An inhalable microsphere loaded with meropenem and EDTA and its application in the treatment of NDM-1 type pneumonia

Abstract

This invention discloses an inhalable microsphere loaded with meropenem and EDTA and its application in the treatment of NDM-1 type pneumonia. The microspheres are prepared via an emulsification cross-linking process and can be delivered to the lungs via aerosol atomization. They also utilize the responsive characteristics of the infection microenvironment for intelligent drug release within the infection microenvironment. This formulation exhibits good biocompatibility, synergistically inactivating NDM-1 enzymes, disrupting bacterial biofilms, killing drug-resistant bacteria, and moderately regulating the inflammatory microenvironment of the lungs. It demonstrates a long-lasting targeted therapeutic effect against NDM-1 resistant Klebsiella pneumoniae pneumonia, exhibiting excellent in vitro and in vivo therapeutic efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and anti-infective therapy technology, specifically relating to an inhalable microsphere loaded with meropenem and EDTA and its application in the treatment of NDM-1 pneumonia. Background Technology

[0002] Antimicrobial resistance (AMR) has become one of the most serious challenges in global public health. The incidence and mortality rates of drug-resistant bacterial infections are rising year by year, putting unprecedented pressure on clinical anti-infective treatment. Among the many drug-resistant Gram-negative bacteria, carbapenem-resistant Klebsiella pneumoniae (CRKP) has become the most threatening pathogen in nosocomial and community-acquired infections due to its high resistance to carbapenems, the severity of infection, and the extreme lack of treatment options. Among them, New Delhi metallo-β-lactamase (NDM-1)-producing Klebsiella pneumoniae is the most typical and widespread subtype of CRKP. This enzyme can efficiently hydrolyze almost all clinically used β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems, directly causing the complete ineffectiveness of first-line carbapenem drugs such as meropenem, leaving these infections in a desperate situation of "no drugs available".

[0003] Even more challenging is that drug-resistant bacteria producing NDM-1 enzymes readily form dense and highly stable bacterial biofilms after invading the lungs. These biofilms not only act as physical barriers, preventing antibiotics from penetrating the bacterial cells, but also induce a metabolic dormancy state, significantly reducing their sensitivity to antimicrobial drugs. Simultaneously, they protect the bacteria from the host's immune system's recognition and clearance, leading to chronic infections, recurrent infections, refractory pneumonia, and severe pneumonia. Conventional anti-infective treatments are often completely ineffective against these biofilm-related infections.

[0004] Currently, the clinical treatment of lung infections caused by NDM-1 enzyme-resistant bacteria mainly relies on "last-line" antibiotics such as polymyxin and tigecycline. However, these drugs have limited efficacy when used alone or in combination, and are accompanied by significant nephrotoxicity, neurotoxicity, and heterogeneous drug resistance, which severely limits their long-term and high-dose use in clinical practice.

[0005] Meropenem (MEM), a broad-spectrum, highly effective, and low-toxicity carbapenem antibiotic, exhibits strong bactericidal activity against most Gram-negative bacteria, but it is completely ineffective against NDM-1 enzyme-producing strains and cannot be directly used to treat such infections. Ethylenediaminetetraacetic acid (EDTA), a highly effective metal ion chelator, has been shown in recent studies to specifically chelate Zn, an essential element for the active site of the NDM-1 enzyme. 2+ This disrupts the enzyme's spatial structure and eliminates its ability to hydrolyze antibiotics, thereby restoring meropenem's antibacterial activity; simultaneously, EDTA can also chelate Ca2+, which maintains the stability of biomembrane structures.2+ Mg 2+ Divalent cations disrupt the extracellular matrix structure of biomembranes, breaking down biomembrane barriers and significantly enhancing the bactericidal effect of antibiotics on bacteria encapsulated within membranes. However, when free EDTA is directly delivered to the lungs, it can cause strong irritation to the respiratory tract and lung tissue, easily inducing systemic toxicity reactions such as hypocalcemia. Furthermore, it suffers from problems such as insufficient local drug concentration in the lungs, rapid metabolism, and short retention time, severely hindering its clinical translation and application.

