A cobalt-crosslinked sodium alginate inhalable microsphere loaded with meropenem and its application in the treatment of carbapenem-resistant Klebsiella pneumoniae pneumonia.

Abstract

This invention discloses a cobalt-crosslinked sodium alginate microsphere loaded with meropenem and its application in the treatment of carbapenem-resistant Klebsiella pneumoniae pneumonia, belonging to the field of biomedicine and anti-infective drugs. The microspheres are prepared using microfluidic technology, by crosslinking sodium alginate with cobalt ions and loading it with meropenem (MEM), with a particle size of 2–5 μm, and can deposit deep in the alveoli of the lungs. 2+ It has dual antibacterial effects: it can replace the Zn active site of NDM-1 in a stoichiometric ratio. 2+ This microsphere restores the efficacy of meropenem and simultaneously interferes with CRKP iron metabolism, inhibiting KPC-2 and OXA-48 resistant strains. In vitro and in vivo experiments show that the microsphere has a significant antibacterial effect against various carbapenemase-producing CRKP strains, reducing pulmonary bacterial load, regulating pulmonary immunity, alleviating inflammation, promoting tissue repair, and improving the survival rate of infected animals, providing a new treatment option for CRKP pneumonia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and anti-infective drug technology, specifically relating to a cobalt-crosslinked sodium alginate inhalable microsphere loaded with meropenem and its application in the treatment of carbapenem-resistant Klebsiella pneumoniae pneumonia. Background Technology

[0002] Carbapenem antibiotics are the last line of defense in the clinical treatment of severe Gram-negative bacterial infections. However, with the widespread availability of carbapenemases, carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a clinically refractory pathogen. CRKP strains can carry different carbapenemase types, such as KPC-2 (Ambler A), NDM-1 (Ambler B), and OXA-48 (Ambler D), which can hydrolyze carbapenem antibiotics through differential catalytic mechanisms, directly leading to the failure of conventional anti-infective treatment regimens. Among them, NDM-1 is a metallo-β-lactamase, which relies on the active site Zn to mediate the catalytic hydrolysis reaction, and there are currently no approved specific inhibitors for it. Serine-type carbapenemases such as KPC-2 and OXA-48 are generally insensitive to existing commercially available enzyme inhibitors, and CRKP strains are prone to superimposing multiple drug resistance genes, forming a pan-drug-resistant phenotype, which greatly increases the difficulty of clinical treatment of CRKP pneumonia.

[0003] To address bacterial resistance, synergistic therapy with antibiotics and functional adjuvants is currently the mainstream research direction for reversing bacterial resistance and restoring the antibacterial activity of antibiotics. Among them, metal-based adjuvants have significant advantages in the intervention of drug-resistant bacterial infections due to their multi-target antibacterial mechanisms, low resistance induction, and good biocompatibility. Existing studies have confirmed that bismuth-based and gold-based metal compounds can effectively restore the bactericidal ability of carbapenem antibiotics by inhibiting the activity of metallo-β-lactamases by replacing the Zn active site of the NDM-1 enzyme. However, existing technologies still have significant limitations: these metal adjuvants are only specifically targeted at NDM-1 type metallocarbapenemases and cannot effectively overcome drug resistance mediated by serine carbapenemases such as KPC-2 and OXA-48. Their antibacterial spectrum is limited and cannot cover CRKP strains with complex clinical subtypes. At the same time, existing synergistic treatment regimens are mostly administered systemically, resulting in poor drug accumulation in the lungs and low local bioavailability. This requires increasing the dosage to improve efficacy, which can easily cause systemic toxic side effects and has poor safety, failing to meet the clinical demand for highly effective and low-toxicity treatment of CRKP pneumonia.

[0004] Compared to systemic drug delivery, local inhalation in the lungs allows for direct targeting of the infection site, effectively increasing drug concentration in the alveoli and reducing systemic drug exposure, making it the preferred route of administration for treating lung infections. Inhalable microspheres, with their advantages of controllable particle size, excellent lung deposition, stable sustained drug release, and good biocompatibility, have become ideal carriers for targeted drug delivery to the lungs. Currently, existing inhalable anti-infective drug delivery systems still have significant technical shortcomings. Most formulations only load a single antibiotic, relying solely on the antibiotic's single mechanism of action to kill bacteria. They cannot achieve broad-spectrum resistance reversal against the complex multi-type and multi-mechanism resistance characteristics of CRKP, and cannot simultaneously address the treatment needs of different types of carbapenemase-positive resistant strains. Furthermore, conventional inhaled formulations lack a co-delivery design with resistance-regulating adjuvants, failing to fundamentally address bacterial resistance issues and resulting in limited clinical treatment efficacy. Given these technological deficiencies, there is an urgent need to develop a novel inhaled therapeutic formulation that can achieve targeted co-delivery of dual active ingredients to the lungs, broad-spectrum reversal of multi-type CRKP resistance, and is low in toxicity and highly effective. Summary of the Invention

