; Figure 10 is a magnetocaloric property of the synthesized composite material under different proportions in the application; Figure 11 The sonodynamic performance of the synthesized composite material at different proportions of the application; Figure 12 The TEM image of the DNH of the application; Figure 13 The element mapping image of the DNH of the application; Figure 14 The Nd 0.03 Fe 0.97 The FT-IR spectrum of Se2, DN, DNH of the application; Figure 15 The magnetic response evaluation of FeSe2, Nd 0.03 Fe 0.97 Se2, DNH of the application; Figure 16 The temperature rising ability of the DNH of the application under AMF; Figure 17 The ROS generation ability of the application under US stimulation at different temperatures; Figure 18 The ROS generation ability of the application under NIR-Ⅱ stimulation at different temperatures; Figure 19 The ROS generation ability of the DNH of the application under different stimulation conditions at 25℃; Figure 20 The ROS generation ability of the DNH of the application under different stimulation conditions at 37℃; Figure 21 The ROS generation ability of the DNH of the application under different stimulation conditions at 43℃; Figure 22 The synergistic ROS generation of the DNH of the application under different stimulations; Figure 23 The ROS generation ability of the application under US stimulation at different temperatures detected by ESR; Figure 24 The ROS generation ability of the application under NIR-Ⅱ stimulation at different temperatures detected by ESR; Figure 25 The synergistic ROS generation of the DNH of the application under different stimulations; Figure 26 The POD-like activity of the DNH of the application under different pH values; Figure 27 The synergistic ·OH generation of the DNH of the application under different pH conditions at 25℃; Figure 28 The synergistic ·OH generation of the DNH of the application under different pH conditions at 37℃; Figure 29is the ·OH generated by DNH under different pH conditions when the application is applied under a stimulating condition at 43℃; Figure 30 is the H2Se gas release standard curve of the application; Figure 31 is the amount of H2Se released under different pH and stimulating conditions by fluorescence detection of the application; Figure 32 is the mucus penetration of DN, DNH within 12h of the application; Figure 33 is the mucus penetration quantification of DN, DNH within 12h of the application; Figure 34 is the mucus penetration of DNH under the synergy of external field of the application; Figure 35 is the mucus penetration quantification of DNH under the synergy of external field of the application; Figure 36 is the band gap width of DNH evaluated by UV-Vis diffuse reflectance spectroscopy and Tauc Plot method of the application; Figure 37 is the flat band potential of DNH determined by Mott-Schottky plot of the application; Figure 38 is the energy level diagram of DNH of the application; Figure 39 is the agar plate and bacterial survival rate quantification chart of MRSA and MDR KP. after different stimulating conditions of the application; Figure 40 is the crystal violet staining image and absorbance value after dissolution of MRSA and MDR KP. biofilm after different stimulating conditions of the application; Figure 41 is the agar plate and bacterial survival rate quantification chart of MRSA and MDR KP. killed by H2Se gas of the application; Figure 42 is the crystal violet staining image and absorbance value after dissolution of MRSA and MDR KP. biofilm after H2Se gas treatment of the application; Figure 43 is the electron transfer between DNH and MRSA / MDR KP. of the application; Figure 44 is the agar plate and bacterial survival rate quantification chart of MRSA and MDR KP. killed by DNH destroying ETC of the application; Figure 45 is the electron transfer between DNH and EPS of the application; Figure 46 is the crystal violet staining image and absorbance value after dissolution of MRSA and MDR KP. biofilm killed by DNH destroying ETC of the application; Figure 47 These are photographs of bacterial colonies on homogenate plates of the lungs of BALB / c mice after different treatments in this invention. Figure 48 This is a statistical diagram of bacterial colonies on homogenate plates of the lungs of BALB / c mice after different treatments in this invention. Detailed Implementation

[0014] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0015] This design presents a novel, multimodal, externally responsive nanotherapeutic platform—DNH—which utilizes AMF, US, and NIR-II to achieve highly efficient treatment of bacterial pneumonia. The mild thermal effect of DNH under AMF increases bacterial membrane fluidity and induces structural defects, mitigating the risk of damage to normal tissues and cells. Simultaneously, the ROS generated by US and NIR-II causes irreversible oxidative damage to bacteria. The triple-field response enhances DNH's mucus penetration and bacterial targeting, synergistically disrupting bacterial energy and metabolic homeostasis through the interaction between DNH and bacteria and the release of H2Se, achieving highly efficient sterilization. The DNH platform, through its multimodal treatment mode inducing membrane structural defects, oxidative stress, metabolic interference, and energy blockade, combined with its integrated therapeutic design, provides a highly efficient new strategy for the treatment of bacterial pneumonia. Specific examples are shown below: Example 1