[0006] During the immune regulation process of bacterial infection, the lung's innate immune system can initiate an anti-infection immune response through macrophage polarization and T cell and NK cell activation. Studies have shown that effectively clearing infection and suppressing excessive inflammatory responses requires the simultaneous achievement of bacterial eradication, biofilm disruption, and immune microenvironment remodeling. However, existing drug delivery systems struggle to achieve targeted co-delivery of EDTA and meropenem to the lungs, failing to synergistically inactivate drug-resistant bacteria, clear biofilms, and regulate pulmonary immune balance.

[0007] In recent years, biodegradable polymeric microspheres have provided a new direction for targeted anti-infection treatment of the lungs. Gelatin microspheres, as natural polymeric carriers, possess excellent biocompatibility, in vivo degradability, high drug loading flexibility, and advantages in lung aerosol deposition. They can achieve drug enrichment, retention, and controlled release at the site of infection, effectively solving the problems of low delivery efficiency, high toxicity, and insufficient local concentration of free drugs. Based on this, a gelatin microsphere delivery system co-loaded with EDTA and meropenem, possessing pH-responsive controlled-release characteristics in the infection microenvironment, was constructed. This system simultaneously achieves NDM-1 enzyme inhibition, biofilm disruption, synergistic bactericidal activity, and remodeling of the lung immune microenvironment. This has crucial theoretical significance and clinical application value for overcoming NDM-1-resistant pneumonia, clearing refractory pulmonary biofilm infections, and improving the efficacy of clinical anti-infective treatments. Summary of the Invention

[0008] This invention provides an inhalable microsphere loaded with meropenem and EDTA, its preparation method, and its application, aiming to solve the technical problems in the prior art, such as the lack of effective treatment for NDM-type drug-resistant bacterial infections, poor safety of EDTA delivery, difficulty in clearing biofilms, and imbalance of the pulmonary immune microenvironment.

[0009] To solve the technical problem, the present invention adopts the following technical solution: A method for preparing inhalable microspheres loaded with meropenem and EDTA includes the following steps: mixing meropenem, EDTA, gelatin and deionized water, stirring until completely dissolved to obtain an aqueous solution; mixing liquid paraffin with Span-80, and homogenizing at high speed until the system is uniform to obtain an oil solution; slowly adding the aqueous solution dropwise into the oil solution, and continuing to homogenize at high speed for emulsification; subsequently adding a crosslinking agent solution dissolved in dimethyl sulfoxide, and stirring at low speed for crosslinking reaction; after the reaction is completed, washing with n-hexane to remove unencapsulated free drug, and then freeze-drying to obtain inhalable microspheres loaded with meropenem and EDTA.

[0010] Preferably, in the aqueous solution, the mass ratio of gelatin, EDTA, and meropenem is 200:10-20:10-20.

[0011] Preferably, in the oil phase solution, the volume ratio of liquid paraffin to Span-80 is 40-50:8-10, which can construct a stable emulsion system to ensure the uniformity of microsphere molding.

[0012] Preferably, the high-speed homogenizing stirring speed is 10,000 to 15,000 rpm, the emulsification treatment time is 20 to 40 min, the crosslinking reaction stirring speed is 500 to 1,000 rpm, the reaction time is 12 to 24 h, and the reaction temperature is room temperature.

[0013] Preferably, the crosslinking agent is any one of genipin, glutaraldehyde, and calcium chloride, which has high biosafety.

[0014] The inhalable microspheres prepared by this invention have a particle size of 2-5 μm and excellent responsive drug release performance. They can rapidly release drugs in sputum and achieve targeted and controllable drug release at the lesion site.

[0015] These inhalable microspheres effectively inactivate NDM-1 metallo-β-lactamases, reversing the drug resistance characteristics of resistant bacteria. Simultaneously, they efficiently dismantle the dense biofilm barrier formed by resistant bacteria, relieving biofilm-mediated drug resistance and persistent infection. Furthermore, these microspheres effectively reshape the pulmonary immune microenvironment for infection by promoting macrophage polarization towards the M1 type, upregulating IFN-γ secretion levels, activating the body's innate local anti-infective immune response, and synergistically enhancing the body's ability to clear drug-resistant bacteria from the lungs.