[0005] To address the shortcomings of existing clinical treatments for CRKP pneumonia, such as narrow antibacterial spectrum, low lung-targeted drug concentration, significant systemic toxicity, and difficulty in simultaneously achieving the elimination of drug-resistant bacteria, regulation of lung inflammation, and repair of damaged lung tissue, this invention aims to provide a cobalt-crosslinked sodium alginate inhalable microsphere loaded with meropenem, its preparation method, and its application. This invention utilizes microfluidic technology to construct a cobalt ion-crosslinked sodium alginate inhalable drug delivery system, achieving targeted lung-targeted co-delivery of meropenem and Co. It leverages the dual resistance reversal mechanism of Co—zinc ion replacement to inactivate NDM-1 enzyme and interference with bacterial iron metabolism to inhibit serine carbapenemase—to broadly overcome the drug resistance characteristics of different CRKP subtypes. Simultaneously, by regulating the lung immune microenvironment and activating lung tissue repair-related genes, it achieves a synergistic therapeutic effect of highly efficient bactericidal, anti-inflammatory, and lung damage repair, providing a novel, low-toxicity, highly effective, and universally applicable treatment strategy for clinically refractory CRKP drug-resistant pneumonia.

[0006] To solve the technical problem, the present invention adopts the following technical solution: A method for preparing cobalt-crosslinked sodium alginate inhalable microspheres loaded with meropenem, characterized by comprising the following steps: Meropenem and sodium alginate were added to deionized water and stirred at room temperature until completely dissolved to prepare aqueous solution A. Cobalt salt was added to the oil phase and ultrasonically dispersed to prepare oil phase solution B. Aqueous solution A was injected into the central channel of a microfluidic chip at a set flow rate, while pure oil phase without cobalt salt was introduced into the channels on both sides of the chip at a set flow rate. The chip outlet was immersed in oil phase solution B containing cobalt salt. The two phases formed uniform water-in-oil droplets through flow focusing shear at the junction. Oil phase solution B containing droplets was allowed to stand at room temperature and in the dark to allow sodium alginate and Co to react. 2+Fully cross-linked and cured; the cured microspheres are repeatedly washed with cyclohexane to remove excess uncross-linked raw materials until the supernatant is clear and the microspheres are collected. Finally, they are freeze-dried to obtain cobalt cross-linked sodium alginate inhalable microspheres loaded with meropenem.

[0007] Preferably, the meropenem is a carbapenem broad-spectrum antibiotic, which is a white to slightly yellow crystalline powder that can inhibit bacterial cell wall synthesis and exert a bactericidal effect.

[0008] Preferably, the cobalt salt is one of cobalt chloride hexahydrate, cobalt sulfate, cobalt nitrate, and cobalt acetate, among which cobalt chloride hexahydrate has the best loading effect and cross-linking stability, providing bioactive Co to the microspheres. 2+ It also acts as a crosslinking agent to form a stable coordination structure with sodium alginate.

[0009] Preferably, in the aqueous solution A, the mass ratio of meropenem to sodium alginate is 1:2 to 4.

[0010] Preferably, the mass concentration of cobalt salt in the oil phase solution B is 7–9 mg / mL, ensuring the Co... 2+ Effective load and crosslinking effect.

[0011] Preferably, the oil phase is at least one of the following: droplet-forming oil phase, cyclohexane, liquid paraffin, and mineral oil, which can help form a stable water-in-oil droplet system and ensure the uniformity of microsphere particle size.

[0012] Preferably, the microfluidic chip channel size is 20 μm to 200 μm; the flow rate of aqueous solution A is 10 to 20 μL / min, and the flow rate of pure droplet oil phase is 50 to 70 μL / min. Most preferably, the microfluidic chip channel size is 20 μm, the flow rate of aqueous solution A is 15 μL / min, and the flow rate of pure oil phase is 60 μL / min, which is suitable for preparing microspheres with a particle size of 2 to 5 μm, meeting the requirements for deep deposition in the lungs.