[0016] like Figure 1 As shown, a method for preparing DCNPs includes the following steps: Take 296 mg of NH4F, 200 mg of NaOH dissolved in 10 mL, 5 mL of anhydrous methanol solution respectively; take 486 mg of YCl3-6H2O, 140 mg of YbCl3-6H2O, 15.3 mg of ErCl3-6H2O in a three-necked flask, add 12 mL of oleic acid and 30 mL of octadecene solution to the flask, stir uniformly at room temperature (20℃~25℃), under argon protection, warm up to 75℃±3℃, under stirring, vacuumize to no bubble generation, to remove water in the reaction system. Under argon atmosphere, raise the temperature to 160℃±3℃, keep for 30 min, the solid is completely dissolved, to get clear and transparent reaction solution. Lower the temperature of the obtained reaction solution to 45℃±3℃, add NH4F and NaOH dissolved in anhydrous methanol, keep for 45 min under high speed stirring (600 rpm / min±50 rpm / min). Then raise the temperature to 75℃±3℃, vacuumize again to no bubble generation, to remove water and methanol in the reaction system. Under argon protection, warm up to 300℃±3℃, keep for 60 min. Finally, cool down the reaction system to room temperature, add 50 mL of anhydrous ethanol to make the nanoparticles precipitate, centrifuge at 12000 rpm / min for 20 min, to get white precipitate. Wash 3 times using cyclohexane and anhydrous ethanol mixed solution with volume ratio of 1:2, finally disperse the obtained white nanoparticles in 10 mL of cyclohexane solution.

[0017] Take 296 mg of NH4F, 200 mg of NaOH dissolved in 10 mL, 5 mL of anhydrous methanol solution respectively; take 486 mg of YCl3-6H2O, 140 mg of YbCl3-6H2O, 15.3 mg of ErCl3-6H2O in a three-necked flask, add 12 mL of oleic acid and 30 mL of octadecene solution to the flask, stir uniformly at room temperature (20℃~25℃), under argon protection, warm up to 75℃±3℃, under stirring, vacuumize to no bubble generation, to remove water in the reaction system. Under argon atmosphere, raise the temperature to 160℃±3℃, keep for 30 min, the solid is completely dissolved, to get clear and transparent reaction solution. Lower the temperature of the obtained reaction solution to 45℃±3℃, add NH4F and NaOH dissolved in anhydrous methanol, keep for 45 min under high speed stirring (600 rpm / min±50 rpm / min). Then raise the temperature to 75℃±3℃, vacuumize again to no bubble generation, to remove water and methanol in the reaction system. Under argon protection, warm up to 300℃±3℃, keep for 60 min. Finally, cool down the reaction system to room temperature, add 50 mL of anhydrous ethanol to make the nanoparticles precipitate, centrifuge at 12000 rpm / min for 20 min, to get white precipitate. Wash 3 times using cyclohexane and anhydrous ethanol mixed solution with volume ratio of 1:2, finally disperse the obtained white nanoparticles in 10 mL of cyclohexane solution, to get DCNPs.

[0018] Firstly, asFigure 1 As shown, the structure of DCNPs was characterized in this technical solution. X-ray diffraction (XRD) patterns revealed that all the crystal peaks of the synthesized nanomaterial were completely consistent with the standard hexagonal β-NaYF4 crystal diffraction (PDF#01-072-4799, RDB), indicating the successful preparation of DCNPs nanomaterials and their excellent crystallinity.

[0019] like Figure 2 As shown, the fluorescence properties of DCNPs in this technical solution are characterized using a near-infrared fluorescence spectrometer. The fluorescence spectroscopy reveals that DCNPs exhibit good fluorescence emission under 980 nm laser excitation, with peak values ​​at approximately 1525 nm and 1550 nm. Example 2

[0020] like Figures 3-4 As shown, a kind of Nd 0.03 Fe 0.97 The method for preparing Se2 includes the following steps: 253 mg of selenium powder was weighed and dissolved in 8 mL of oleylamine. 34.4 mg of NdCl3·6H2O and 617 mg of FeCl2·4H2O were weighed into a three-necked flask, and 5 mL of octadecene and 30 mL of oleylamine were added. The temperature was raised to 120℃±3℃, and a vacuum was applied until no bubbles were generated to remove moisture from the reaction system. The temperature was maintained at 120℃±3℃ for 30 min, and the selenium powder dissolved in oleylamine was slowly added (1 mL / min±0.1 mL / min). The temperature was maintained at 120℃±3℃ for 10 min, then raised to 150℃±3℃ and maintained for 60 min. Finally, the reaction system was cooled to room temperature, and 50 mL of anhydrous ethanol was added to allow the nanoparticles to settle. The mixture was centrifuged at 12000 rpm / min for 15 min to obtain a black precipitate. The precipitate was washed three times with a 1:2 (v / v) mixture of cyclohexane and anhydrous ethanol, and then placed in a 60℃ vacuum oven for 12 h to obtain NdCl3. 0.03 Fe 0.97 Se2.