[0016] The inhalable microspheres containing meropenem and EDTA described in this invention can be used to prepare targeted therapeutic drugs for treating pneumonia caused by NDM-1 enzyme-resistant bacteria and pulmonary bacterial biofilm infections. The drug can be administered via aerosol nebulization, which can directly act on the pulmonary lesions, achieving precise local drug delivery, improving therapeutic efficacy, and reducing systemic toxic side effects.

[0017] Compared with existing technologies, the beneficial effects of this invention are reflected in: 1. This invention is the first to construct an inhalable microsphere delivery system for genipin-crosslinked gelatin co-loaded with meropenem and EDTA. It is prepared by an emulsification crosslinking process. The overall process is simple and controllable, the reaction conditions are mild, and no complex equipment is required. The prepared microspheres have regular morphology, uniform particle size, good reproducibility, and low preparation cost, and have good prospects for industrial scale-up.

[0018] 2. The microspheres of this invention have good biocompatibility and significant drug release characteristics in response to the infection microenvironment. They can accelerate drug release in the acidic microenvironment of lung infection and slowly release drugs in the normal physiological environment. Combined with aerosol nebulization inhalation, they can achieve targeted deposition and localized controlled drug release in lung lesions, effectively increase the drug concentration in the lesions, reduce systemic drug exposure, and have significantly better drug safety and targeting than free drugs.

[0019] 3. This invention utilizes EDTA continuously released from microspheres to precisely chelate Zn in the active center of the NDM-1 enzyme. 2+ It disrupts the spatial conformation of enzymes and completely inactivates them, effectively reversing the resistance of drug-resistant bacteria to carbapenem antibiotics; at the same time, EDTA can chelate Ca, which maintains the stability of biofilm structure. 2+ Mg 2+ Divalent cations disrupt the extracellular matrix structure of bacterial biofilms, break down the physical barriers of biofilms, and achieve dual and efficient removal of drug-resistant bacteria and stubborn biofilms.

[0020] 4. This invention restores the bactericidal activity of meropenem through EDTA, achieving a synergistic antibacterial effect of "enzyme inhibition-membrane disruption-bactericidal action"; at the same time, it can effectively regulate the lung infection microenvironment, promote macrophage polarization towards the M1 type, increase the secretion level of IFN-γ cytokines in the lungs, activate the local innate immune response, and enhance the body's own anti-infection ability, thus synergistically achieving antibacterial and bactericidal action and immune microenvironment remodeling, providing a new, efficient, and safe treatment strategy for clinically refractory NDM-1 type drug-resistant bacterial lung biofilm infections. Attached Figure Description

[0021] Figure 1 The physicochemical characterization results of the GEM microspheres prepared in Example 1 are shown, including: (A, B) Scanning electron microscope (SEM) images at different magnifications; (C) Particle size distribution and Zeta potential diagram; (D) X-ray diffraction (XRD) pattern.

[0022] Figure 2 The in vitro drug release curves of the GEM microspheres prepared in Example 1 in different media (PBS and artificial sputum) are shown, including: (A) Meropenem (MEM) release curve; (B) EDTA release curve.

[0023] Figure 3The results of the synergistic antibacterial experiment of ferropenem and EDTA in Example 2 are as follows: (A) Antibacterial thermogram of checkerboard dilution method; (B) Colony photograph of plate coating of key concentration combination; (C) Synergistic curve of fractionated inhibitory concentration index (FICI).

[0024] Figure 4 The in vitro killing effect of GEM microspheres and control materials on NDM-1 enzyme-producing Klebsiella pneumoniae in Example 2 is shown in the following figures: (A) bacterial growth curves of different treatment groups; (B) thermogram of antibacterial effect at 12 h; and (C) photographs of colonies on plate at key time points.

[0025] Figure 5 The results show the inhibitory effect and molecular mechanism of GEM microspheres on NDM-1 enzyme, including: (A) Endogenous Zn in NDM-1 enzyme after treatment with different concentrations of GEM microspheres. 2+ (B) Changes in content; (C) Time-dependent activity curve of NDM-1 enzyme hydrolyzing meropenem under GEM microsphere intervention; (D) Supplementation of exogenous Zn 2+ The activity curve of NDM-1 enzyme after exogenous Zn supplementation; (D) for exogenous Zn supplementation 2+ (E) Statistical results of NDM-1 enzyme activity after successive passages; (F) MIC fluctuations of the strain to meropenem after successive passages; (G) Volcano plot of differentially expressed genes in bacteria; (G) GO functional enrichment and KEGG pathway enrichment results of differentially expressed genes.