[0013] Preferred conditions: Crosslinking curing is performed at room temperature in the dark for 10–16 hours; freeze-drying conditions are: temperature -45–55°C, vacuum degree ≤10 Pa, time 20–28 hours, to remove solvent from microspheres and maintain the porous structure of microspheres. The inhalable microspheres prepared in this invention have a particle size of 2–5 μm, with an average particle size of approximately 4 μm, making them suitable for deep deposition in the lungs. The meropenem loading efficiency (i.e., the mass percentage of meropenem in the microspheres) of the inhalable microspheres is 2–8%, and the Co loading efficiency (i.e., the mass percentage of Co in the microspheres) is 30–40%. The inhalable microspheres exhibit pH-responsive drug release characteristics: rapid drug release in the acidic microenvironment (pH 6.2) of lung bacterial infection, with meropenem and Co reacting within 2 hours. 2+The release rate is ≥80% and ≥90% after 72 hours; it is slowly released in a normal physiological microenvironment (pH 7.4), with a release rate of about 60% after 72 hours.

[0014] The cobalt-crosslinked sodium alginate inhalable microspheres loaded with meropenem prepared in this invention can be administered via nebulization and are suitable for treating lung infections caused by carbapenem-producing NDM-1, KPC-2, OXA-48 type carbapenemases and clinically multidrug-resistant carbapenem-resistant Klebsiella pneumoniae.

[0015] Compared with existing technologies, the beneficial effects of this invention are reflected in: 1. This invention is the first to prepare cobalt-crosslinked sodium alginate inhalable microspheres loaded with meropenem. The preparation method is based on microfluidic technology, with mild reaction conditions and simple operation. The resulting microspheres have uniform particle size and stable drug loading efficiency. The raw materials are readily available and the production cost is low, making it suitable for industrial-scale production.

[0016] 2. The microspheres prepared in this invention achieve targeted lung delivery through atomized inhalation. Their particle size of 2–5 μm allows for deep deposition in the lungs, significantly increasing drug concentration at the site of lung infection and reducing CO2 levels. 2+ And the full-body exposure of meropenem; Co 2+ It mainly accumulates in the lungs, is rapidly excreted after being metabolized by the liver, and does not cause significant pathological damage to major organs such as the heart, liver, and kidneys. Its systemic toxicity is significantly lower than that of intravenous administration, thus overcoming the technical shortcomings of traditional intravenous administration, such as poor targeting and strong toxicity.

[0017] 3. This invention utilizes Co 2+ Its dual antibacterial mechanism achieves broad-spectrum resistance against carbapenem-resistant Klebsiella pneumoniae. 2+ Zn can be irreversibly replaced at the active site of NDM-1 in a 1:1 stoichiometric ratio. 2+ To restore the bactericidal effect of meropenem against metallo-β-lactamase-positive strains; and at the same time, Co 2+ Interfering with the iron metabolism of KPC-2 and OXA-48 positive strains, causing bacterial energy crisis and nutrient deficiency, significantly enhances their sensitivity to meropenem, and achieves highly efficient killing of different types of carbapenemase-mediated resistant strains.

[0018] 4. The microspheres prepared by this invention have pH-responsive drug release characteristics: they rapidly release drugs in the acidic microenvironment (pH 6.2) of lung bacterial infection, with a release rate of ≥80% within 2 hours, and quickly exert bactericidal effects; they release drugs slowly in the normal physiological microenvironment (pH 7.4), with a release rate of about 60% after 72 hours, continuously maintaining an effective drug concentration in the lungs, inhibiting the evolution of bacterial drug resistance, and achieving long-lasting antibacterial effects.

[0019] 5. The microspheres of this invention can achieve synergistic therapeutic effects of sterilization, anti-inflammation, and lung tissue repair. While efficiently eliminating drug-resistant bacteria in the lungs, they can precisely regulate the lung's immune microenvironment, inhibit the infiltration of excessive neutrophils into the lungs, promote the transformation of macrophages from the M1 pro-inflammatory phenotype to the M2 repair phenotype, and simultaneously retain effector CD8. + It can protect the immune function of T cells and NK cells; it can also activate lung tissue repair-related genes, reverse infection-induced gene expression disorders, reduce lung inflammation damage, and promote the recovery of lung tissue structure and function. Attached Figure Description

[0020] Figure 1 This is an optical micrograph of the SCM microspheres prepared in Example 1 in a microfluidic chip.

[0021] Figure 2 This is a scanning electron microscope (SEM) image of the SCM microspheres prepared in Example 1.

[0022] Figure 3 This is an elemental mapping diagram of the SCM microspheres prepared in Example 1.

[0023] Figure 4 Co in SCM microspheres in phosphate buffer solutions of different pH values ​​in Example 2 2+ ( Figure 4 (a) and Meropenem ( Figure 4 (b) Release curve.