[0021] like Figure 3 As shown, this technical solution uses XRD to analyze Nd with different doping levels. X Fe 1-X The structure of Se2 was characterized. XRD patterns showed that doping with different Nd contents did not significantly affect the crystal peak shape of FeSe2, indicating the successful preparation of the nanomaterial.

[0022] Meanwhile, scanning electron microscope (SEM) images show that Nd doping did not significantly alter the morphology of FeSe2.

[0023] like Figure 4 As shown, this technique evaluated the effect of different Nd doping concentrations on the magnetocaloric properties of FeSe2. The results showed that 5 mg / mL FeSe2 could be heated to approximately 53 °C within 15 min. Nd doping decreased the magnetocaloric properties of FeSe2, with the smallest decrease observed at a doping concentration of 3%. Furthermore, we used 1,3-diphenylisobenzofuran (DPBF) as a ROS probe to evaluate the ROS generation capacity of FeSe2 with different Nd doping contents under US stimulation. The results showed that Nd doping significantly improved the ROS generation capacity of FeSe2, with 3% doping yielding the best result. Example 3

[0024] like Figures 7-11 As shown, this embodiment discloses a DCNPs@Nd based on embodiment one. 0.03 Fe 0.97 The method for preparing Se2 includes the following steps; Weigh 253 mg of selenium powder and dissolve it in 8 mL of oleylamine. Weigh 34.4 mg of NdCl3·6H2O and 617 mg of FeCl2·4H2O into a three-necked flask, add 5 mL of octadecene and 30 mL of oleylamine, heat to 120℃±3℃, and evacuate until no bubbles are generated to remove water from the reaction system. Cool to 45℃±3℃, add white nanoparticles (DCNPs) dispersed in cyclohexane solution, heat to 75℃±3℃, and evacuate again until no bubbles are generated to remove cyclohexane and water from the reaction system. Then raise the temperature to 120℃±3℃ and hold for 30 min. Slowly (1 mL / min±0.1 mL / min) add the selenium powder dissolved in oleylamine, hold at 120℃±3℃ for 10 min, then raise the temperature to 150℃±3℃ and hold for 60 min. Finally, the reaction system was cooled to room temperature, and 50 mL of anhydrous ethanol was added to allow the nanoparticles to precipitate. The mixture was centrifuged at 12000 rpm for 20 min to obtain a black precipitate. After washing three times with a 1:2 mixture of cyclohexane and anhydrous ethanol, the resulting black precipitate was placed in a 60 °C vacuum oven for 12 h to obtain DCNPs@Nd 0.03 Fe 0.97 Se2 (hereinafter referred to as DN).

[0025] like Figure 7 As shown, this technical solution characterizes DCNPs@Nd using XRD patterns. 0.03 Fe 0.97The results of the Se2 composite analysis showed that the synthesized nanomaterials possessed all the crystal peaks present in the standard hexagonal β-NaYF4 and FeSe2 standard cards (PDF#01-089-4075, RDB), proving the successful preparation of the nanomaterials. The peak intensities varied depending on the proportion of DN used in the composite.

[0026] like Figures 8-11 As shown, the fluorescence properties of composite nanomaterials at different ratios were characterized using near-infrared fluorescence spectroscopy and a small animal in vivo imaging system. The results showed that the fluorescence imaging capability of the nanomaterials was positively correlated with the proportion of DCNPs; that is, the higher the proportion of DCNPs, the stronger the imaging capability. However, when the ratio was below 1:1, the imaging effect was poor and could not meet the requirements for subsequent in vivo animal imaging. (DN in DN...) 0.03 Fe 0.97 Se2 is the effective component in AMF that mediates localized heating; the heating capacity of the composite material is Nd 0.03 Fe 0.97 The decrease in Se2 content leads to a reduction in ROS production. Similar to heating in AMF, the ability of the composite material to generate ROS under US stimulation is related to Nd... 0.03 Fe 0.97 The content of Se2 is directly proportional to that of Nd, i.e. 0.03 Fe 0.97 The higher the Se2 content, the stronger the ROS generation capacity. Example 4

[0027] like Figures 11-15 As shown, this embodiment discloses a method for preparing a multi-field responsive nanotherapeutic agent based on the above embodiments, including the following steps: Weigh 20mg DCNPs@Nd 0.03 Fe 0.97 Se2 was dispersed in 20 mL of dimethyl sulfoxide (DMSO). 60 mg of HA was weighed and dissolved in 10 mL of deionized water. The HA solution was slowly added dropwise to the DMSO solution under sonication (1 mL / min ± 0.1 mL / min). The mixture was then transferred to an oven and reacted at 37 °C and 180 rpm for 24 h. 50 mL of anhydrous ethanol was added, and the mixture was centrifuged at 12000 rpm for 20 min. The precipitate was washed three times with deionized water and then freeze-dried to obtain DCNPs@Nd. 0.03 Fe 0.97 Se2 / HA (hereinafter referred to as DNH) is a multifield responsive nanotherapeutic agent.