[0026] Figure 6 The in vivo treatment effect of the mouse NDM-1 pneumonia model in Example 4 was evaluated, including: (A) survival curves of mice in each group within 7 days; (B) weight change curves of mice in each group; and (C) weight change trajectory of individual mice.

[0027] Figure 7 The results of pathological examination and immunohistochemical staining of mouse lung tissue in Example 4 are as follows: (A) HE-stained lung tissue section; (B) IFN-γ immunohistochemical staining results; (C) IL-6 immunohistochemical staining results; (D) TNF-α immunohistochemical staining results.

[0028] Figure 8 The images show HE staining results of tissue sections from the heart, liver, spleen, lung, and kidney of healthy mice after administration of GEM microspheres in Example 4. Detailed Implementation

[0029] 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.

[0030] Example 1 In this embodiment, inhalable microspheres loaded with meropenem and EDTA were prepared according to the following steps: Accurately weigh 200 mg of gelatin, 10 mg of EDTA, and 20 mg of meropenem trihydrate, add them together to 5 mL of deionized water, and stir at room temperature until completely dissolved to obtain a homogeneous and transparent aqueous solution.

[0031] Mix 50 mL of liquid paraffin with 8 mL of Span-80 in a beaker and homogenize using a high-speed homogenizer at 10,000 rpm for 5 min to obtain a homogeneous and stable oil phase system.

[0032] The aqueous solution was slowly added dropwise to the oil phase system, and high-speed homogenization and emulsification was continued at 10,000 rpm for 20 min to form a homogeneous and stable oil-water emulsion. Then, 5 mg of genipin was weighed and fully dissolved in 1.5 mL of dimethyl sulfoxide, and slowly added dropwise to the emulsion system. The mixture was stirred at a low speed of 500 rpm for 12 h at room temperature to complete the crosslinking reaction.

[0033] After the reaction was completed, the product was washed multiple times with n-hexane to remove unloaded free drug. After freeze-drying at -40℃ for 24 h, genipin crosslinked gelatin microsphere powder loaded with meropenem and EDTA was obtained (denoted as GEM).

[0034] The morphology of the prepared GEM microspheres was observed using a Carl Zeiss Gemini SEM 300 scanning electron microscope. Figure 1 As can be seen from (A) and (B), the prepared microspheres are generally spherical, have good dispersibility, no obvious agglomeration, smooth surface, and regular shape, indicating that the emulsification crosslinking process can effectively prepare microsphere carriers with uniform morphology.

[0035] The hydrated particle size of GEM microspheres was analyzed using a 90 Plus PALS particle size analyzer, and the results are as follows: Figure 1 As shown in (C), the microspheres exhibit a unimodal normal distribution in particle size, with a hydrated particle size of approximately 2.2 μm. This concentrated distribution meets the particle size requirements (2-5 μm) for nebulized inhalation administration, facilitating effective deposition in lung lesions. The zeta potential of the microspheres is -16.34 mV, indicating good colloidal stability of the system.

[0036] The crystal structure of GEM microspheres was analyzed using a DX-2700BH X-ray diffractometer, and the results are as follows: Figure 1 As shown in (D), within the diffraction angle range of 10° to 90°, the GEM microspheres did not exhibit obvious characteristic lattice diffraction peaks, but only a broadened diffuse peak, indicating that the prepared microspheres have an amorphous structure. This suggests that the drug is dispersed in the gelatin matrix in an amorphous or molecular state without crystallization, which is beneficial for improving drug dissolution and release performance.

[0037] Figure 2 The images show the in vitro drug release curves of GEM microspheres in different media (PBS and artificial sputum), where (A) is the meropenem (MEM) release curve and (B) is the EDTA release curve. The GEM microspheres prepared in this invention exhibit sustained drug release behavior in both PBS and artificial sputum media. Compared to PBS, the release rate of meropenem and EDTA from the microspheres is significantly accelerated in artificial sputum media, with the cumulative drug release rate within 24 hours being significantly higher than that in the PBS group. These results indicate that GEM microspheres possess good drug release characteristics in response to the infection microenvironment. They can accelerate drug release in the sputum microenvironment of lung infection lesions, while the release is relatively gradual in normal physiological environments, which is beneficial for achieving targeted and controllable drug release, increasing local drug concentration, and reducing the risk of systemic exposure.