[0024] Figure 5 The results of in vitro antibacterial detection of SCM microspheres in Example 3 are shown below. Among them, (ad) are the growth curves of KPC-2, OXA-48, NDM-1 and clinical multidrug-resistant CRKP strains, respectively; (e) are the colony photos of KPC-2 and NDM-1 type CRKP plates; (fg) are the quantitative count results of KPC-2 and NDM-1 type CRKP plate colonies, respectively; and (h) is the quantitative table of the logarithmic reduction value of the bactericidal effect of each group on KPC-2 and NDM-1 type CRKP.

[0025] Figure 6 The results of the detection of the CRKP resistance reversal mechanism of SCM microspheres in Example 4 are shown; (a) is the NDM-1 enzyme activity detection graph, and (b) is the result of exogenous Zn supplementation. 2+ The NDM-1 enzyme activity recovery diagram is shown below. (c) shows the intraenzyme Zn content. 2+ / Co 2+ The results of ion exchange quantification are as follows: (d) shows the detection of siderophore content in the strain, (e) shows the detection of intracellular ATP content in the strain, and (fh) shows the results of transcriptome sequencing analysis of the strain.

[0026] Figure 7The results of in vivo efficacy evaluation of CRKP pneumonia in mice in each experimental group in Example 7 are shown in (a) 7-day survival curve of mice, (b) average weight change curve of mice, and (c) scatter plot of individual weight change of mice.

[0027] Figure 8 HE and Ki67 staining images of lung tissues from mice in the Blank group, Control group, and SCM group at different time points in Example 7.

[0028] Figure 9 The images show the staining of TNF-α, IL-10, iNOS, ARG-1, and PD-1 in the lung tissues of mice in the Blank group, Control group, and SCM group at different time points in Example 7.

[0029] Figure 10 HE staining images of major organs (heart, liver, spleen, lung, and kidney) of healthy mice in Example 7 after treatment with SCM microspheres. Detailed Implementation

[0030] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0031] Example 1 In this embodiment, cobalt-crosslinked sodium alginate inhalable microspheres (SCM) loaded with meropenem were prepared according to the following method: Weigh 20 mg of sodium alginate and 5 mg of meropenem and add them to 5 mL of deionized water. Stir magnetically for 30 min at room temperature in the dark until completely dissolved to form a homogeneous and transparent aqueous solution A. Separately, add 40 mg of cobalt chloride hexahydrate to 5 mL of droplet-generated oil phase and sonicate at room temperature for 20 min (power 200 W, 3 s working interval 5 s interval) to prepare oil phase solution B. Inject aqueous solution A into the middle channel of a 20 μm microfluidic chip at a flow rate of 15 μL / min. Flow pure droplet-generated oil phase into the channels on both sides of the chip at a flow rate of 60 μL / min. Immerse the chip outlet in oil phase solution B to form homogeneous water-in-oil droplets. Place the droplet-containing oil phase solution B at room temperature in the dark for 12 h to allow the sodium alginate and Co to react. 2+ Fully cross-linked and cured; add cyclohexane to the cured microsphere suspension and wash 3 times, each time letting it stand for 30 minutes and then removing the supernatant until the supernatant is clear and free of impurities, and collect the microspheres; place the microspheres in a freeze dryer at -50℃ and freeze-dry for 24 hours to obtain cobalt cross-linked sodium alginate inhalable microspheres loaded with meropenem (denoted as SCM), and store them sealed at 4℃ protected from light.

[0032] Figure 1The image shows an optical image of the SCM microspheres prepared in this embodiment. It can be observed from the image that the microspheres are regular spherical, with uniform particle size distribution, no obvious agglomeration, and good dispersibility.

[0033] Figure 2 The scanning electron microscope (ZEISS Gemini SEM 300 or Hitachi Regulus 8100) images of the SCM microspheres prepared in this embodiment clearly show that the surface of the microspheres is smooth and the average particle size is about 4 μm, which is a micron-sized particle size suitable for deep deposition in the lungs.

[0034] Figure 3 This is an elemental mapping distribution diagram of the SCM microspheres prepared in this embodiment. The diagram shows that C, O, Na, Cl, and Co elements are uniformly distributed on the surface of the microspheres, proving that Co... 2+ The microspheres successfully crosslinked with sodium alginate, achieving effective meropenem loading. Testing showed that the meropenem loading efficiency of the SCM microspheres obtained in this embodiment was 3.1%, and the Co loading efficiency was 37.9%.