[0028] The DCNPs@Nd 0.03 Fe 0.97Se2 / HA consists of a core layer, an intermediate shell layer, and an outer wrapping layer; The core layer consists of DCNPs (NaYF4:Yb,Er nanoparticles), which have fluorescence emission capabilities, enabling real-time imaging of the infected site. The intermediate shell is Nd2O3 wrapped around the surface of DCNPs. 0.03 Fe 0.97 Se2 is used to generate mild heat therapy (43-45℃) in response to AMF, to generate ROS in response to US / NIR-II, and to release H2Se; The outer coating layer is wrapped around Nd 0.03 Fe 0.97 The HA on the outer side of Se2 has mucus penetration and CD44 receptor targeting (CD44 overexpression at the infection site), which increases the lesion enrichment rate.

[0029] like Figures 12-15 As shown, this technical solution first uses TEM to characterize DNH. TEM results show that DNH is a core-shell nanoparticle, where DCNPs are the core and Nd... 0.03 Fe 0.97 Se2 forms the shell, with a crystal size of approximately 30 nm. Elemental mapping results show that Na, F, Y, Yb, Er, Nd, Fe, and Se are uniformly distributed on the surface of the nanoparticles, proving the successful preparation of the nanomaterial. Fourier transform infrared spectroscopy (FT-IR) results show that at 1562 cm⁻¹... -1 1374cm -1 1043cm -1 Absorption peaks for CO-NH2, CH, and C=O were observed at the respective locations, indicating that HA was successfully coated onto the surface of the nanomaterial. Simultaneously, the magnetic responsiveness of DNH was tested using a vibrating sample magnetometer (VSM), revealing that DNH exhibits superparamagnetism.

[0030] like Figures 16-25As shown, this technical solution tested the local heating capacity of DNH under AMF. The results showed that 5 mg / mL DNH could raise the temperature to approximately 45°C within 15 min, meeting the requirements for mild hyperthermia (43~45°C). The ROS generation capacity of DNH under different stimulation conditions was detected by DPBF. The results showed that the ROS generation capacity of DNH under US / NIR-II stimulation increased with increasing temperature; at the same temperature, the ROS generation capacity under synergistic stimulation of US and NIR-II was higher than that under single US or NIR-II stimulation. Furthermore, we also used electron spin resonance (ESR) spectroscopy to detect the types of ROS generated by DNH nanomaterials and their ROS generation capacity under different stimulation conditions; the results were consistent with the DPBF results.

[0031] like Figures 26-29 As shown, Fe in DNH 2+ TMB can react with H₂O₂ to produce ·OH, increasing ROS production and further enhancing bacterial oxidative stress, thus promoting bacterial growth. The reaction of TMB with ·OH produces a blue color. The absorbance at 632 nm, measured using an ultraviolet-visible spectrophotometer (UV-Vis), characterizes the production of ·OH, i.e., peroxidase-like (POD-like) activity. Results show that the higher the absorbance, the stronger the POD activity, with decreasing pH, increasing temperature, and the addition of stimulating conditions. This technique further measures the peak intensity of ·OH using ESR, and the results are consistent with the TMB colorimetric experiment, both demonstrating its excellent POD-like performance and providing favorable conditions for generating ROS to kill bacteria in slightly acidic inflammatory lung regions.

[0032] like Figures 30-35As shown, DNH releases H2Se gas under acidic conditions, which interferes with bacterial sulfur-related metabolic processes, leading to protein dysfunction and metabolic disorders. We detected this gas using quantitative fluorescence. The results showed that, under otherwise identical conditions, the release of H2Se increased with decreasing pH and increasing temperature. The release was further enhanced by the application of stimuli, exhibiting a synergistic effect. Pulmonary infection leads to excessive mucin secretion, further increasing lung mucus viscosity, which constitutes a major obstacle to nanomedicine delivery. HA possesses ideal mucus permeability, enabling efficient drug delivery by overcoming the mucus barrier. Furthermore, the H2Se released by DNH in slightly acidic pneumonia provides the driving force, and under the influence of the external field of AMF / US / NIR-II, HA synergistically achieves mucus barrier penetration. Among them, the penetration ability of the double stimulation condition group, namely AMF+US(A+U), AMF+NIR-II(A+N) and US+NIR-II(U+N) group, was significantly stronger than that of the single stimulation condition group, and the penetration ability of the triple stimulation condition (AMF+US+NIR-II, A+U+N) was the strongest.