[0038] Comparative Example 1 This comparative example prepared single-drug-loaded gelatin microspheres containing only meropenem. Except for the absence of EDTA, all other raw material ratios, process parameters, and preparation steps were completely consistent with those in Example 1, and were used for comparative analysis of single-drug-loaded systems.

[0039] Comparative Example 2 This comparative example prepared single-drug-loaded gelatin microspheres containing only EDTA. Except for the absence of meropenem trihydrate, all other raw material ratios, process parameters, and preparation steps were completely consistent with those in Example 1, and were used for comparative analysis of single-drug-loaded systems.

[0040] Example 2 This embodiment evaluates the in vitro antibacterial activity of meropenem combined with EDTA and GEM microspheres.

[0041] (I) Experiment 1: Evaluation of the synergistic antibacterial effect of meropenem and EDTA using the checkerboard dilution method This section uses the checkerboard dilution method to evaluate the synergistic antibacterial effect of meropenem (MEM) and EDTA in combination against NDM-1 enzyme-producing Klebsiella pneumoniae, calculates the minimum inhibitory concentration (MIC) and fractionated inhibitory concentration index (FICI), and verifies the synergistic antibacterial effect of the two.

[0042] (1) Experimental method: Frozen NDM-1 enzyme-producing Klebsiella pneumoniae was inoculated into sterile TSB liquid medium and cultured in a constant temperature shaker at 37℃ and 200 rpm until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶. 5 CFU / mL available for use.

[0043] EDTA stock solution and meropenem stock solution with a concentration of 256 μg / mL were prepared separately. After filtration and sterilization, they were serially diluted with TSB medium to obtain a series of working solutions with different concentrations: EDTA (final concentration 1-64 μg / mL) and meropenem (final concentration 1-128 μg / mL).

[0044] A checkerboard layout was used to conduct a combined antimicrobial susceptibility test in a 96-well plate: 200 μL of a mixture of material and bacteria was added to each well, resulting in a final bacterial concentration of 5 × 10⁻⁶. 3 CFU / mL; a control group (bacteria + culture medium) and a single-drug control group (EDTA only or meropenem only) were also set up. After sealing, the mixture was incubated at 37°C for 12 h, and the OD of each well was measured using a microplate reader. 600 The value was measured, and the turbidity inside the well was observed with the naked eye, with no obvious bacterial growth and OD value. 600 The lowest drug concentration significantly lower than that of the bacterial control group was determined as the MIC value, and the FICI index was calculated.

[0045] (2) Results Figure 3 The results of the checkerboard dilution method combined with antibacterial experiments include: (A) antibacterial heatmap (the darker the color, the more obvious the bacterial growth; the lighter the color, the stronger the antibacterial effect); (B) plate swab photographs of key concentration combinations ("x:y" represents the concentration of meropenem:EDTA, in μg / mL); and (C) FICI index calculation results.

[0046] The results show that: When meropenem was used alone (EDTA=0 μg / mL), bacteria still grew extensively at a higher test concentration (64 μg / mL), while at the highest test concentration (128 μg / mL), the bacteria were almost completely eliminated, indicating that the NDM-1 enzyme-producing strain was highly resistant to meropenem, with a single-drug MIC of 128 μg / mL.

[0047] When EDTA was used alone (meropenem = 0 μg / mL), it did not show a significant antibacterial effect even at the highest concentration (64 μg / mL), with a single-drug MIC > 64 μg / mL.

[0048] The two drugs showed a significant synergistic effect when used together: when meropenem concentration was ≥2 μg / mL and EDTA concentration was ≥4 μg / mL, bacterial growth was completely inhibited without visible turbidity or colony formation; when the concentration was 2:4 or higher, the antibacterial effect increased with increasing drug concentration.

[0049] The FICI of this strain was calculated as follows: FICI = FIC(MEM) + FIC(EDTA) = (2 / 128) + (4 / 128) = 0.046875 (<0.1). The judgment criteria are as follows: FICI ≤ 0.5 indicates synergistic effect, 0.5 < FICI ≤ 1 indicates additive effect, 1 < FICI ≤ 2 indicates no relevant effect, and FICI > 2 indicates antagonistic effect.