[0035] This embodiment also prepared a material loaded only with Co. 2+ Cobalt-crosslinked sodium alginate microspheres were used as a control: 20 mg of sodium alginate was added to 5 mL of deionized water and stirred at room temperature in the dark for 30 min to obtain an aqueous solution free of meropenem. All subsequent process parameters for oil phase solution preparation, microfluidic droplet preparation, crosslinking and curing, cyclohexane washing, and freeze-drying were consistent with those for SCM microsphere preparation, ultimately yielding a Co-loaded... 2+ Cobalt cross-linked sodium alginate microspheres (denoted as SC microspheres).

[0036] Example 2 This embodiment is used to detect the in vitro pH-responsive drug release characteristics of SCM microspheres: The SCM microspheres prepared in Example 1 were prepared into a 1 mg / mL suspension, and 1 mL of each suspension was placed into a dialysis bag (MWCO: 8-14 kDa). The dialysis bag was immersed in a centrifuge tube containing 20 mL of phosphate-buffered saline (PBS) solution at pH 6.2 (simulating the bacterial infection microenvironment) or 7.4 (simulating the normal physiological environment), and the tube was shaken at 100 rpm in a 37°C constant temperature shaker. Samples were taken at 1 h, 2 h, 4 h, 8 h, 12 h, 1 day, 3 days, and 7 days. The absorbance of the sample solution at 297 nm was measured using a UV spectrophotometer, and the drug release at different time points was calculated using the meropenem standard curve. Co was detected using ICP-MS. 2+ The release concentration was determined, and a release curve was plotted.

[0037] Figure 4The figures for meropenem release curves and Co from SCM microspheres under different pH conditions in this embodiment are shown. 2+ The release curves, as shown in the figure, reveal that SCM microspheres exhibit significant pH-responsive release characteristics: the drug release rate is significantly faster at pH 6.2, reaching over 80% within 2 hours and approaching 90% after 3 days; while at pH 7.4, the drug release rate is slower, with approximately 60% after 3 days. These results demonstrate that SCM microspheres can rapidly release drugs in the acidic microenvironment of lung infections, achieving targeted and precise drug delivery to the infection site, while maintaining a low drug release rate in normal physiological environments, reducing systemic exposure.

[0038] Example 3 This embodiment aims to evaluate the in vitro antibacterial activity of SCM microspheres against carbapenem-resistant Klebsiella pneumoniae producing different carbapenemases (KPC-2, NDM-1, OXA-48, and clinical CRKP): KPC-2, NDM-1, OXA-48, and clinical CRKP strains were inoculated into sterile TSB medium and cultured at 37°C and 200 r / min with shaking until the logarithmic growth phase. The cultures were then diluted with fresh TSB medium to a concentration of 1×10⁻⁶. 5 The bacterial suspension was prepared at CFU / mL. Experimental setups included a blank control group (Control), meropenem monotherapy group (MEM 6.2 μg / mL), SC microsphere group (200 μg / mL), and SCM microsphere group (containing 1 / 4 dose 50 μg / mL, 1 / 2 dose 100 μg / mL, and full dose 200 μg / mL SCM), with 3 replicates per group. 50 μL of bacterial suspension was added to each well of a 96-well plate, followed by equal volumes of sterile PBS (blank control), meropenem solution (final concentration 6.2 μg / mL), and SC microsphere solution (CFU / mL). 2+ Final concentration 75.8 μg / mL), SCM microsphere suspension (full dose group, final concentration corresponding to meropenem 6.2 μg / mL + Co) 2+ (75.8 μg / mL), after thorough mixing, incubated in a 37℃ incubator. OD values ​​were measured at 0, 2, 4, 6, 8, 10, 12, and 24 hours. 600 The absorption peaks were observed, and growth curves were plotted. At 12 h, the bacterial cultures of KPC-2 and NDM-1 Klebsiella pneumoniae were serially diluted, and 50 μL of each dilution was spread on TSB solid agar plates. After incubation at 37 °C for 24 h, colony counts were performed, and the relative survival rate of bacteria was calculated.

[0039] Figure 5The results of in vitro antibacterial experiments on SCM microspheres against KPC-2, NDM-1, OXA-48 and clinically multidrug-resistant CRKP strains include bacterial growth curves, plate colony photographs and quantitative statistical data.

[0040] (ad) Growth curve results showed that the bacteria in the blank control group grew vigorously, with OD... 600 The value rose rapidly; the meropenem (MEM) monotherapy group and the cobalt-only SC microsphere group could only mildly inhibit the growth of the strains, and the bacteria still showed a significant proliferation trend; while the SCM microsphere group could significantly delay and inhibit bacterial growth, and the antibacterial effect increased in a dose-dependent manner. The full-dose SCM group showed extremely strong growth inhibition on all strains, and the bacteria hardly proliferated within 24 hours.