[0033] like Figures 36-38 As shown, Figure 38 The light red area represents BRP; ETC is a key physiological process in bacteria, supporting core functions such as respiration, energy production, and growth. By constructing an abiotic-biological interface, an artificial electron transport channel is established from the bacterial membrane to the nanomaterial itself. This targeted electron flow redirection strategy interrupts the natural electron transport chain of bacteria, effectively disrupting its ability to maintain normal physiological activities. Based on this, this technical solution evaluates the band gap of DNH using UV-visible diffuse reflectance spectra (UV-Vis DRS) and Tauc Plot curves, and determines the flat band potential of DNH using Mott-Schottky (MS) spectroscopy. The n-type semiconductor conduction band (CB) potential is 0.1~0.2V (calculated using 0.1V). The results show that the DNH band gap is 2.05eV, the flat band potential is -0.16eV (vs. NHE), the conduction band potential is -0.26V (vs. NHE), and the valence band (VB) potential is 1.89V. Given that the bacterial biological redox potential (BRP) ranges from -4.12 to -4.84eV (vs. vacuum level), and the DNH potential falls within this range, theoretically confirming the feasibility of electron transfer from bacteria to the material.

[0034] like Figures 39-40As shown, methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Klebsiella pneumoniae (MDR KP.) are the most common Gram-positive and Gram-negative bacteria causing bacterial pneumonia. To evaluate the in vitro bactericidal ability of DNH, this technique used MRSA and MDR KP. as model bacteria to study the in vitro antibacterial effect of DNH. The results showed that AMF / US / NIR-II had no significant bactericidal effect on either bacterium, indicating that the intensity of the applied external field stimulation was within the biosafety range. After incubation with the added material, the number of the two bacteria decreased by 13.69±1.53% and 7.77±1.94%, respectively, due to electron transfer between DNH and bacteria and the bactericidal effect of ·OH generated by POD-Like. The difference may be attributed to the fact that Gram-negative bacteria have a double membrane structure, which hinders the interaction between DNH and transmembrane proteins such as cytochrome C (Cyt c) located on the inner membrane, thus reducing the interference with the bacterial electron transport chain. After AMF application, heat enhanced bacterial cell membrane fluidity and induced structural defects, thereby enhancing the bactericidal ability. MRSA and MDR KP. decreased by 28.19±5.19% and 17.55±1.76%, respectively. In the US / NIR-II group, ROS production was enhanced, and the synergistic effect of electron transfer interference and POD-like generated ·OH on bacterial killing resulted in reductions of MRSA by 38.26±5.24% and 28.86±6.93%, respectively, and MDR KP. by 24.68±2.73% and 13.09±2.08%, respectively. When AMF and US / NIR-II were synergistically stimulated, ROS production increased. Combined with the heat-enhanced bacterial cell membrane fluidity and induced structural defects, this further amplified the ROS killing effect. MRSA decreased by 70.47±3.43% and 78.79±2.97%, respectively, and MDR KP. decreased by 13.09±2.08%, respectively. KP. decreased by 72.45±2.08% and 53.62±1.81%, respectively; when US and NIR-II were synergistically stimulated, a large amount of ROS mediated bacterial oxidative stress was generated, and the mortality rates of the two bacteria were 73.96±6.77% and 61.60±3.81%, respectively; when AMF / US / NIR-II triple external field synergistically stimulated, bacterial colonies could no longer be observed on the agar plate.

[0035] Biofilms are complexes formed by densely packed bacteria encapsulated in extracellular polymeric substances (EPS). EPS components include polysaccharides, proteins, lipids, and extracellular DNA (eDNA). This EPS barrier effectively resists attacks from antibacterial agents and immune cells. Furthermore, the cross-linking of eDNA with extracellular polysaccharides in the biofilm microenvironment (BME) stabilizes the biofilm structure and prevents antibacterial agent penetration, thus increasing the difficulty of treatment. After entering the respiratory tract, bacteria readily adhere to the respiratory tract and alveoli to form biofilms. Based on this, we used crystal violet staining to assess the destructive effect of DNH on biofilms. The results from the agar plate tests were similar; under the synergistic effect of triple stimulation, the biofilms formed by the two types of bacteria disintegrated.