[0050] FICI < 0.1, which confirmed that the combination of meropenem and EDTA had a significant synergistic antibacterial effect. The mechanism was that EDTA chelated Zn at the active center of the NDM-1 enzyme 2+ , inactivated the enzyme, restored the antibacterial activity of meropenem against Klebsiella pneumoniae producing NDM-1 enzyme, and achieved the synergistic effect of "enzyme inhibition + antibiotic bactericidal".

[0051] (2) Experiment 2: In vitro killing effect of GEM microspheres on Klebsiella pneumoniae producing NDM-1 enzyme In this part, the in vitro killing activity and dose-dependence of GEM microspheres on Klebsiella pneumoniae producing NDM-1 enzyme were verified through bacterial growth curve and plate coating experiments.

[0052] (1) Experimental method A control group (Control), a single-drug EDTA group (8 μg / mL), a single-drug meropenem group (6.4 μg / mL), a 1 / 4-dose GEM group (25 μg / mL), a 1 / 2-dose GEM group (50 μg / mL), and a full-dose GEM group (100 μg / mL) were set up and co-incubated with Klebsiella pneumoniae producing NDM-1 enzyme in the logarithmic growth phase. The OD 600 value was measured at 0, 2, 4, 6, 8, 10, 12, and 24 h respectively to draw the bacterial growth curve; and plate coating experiments were carried out at 0, 2, 4, 8, and 12 h to observe the change in the number of viable bacteria.

[0053] (2) Results Figure 4 For the comparison of in vitro antibacterial effects of different treatment groups, including: (A) bacterial growth curve; (B) heat map of antibacterial effect at 12 h; (C) photos of colonies on the plate at the key time points.

[0054] The results showed that: The growth of bacteria in the control group, the single-drug EDTA group, the single-drug meropenem group, and the 1 / 4-dose GEM group was not significantly inhibited, and the OD 600 value continued to rise with time, and the colonies on the plate were dense; The growth of bacteria in the 1 / 2-dose GEM group was significantly inhibited, the OD 600 value increased slowly, and the number of colonies on the plate decreased significantly; The OD of the full-dose GEM group600 The value remained at an extremely low level, with no obvious colony growth on the plates at any time point, demonstrating the strongest antibacterial effect and showing a clear dose-dependent effect.

[0055] Example 3 This embodiment aims to evaluate the in vitro enzyme activity of GEM microspheres against NDM-1-producing carbapenem-resistant Klebsiella pneumoniae: (1) Experimental methods NDM-1 enzyme was extracted from NDM-1 bacteria by centrifugation. After co-incubation with different concentrations of GEM microspheres (0, 5, 10, 20, 40, 80 μg / mL) for 12 h, unbound metal ions were dialyzed off using 8-14 kDa dialysis bags. The NDM-1 enzyme in the dialysis bags was then hydrolyzed, and the Zn content in the system was determined using inductively coupled plasma (ICP) assay. 2+ Content, calculation of Zn 2+ Chelation rate; NDM-1 enzyme was extracted from NDM-1 bacteria by centrifugation. After mixing with different concentrations of GEM microspheres (25, 50, 100 μg / mL) or EDTA solution (8 μg / mL), the following assays were performed: The characteristic absorption peak at 297 nm was detected within 4 hours after the addition of meropenem, and the NDM-1 enzyme activity was calculated. After incubation for 4 hours following mixing, an excess of Zn was added. 2+ The characteristic absorption peak at 297 nm was detected within 4 hours to verify Zn. 2+ The direct association between chelation and enzyme activity inhibition was investigated. Transcriptome sequencing was performed on bacteria before and after treatment with GEM microspheres (80 μg / mL) to analyze differentially expressed genes and enrichment pathways.

[0056] (2) Results Figure 5 The results show the inhibitory effect and molecular mechanism of GEM microspheres on NDM-1 enzyme, including: (A) Endogenous Zn in NDM-1 enzyme after treatment with different concentrations of GEM microspheres. 2+ (A) Changes in content; (B) Time-dependent activity curve of NDM-1 enzyme hydrolyzing meropenem under GEM microsphere intervention; (C)-(D) Activity curves and statistical results of NDM-1 enzyme under GEM microsphere treatment after zinc ion supplementation; (E) MIC fluctuations of the strain after continuous subculturing; (F) Volcano plot of differentially expressed genes in bacteria; (G) GO functional enrichment and KEGG pathway enrichment results of differentially expressed genes.