[0041] (eh) The results of plate colony counting and quantitative statistics were consistent with the growth curve trend: the blank control group had the highest number of colonies; the MEM single drug group and SC microsphere group could only slightly reduce the number of colonies; the number of colonies in the SCM microsphere group decreased significantly with increasing dose, and the full-dose SCM group had almost no colony growth, with the decrease in bacterial count being much greater than that in each single drug group.

[0042] The above results confirm that Co 2+ Meropenem exhibits significant synergistic antibacterial effects against various carbapenem-resistant CRKP strains, with the strongest synergistic effect against NDM-1 strains (FICI=0.25). It also shows strong bactericidal effects against KPC-2, OXA-48, and clinically multidrug-resistant CRKP, fully demonstrating the broad-spectrum inhibitory ability of SCM microspheres against multiple types of drug-resistant CRKP.

[0043] Example 4 This embodiment aims to elucidate the molecular mechanism by which SCM microspheres reverse carbapenem-resistant Klebsiella pneumoniae (CRKP) resistance, and to evaluate their inhibitory effect on NDM-1 metallo-β-lactamase and their impact on bacterial metabolism.

[0044] Figure 6 In vitro experimental results regarding the mechanism of action of SCM microspheres: (a-b) NDM-1 enzyme activity inhibition and Zn 2+Supplementary validation: NDM-1 enzyme was extracted from NDM-1 type CRKP strain by centrifugation. Different materials were added, and enzyme activity was calculated by detecting the absorbance of meropenem hydrolysis products at 297 nm. The experiment included a blank control group (Control), SC microsphere group (200 μg / mL), and SCM microsphere group (containing 1 / 4 dose 50 μg / mL, 1 / 2 dose 100 μg / mL, and full dose 200 μg / mL SCM group). The experiment was divided into two groups: one group received meropenem directly, and the characteristic absorption peak at 297 nm was detected within 4 h to evaluate NDM-1 enzyme activity; the other group received an additional excess of Zn after 4 h of incubation. 2+ The characteristic absorption peak at 297 nm was also detected within 4 hours. The results showed that SCM microspheres significantly inhibited NDM-1 enzyme activity in a dose-dependent manner (Figure a); additional Zn... 2+ The inhibitory effect of SCM microspheres on NDM-1 enzyme can be partially reversed (Figure b), confirming that its inhibitory mechanism is related to Co. 2+ Zn at the active site of the replacement enzyme 2+ Directly related.

[0045] (c) Quantitative Detection by Ion Displacement: NDM-1 enzyme was extracted from NDM-1 bacteria by centrifugation. After co-incubation with different concentrations of SCM microspheres (0–100 μg / mL) for 12 h, unbound metal ions were dialyzed out using 8–14 kDa dialysis bags. The NDM-1 enzyme in the dialysis bags was then hydrolyzed, and the metal ion composition of the enzyme active site was analyzed by ICP-MS. The results showed that the Co released from the microspheres… 2+ Zn can significantly replace the enzyme's active site 2+ The replacement efficiency increases with increasing SCM concentration, providing direct evidence for the inactivation of NDM-1 enzyme.

[0046] (de) Detection of bacterial metabolism and energy levels: (1) Detection of siderophores: Klebsiella pneumoniae carrying NDM-1 was inoculated into iron-deficient M9 medium (containing M9 salt, 0.4% glucose, 2 mM MgSO4, 0.1 mM CaCl2, and no FeCl3). The glassware was soaked in 6 M HCl overnight to remove iron. The bacterial culture was adjusted to OD. 600After reaching a final concentration of 0.1, the solution was dispensed into smaller portions. The experimental groups were: blank control (PBS), meropenem monotherapy group (6.2 µg / mL), SC microsphere group (200 µg / mL), chitosan group (200 µg / mL), 1 / 4 SCM microsphere group (50 µg / mL), 1 / 2 SCM microsphere group (100 µg / mL), and full-dose SCM microsphere group (200 μg / mL), totaling 6 groups. Each group was incubated with an equal volume of bacterial suspension at 37℃ and 180 r / min for 24 h. The supernatant was filtered through a 0.22 μm filter and mixed with an equal volume of CAS detection solution (containing chromium azurite S, FeCl3, CTAB, pH 5.6). The mixture was incubated at room temperature in the dark for 30 min, and the OD was measured. 630 Relative siderophore yield = Control group OD 630 / Sample OD 630 (The higher the ratio, the more secretion); quantification was performed using the DFO standard curve, and the results were expressed as deferoxamine equivalents (μmol / L). (2) ATP content detection: KPC-2 type CRKP strain was cultured in TSB medium at 37℃ and 200 rpm to the logarithmic phase, and diluted with TSB to 1×10 5 CFU / mL. Grouping was the same as above, with 50 μL of bacterial culture added to each 1 mL system. After sealing, the mixture was incubated at 37℃ for 12 h. After incubation, the cells were centrifuged at 3000 rpm for 5 min, washed 2–3 times with PBS, and the ATP content of the bacteria was measured using an ATP assay kit. The results showed that SCM microsphere treatment significantly reduced the siderophore content and intracellular ATP level of CRKP, disrupting bacterial iron homeostasis and energy metabolism.