[0036] like Figures 41-42 As reported, unlike antibiotic overuse which leads to increased bacterial resistance, H2Se antibacterial gas therapy enters bacterial cells via free diffusion, interfering with sulfur-related metabolic processes, leading to protein dysfunction and metabolic disorders, inducing the death of multidrug-resistant bacteria, and is less likely to induce bacterial resistance. Experimental results demonstrated that DNH, in conjunction with the slightly acidic environment of pneumonia infection under repeated external field responses, releases H2Se. To evaluate its in vitro antibacterial performance, we co-incubated H2Se with MRSA and MDRKP. and evaluated it using the plate spread method. The results showed that the antibacterial effect of H2Se was concentration-dependent; as the concentration increased, the number of dead bacteria increased. When the H2Se concentration reached 12 μg / mL, the mortality rates of MRSA and MDR KP. were 87.31±2.77% and 43.09±4.58%, respectively. The difference in bacterial killing may be attributed to the differences in the cell membranes of Gram-positive and Gram-negative bacteria. Gram-negative bacteria have a double membrane structure with a cavity between the two membranes. This structure hinders the diffusion of H2Se gas, reduces the amount that enters the bacterial cell, and thus reduces the killing effect.

[0037] Furthermore, we evaluated the biofilm-destructive ability of H₂Se using crystal violet staining. The results showed that H₂Se gas enters the biofilm via free diffusion and exerts an antibacterial effect within bacterial cells. Similar to the results from agar plate coating, the biofilm-destructive effect improved with increasing H₂Se concentration. When the H₂Se concentration reached 12 μg / mL, the absorbance values ​​of MRSA and MDRKP. biofilms decreased by 40.13 ± 0.18% and 47.98 ± 0.58%, respectively, after crystal violet dissolution. Unlike the results of killing free bacteria, H₂Se showed slightly better killing effects on MDR KP. biofilms than on MRSA biofilms. This may be due to differences in the EPS components within the two biofilms, leading to differences in the structural density of MRSA biofilms and thus different inhibition efficiencies against H₂Se penetration.

[0038] like Figures 43-46 As shown, the physiological functions of organisms, such as energy conversion, biocatalysis, and signal transduction, all depend on the electrochemical current (ETC) between biological structures. Interfering with this process can disrupt the electronic balance of bacteria, thereby achieving a bactericidal effect. Based on this, this technical solution uses an electrochemical workstation to conduct cyclic voltammetry (CV) experiments. The results show that compared with untreated MRSA / MDR KP., the current of treated MRSA / MDR KP. is enhanced, indicating that the addition of DNH disrupts the electronic balance of bacteria. Specifically, the current of DNH-treated MRSA is greater than that of treated MDR KP., which may be because Gram-negative bacteria have a double-membrane structure, hindering the interaction between DNH and transmembrane proteins such as Cyt c located on the inner membrane, thus reducing the interference with bacterial ETC. Furthermore, this technical solution evaluates the results by co-incubating DNH with MRSA and MDR KP. using a plate coating method. The results showed that the antibacterial effect of DNH by interfering with ETC was concentration-dependent. As the concentration increased, the number of dead bacteria increased. When the DNH concentration reached 400 μg / mL, no colonies were observed in the agar plate.

[0039] Furthermore, to evaluate the electron transfer capability of DNH in EPS, we extracted EPS from MRSA / MDR KP. and performed CV experiments with DNH. The results showed that the current of EPS treated with DNH was enhanced, indicating that DNH can transfer electrons with EPS and disrupt the biofilm. This technique further evaluated the biofilm-killing effect of DNH on ETC disruption using crystal violet staining. The results were similar to those of plate coating, showing that the effect of DNH in disrupting ETC and thus thus disrupting the biofilm was concentration-dependent. Example 5

[0040] like Figures 47-48As shown, to further evaluate the in vivo antibacterial and anti-inflammatory capabilities of DNH, this embodiment uses BALB / c mice to establish an acute pneumonia model to test the in vivo antibacterial and anti-inflammatory capabilities of DNH. In this embodiment, mice were divided into 11 groups: 1-Health, 2-NS, 3-NS+AMF+US+NIR-II, 4-DNH, 5-DNH+AMF, 6-DNH+US, 7-DNH+NIR-II, 8-DNH+AMF+US, 9-DNH+AMF+NIR-II, 10-DNH+US+NIR-II, and 11-DNH+AMF+US+NIR-II. Except for the Health group, all other groups used MDR-KP to infect the lungs of mice to establish a bacterial pneumonia model. Appropriate treatment was administered 24 hours after successful infection. On day 4, lung homogenate plating and counting, as well as hematoxylin and eosin staining (H&E stains) of major organs, were performed to evaluate the treatment. The experimental results showed that a large number of bacteria were still present in the lung lesions of the 2-NS and 3-NS+AMF+US+NIR-II groups on the fourth day, while no bacterial colonies were observed on the homogenate plate of the 11-DNH+AMF+US+NIR-II group. DNH showed excellent in vivo antibacterial properties.

[0041] In summary, this technical solution proposes DCNPs@Nd 0.03 Fe 0.97 Se2 / HA (DNH) nanocomposites, through doping with the rare earth element neodymium (Nd), enhance the sonodynamic properties of nanomaterials, thereby improving the therapeutic effect on deep bacterial infections. Real-time imaging of the infection site is achieved through DCNPs. Furthermore, this technical solution proposes that DNH targets bacteria and interacts with them, capturing protons / electrons from the bacterial cell membrane, disrupting the bacterial respiratory chain, and blocking energy metabolism. In synergy with heat, ROS, and H2Se, it achieves a multimodal therapy of membrane structure defect induction, oxidative stress, metabolic interference, and energy blockade, enabling highly efficient treatment of bacterial infections. HA-coated DNH exhibits good mucus barrier penetration, improving its retention capacity in lung infection lesions and achieving efficient drug delivery. This can be applied to the preparation of drugs for treating bacterial infections, and more specifically, it can be well applied to the preparation of drugs for treating bacterial pneumonia caused by multidrug-resistant (MDR) bacteria. The drugs can be solid drugs or pharmaceutical preparations.

[0042] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained in this invention are implemented according to conventional methods in the art unless otherwise specified or limited.

Claims

1. A multi-field responsive nanotherapeutic agent, characterized in that: It includes a core layer, an intermediate shell layer, and an outer wrapping layer; The core layer is DCNPs; The intermediate shell is Nd2O3 wrapped around the surface of DCNPs. 0.03 Fe 0.97 Se2; The outer coating layer is wrapped around Nd 0.03 Fe 0.97 HA outside Se2.

2. The preparation method and application of a multi-field responsive nanotherapeutic agent according to claim 1, characterized in that: The Nd 0.03 Fe 0.97 The Nd doping content in Se2 is 3%.

3. A method for preparing a multi-field responsive nanotherapeutic agent, used to prepare the multi-field responsive nanotherapeutic agent according to any one of claims 1-2, characterized in that: Includes the following steps: Step 1, Preparation of DCNPs: YCl3·6H2O, YbCl3·6H2O, ErCl3·6H2O, oleic acid and octadecene were mixed and heated under argon protection. NH4F and NaOH dissolved in anhydrous methanol were added. After high-temperature reaction and centrifugation and washing, the resulting white nanoparticles were dispersed in a cyclohexane solution. YCl3·6H2O, oleic acid and octadecene solution were mixed and heated under argon protection. NH4F and NaOH dissolved in anhydrous methanol were added. After high-speed stirring, white nanoparticles dispersed in cyclohexane solution were added. After high-temperature reaction and centrifugation washing, the white nanoparticles were dispersed in cyclohexane solution to obtain DCNPs. Step 2: Preparation of DCNPs@Nd 0.03 Fe 0.97 Se2: NdCl3·6H2O, FeCl2·4H2O, octadecene, and oleylamine were mixed, and after vacuum dehydration, white nanoparticles dispersed in cyclohexane solution were added. After vacuum dehydration again, selenium powder dissolved in oleylamine was added. The mixture was then heated, reacted, centrifuged, and dried to obtain DCNPs@Nd 0.03 Fe 0.97 Se2; Step 3: Preparation of DCNPs@Nd 0.03 Fe 0.97 Se2 / HA: DCNPs@Nd 0.03 Fe 0.97 Se2 was dispersed in DMSO and reacted with an aqueous HA solution. After centrifugation, washing, and freeze-drying, DCNPs@Nd were obtained. 0.03 Fe 0.97 Se2 / HA.