[0047] (fh) Transcriptome sequencing analysis: Transcriptome sequencing was performed on bacteria before and after treatment with SEM microspheres (100 μg / mL) to analyze differentially expressed genes and enriched pathways. GO functional enrichment and metabolic pathway analysis showed that SEM microsphere treatment significantly regulated CRKP metabolism, especially pathways related to organic acid catabolism and energy metabolism; differential gene volcano plots showed that bacterial Zn... 2+ / Fe 2+ Significant changes in the expression of transport-related genes (such as znuB and zntA) confirm that SCM microspheres can interfere with bacterial ion homeostasis and energy metabolism through a dual mechanism, thereby achieving a broad-spectrum synergistic bactericidal effect against different carbapenemases CRKP.

[0048] Example 5 This embodiment tests the in vivo therapeutic effect of SCM microspheres on NDM-1 type CRKP pneumonia mice: Forty Balb / C mice aged 6-8 weeks were randomly divided into 6 groups of 8 mice each: Blank control group, Control group, Sodium alginate (SA) group, Meropenem (MEM) group, and Cobalt chloride hexahydrate group. 2+ The mice were divided into two groups: the control group and the SCM microsphere group. Except for the blank control group, all other groups of mice received 50 μL of a 1×10⁻⁶ microsphere solution via intratracheal infusion. 8 A mouse model of CRKP pneumonia was established using NDM-1 type CRKP bacterial suspension at CFU / mL. One hour after modeling, mice were divided into three groups: sodium alginate (SA) group (200 μg / mouse), MEM group (6.4 μg / mouse), and groups treated with cobalt chloride hexahydrate and Co. 2+ Group (dosage Co) 2+ Mice in the 75.8 μg / mouse group and the SCM microsphere group (200 μg / mouse) were nebulized with the corresponding microsphere suspensions, while the blank control group and the model group were nebulized with an equal volume of sterile PBS. The drugs were administered once a day for 7 consecutive days.

[0049] During the experiment, the survival status and weight changes of the mice were recorded daily. After 12 hours of treatment, some lung tissue was taken from the mice to detect bacterial load. The results showed that the bacterial load in the lung tissue of the SCM microsphere group was reduced by more than 95% compared with the model group, and the early bactericidal effect was significant. After 7 days of treatment, the surviving mice in each group were sacrificed, and the lung tissue was aseptically harvested. Some lung tissue was used for pathological section preparation, and HE staining was performed to observe the pathological damage of the lung tissue. Immunohistochemical staining was used to detect the expression level of the inflammatory factor TNF-α.

[0050] Figure 7 The following are the survival curves and weight changes of CRKP pneumonia mice in each treatment group in this embodiment: (a) The survival curves show that the 7-day survival rate of mice in the Control group was 62.5% (5 / 8), the survival rate of mice in the SA group and MEM group was slightly improved (6 / 8), while the Co 2+ The 7-day survival rate of mice in the control group and the SCM microsphere group was 100% (8 / 8), and the difference was statistically significant (p<0.01). (bc) The weight change curves showed that mice in all groups experienced varying degrees of weight loss after infection. Among them, the control group and the SA and MEM single-drug groups had a large weight loss and slow recovery, while the SCM microsphere group had the smallest weight loss and the fastest recovery, which was consistent with the survival curve results. This proves that SCM microspheres can significantly improve the survival rate of CRKP pneumonia mice and have a significant therapeutic effect.

[0051] Figure 8The images show HE staining and Ki67 staining of mouse lung tissue from the Blank group, Control group at different time points, and SCM group in this embodiment. As can be seen from the images, the lung tissue of the blank control group mice was structurally intact, without inflammatory infiltration, and Ki67-positive cells were rare, indicating extremely low cell proliferation activity in the lung tissue. The model group mice showed severe alveolar septal thickening, extensive inflammatory cell infiltration, and severe damage to the lung tissue structure. Simultaneously, the number of Ki67-positive cells increased significantly, especially in the inflammatory infiltration areas and around the alveolar septa, suggesting abnormal and active cell proliferation after injury. In the SCM microsphere group mice, the alveolar septa of the lung tissue were basically restored to normal, inflammatory cell infiltration was significantly reduced, the lung tissue structure was intact, and the number of Ki67-positive cells decreased significantly to levels close to the blank control group. This indicates that SCM microspheres can effectively inhibit excessive cell proliferation, promote tissue homeostasis recovery, and thus alleviate lung pathological damage caused by CRKP pneumonia.