4. The method for preparing a multi-field responsive nanotherapeutic agent according to claim 3, characterized in that: Step 1 includes the following steps: Weigh out 296 mg NH4F and 200 mg NaOH and dissolve them in 10 mL and 5 mL of anhydrous methanol solution, respectively; weigh out 486 mg YCl3·6H2O, 140 mg YbCl3·6H2O, and 15.3 mg... ErCl3·6H2O was placed in a three-necked flask. 12 mL of oleic acid and 30 mL of octadecene solution were added to the flask. After stirring, the mixture was heated to 75 °C under argon protection. Vacuum was applied while stirring to remove water from the reaction system. Under argon atmosphere, the temperature was raised to 160 °C and held for 30 min until the solid completely dissolved to obtain a reaction solution. The temperature of the obtained reaction solution was lowered to 45 °C, and NH4F and NaOH dissolved in anhydrous methanol were added. The mixture was stirred at high speed and held for 45 min. The temperature was then raised to 75 °C, and vacuum was applied again to remove water and methanol from the reaction system. Under argon protection, the temperature was raised to 300 °C and held for 60 min. Finally, the reaction system was cooled to room temperature, and 50 mL of anhydrous ethanol was added to allow the nanoparticles to precipitate. The mixture was centrifuged at 12000 rpm / min for 20 min to obtain a white precipitate. The precipitate was washed three times with a 1:2 volume ratio of cyclohexane and anhydrous ethanol mixture. Finally, the obtained white nanoparticles were dispersed in 10 mL of cyclohexane solution. Weigh 296 mg NH4F and 200 mg NaOH and dissolve them in 10 mL and 5 mL of anhydrous methanol solution, respectively. Weigh 608 mg YCl3·6H2O into a three-necked flask, add 12 mL of oleic acid and 30 mL of octadecene solution to the flask, stir well, and then heat to 75 °C under argon protection. Vacuum is applied while stirring to remove moisture from the reaction system. Under argon atmosphere, the temperature is raised to 160 °C and maintained for 30 min until the solid is completely dissolved, yielding the reaction solution. The temperature of the reaction solution is lowered to 45 °C, and NH4F and NaOH dissolved in anhydrous methanol are added. The mixture is stirred at high speed for 45 min. Subsequently, white cyclohexane solution dispersed in cyclohexane is added. Nanoparticles were heated to 75°C and then evacuated again to remove water, anhydrous methanol, and cyclohexane from the reaction system. Under argon protection, the temperature was raised to 300°C and held for 60 min. Finally, the reaction system was cooled to room temperature, and 50 mL of anhydrous ethanol was added to allow the nanoparticles to precipitate. The precipitate was centrifuged at 12000 rpm for 20 min to obtain a white precipitate. The precipitate was washed three times with a 1:2 volume ratio of cyclohexane to anhydrous ethanol. Finally, the obtained white nanoparticles were dispersed in 10 mL of cyclohexane solution to obtain DCNPs.

5. The method for preparing a multi-field responsive nanotherapeutic agent according to claim 3, characterized in that: Step 2 includes the following steps; Weigh 253 mg of selenium powder and dissolve it in 8 mL of oleylamine; weigh 34.4 mg of NdCl3·6H2O and 617 mg of FeCl2·4H2O into a three-necked flask, add 5 mL of octadecene and 30 mL of oleylamine, heat to 120 °C, and evacuate to remove water from the reaction system; cool to 45 °C, add white nanoparticles dispersed in cyclohexane solution, heat to 75 °C, and evacuate again to remove cyclohexane and water from the reaction system; The temperature was then raised to 120°C and held for 30 minutes. Selenium powder dissolved in oleylamine was added, and the temperature was held at 120°C for 10 minutes. The temperature was then raised to 150°C and held for 60 minutes. Finally, the reaction system was cooled to room temperature, 50 mL of anhydrous ethanol was added to allow the nanoparticles to precipitate, and the mixture was centrifuged at 12000 rpm / min for 20 min to obtain a black precipitate. The black precipitate was washed three times with a 1:2 mixture of cyclohexane and anhydrous ethanol by volume, and then placed in a vacuum oven at 60°C for 12 hours to obtain DCNPs@Nd. 0.03 Fe 0.97 Se2.

6. The method for preparing a multi-field responsive nanotherapeutic agent according to claim 3, characterized in that: Step 3 includes the following steps: Weigh 20mg DCNPs@Nd 0.03 Fe 0.97 Se2 was dispersed in 20 mL DMSO. 60 mg of HA was weighed and dissolved in 10 mL deionized water. The HA solution was added dropwise to the DMSO solution under ultrasonication. The mixture was then transferred to an oven and reacted at 37 °C and 180 rpm / min for 24 h. 50 mL of anhydrous ethanol was added, and the mixture was centrifuged at 12000 rpm / min for 20 min. The precipitate was washed three times with deionized water and then freeze-dried to obtain DCNPs@Nd. 0.03 Fe 0.97 Se2 / HA.

7. The use of the multi-field responsive nanotherapeutic agent according to any one of claims 1-2 in the preparation of a therapeutic agent for bacterial infections.

8. The application according to claim 7, characterized in that: The bacterial infection is bacterial pneumonia caused by multidrug-resistant bacteria.

9. The application according to claim 8, characterized in that: The multidrug-resistant bacteria include methicillin-resistant Staphylococcus aureus and multidrug-resistant Klebsiella pneumoniae.

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