[0052] Figure 9 This image shows staining data for TNF-α, IL-10, iNOS, ARG-1, and PD-1 in the lung tissues of mice in the Blank group, Control group at different time points, and SCM group in this embodiment. The images show that all five indicators were expressed at low levels in the lung tissue of the blank control group mice, indicating that the lungs were in an immune homeostasis state. In the lung tissue of the model group mice, the positive expression of TNF-α, iNOS, and PD-1 gradually increased from 0.5 days after infection, reaching a significant level on day 3, suggesting a pro-inflammatory response and M1 macrophage activity. The occurrence of cell polarization and T cell exhaustion, along with the initial increase and subsequent decrease of IL-10 and ARG-1, indicates that endogenous anti-inflammatory and repair mechanisms are insufficient to inhibit the progression of inflammation. The expression of TNF-α, iNOS, and PD-1 in the lung tissue of mice in the SCM microsphere group was significantly lower than that in the model group at all time points, while IL-10 and ARG-1 remained moderately elevated. This suggests that SCM microspheres can effectively inhibit excessive inflammatory response, promote the M2 anti-inflammatory repair phenotype, and reduce immune exhaustion, thereby synergistically alleviating the immunopathological damage caused by CRKP pneumonia.

[0053] Figure 10 HE staining of the main organ sections of mice treated with SCM microspheres in the Blank group in this embodiment, including the heart, liver, spleen, lung, and kidney. As can be seen from the figure, the material does not cause significant organ damage in mice and has good biocompatibility.

[0054] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing cobalt-crosslinked sodium alginate inhalable microspheres loaded with meropenem, characterized in that, Includes the following steps: Meropenem and sodium alginate were added to deionized water and stirred at room temperature until completely dissolved to obtain aqueous solution A; cobalt salt was added to the oil phase and ultrasonically dispersed to obtain oil solution B. The aqueous solution A is pumped into the middle channel of the microfluidic chip, and the pure oil phase is pumped into the channels on both sides of the chip. The chip outlet is immersed in oil phase solution B, and the aqueous phase and oil phase form monodisperse water-in-oil droplets at the chip junction through flow focusing shearing. The oil phase solution B containing droplets was placed in a room temperature, light-protected environment for cross-linking and curing. After washing with cyclohexane and freeze-drying, cobalt-crosslinked sodium alginate inhalable microspheres loaded with meropenem were obtained.

2. The preparation method according to claim 1, characterized in that: In the aqueous solution A, the mass ratio of meropenem to sodium alginate is 1:2 to 4.

3. The preparation method according to claim 1, characterized in that: The mass concentration of cobalt salt in the oil phase solution B is 7–9 mg / mL.

4. The preparation method according to claim 1, characterized in that: The oil phase is at least one of the following: droplet-forming oil phase, cyclohexane, liquid paraffin, and mineral oil.

5. The preparation method according to claim 1, characterized in that: The microfluidic chip has a channel size of 20μm to 200μm; the flow rate of the aqueous solution A is 10 to 20μL / min, and the flow rate of the pure oil phase is 50 to 70μL / min.

6. The preparation method according to claim 1, characterized in that: The crosslinking and curing conditions are: standing at room temperature in the dark for 10–16 hours, allowing sodium alginate and Co to react. 2+ Fully cross-linked to form a stable microsphere structure; the freeze-drying conditions are: temperature -45 to -55℃, vacuum degree ≤10Pa, time 20 to 28h.

7. Meropenem-loaded cobalt-crosslinked sodium alginate inhalable microspheres prepared by the preparation method according to any one of claims 1 to 6.

8. The inhalable microspheres according to claim 7, characterized in that: The inhalable microspheres have a particle size of 2-5 μm.

9. The inhalable microspheres according to claim 7, characterized in that: The inhalable microspheres have a ferropenem loading efficiency of 2-8% and a Co loading efficiency of 30-40%.

10. An application of the inhalable microspheres according to claim 7, 8 or 9, characterized in that: The microspheres are used to prepare formulations for treating lung infections caused by carbapenem-resistant Klebsiella pneumoniae.

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