[0057] The results show that: 1. Zn 2+ Chelation ( Figure 5 A): As the concentration of GEM microspheres increases, the Zn content in the NDM-1 enzyme increases. 2+The content decreased significantly, and more than 75% of Zn could be chelated at a concentration of 30 μg / mL. 2+ This indicates that GEM microspheres can effectively deprive the NDM-1 enzyme active site of essential Zn. 2+ .

[0058] 2. Enzyme activity inhibition effect ( Figure 5 (B, C, D): After treatment with GEM microspheres, the hydrolytic activity of NDM-1 enzyme against meropenem decreased rapidly over time in a significant dose-dependent manner. The residual enzyme activity in the full-dose GEM group was less than 30%, significantly lower than that in the single-drug EDTA group and the control group, confirming that GEM microspheres can effectively inhibit NDM-1 enzyme activity.

[0059] 3. Reversing drug resistance ( Figure 5 E): In the continuous passage experiment, the MIC of meropenem in the control group NDM-1 bacteria increased rapidly with the number of passages, while the MIC of the GEM microsphere treatment group remained at a low level, indicating that GEM microspheres can effectively reverse bacterial resistance to meropenem and are not likely to induce new resistance mutations.

[0060] 4. Transcriptome analysis ( Figure 5 F, G): Volcano plots showed that GEM microsphere treatment significantly downregulated genes related to NDM-1 enzyme expression, biofilm formation, and metal ion transport. GO and KEGG enrichment analyses further indicated that GEM microspheres could significantly inhibit bacterial transmembrane transport, metal ion homeostasis regulation, and secretion system-related pathways, revealing their mechanism of action in inhibiting drug resistance and biofilm formation at the molecular level.

[0061] In summary, GEM microspheres can chelate Zn from the active center of the NDM-1 enzyme. 2+ This disrupts the enzyme's spatial conformation and eliminates its ability to hydrolyze meropenem, thereby restoring meropenem's antibacterial activity and achieving synergistic bactericidal effects. Simultaneously, it can regulate bacterial-related pathways and inhibit the further development of drug resistance.

[0062] Example 4 This embodiment uses a mouse NDM-1 pneumonia model to evaluate the in vivo therapeutic effect, lung tissue pathological improvement, and biosafety of GEM microsphere aerosol nebulization drug delivery.

[0063] (1) Experimental methods Healthy mice were randomly divided into a blank control group, a model group, and a GEM treatment group, with 8 mice in each group. NDM-1-producing Klebsiella pneumoniae was cultured to the logarithmic growth phase, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; simultaneously, GEM microsphere atomized suspension was prepared.

[0064] Mice in the model group and GEM treatment group underwent endotracheal intubation and were injected with 50 μL of bacterial suspension to establish an NDM-1 pneumonia model; mice in the blank control group were injected with an equal volume of sterile PBS. One hour after modeling, the GEM treatment group was treated with GEM microsphere (100 μg) suspension aerosol nebulization; the blank control group and model group were treated with an equal volume of sterile saline nebulization. Subsequently, the survival status and weight changes of mice in each group were observed and recorded at fixed times every day for 7 consecutive days. After the experiment, lung tissue was collected for HE staining and immunohistochemical analysis of inflammatory factors. At the same time, major organs such as heart, liver, spleen, and kidney were collected for HE staining to evaluate biosafety.

[0065] (2) Results Figure 6 Survival curves and body weight changes in a mouse NDM-1 pneumonia model: (A) Survival curves of mice in each group over 7 days; (B) Body weight change curves of mice in each group; (C) Individual mouse body weight change trajectory. Results showed: The model group mice had a high mortality rate and a significantly lower survival rate within 7 days than the GEM treatment group; while the survival rate of the GEM treatment group mice was close to that of the blank group, indicating that GEM microspheres can effectively control lung infection and reduce infection-induced mortality.

[0066] The mice in the model group experienced a continuous decrease in body weight, exhibiting a clear state of infectious wasting. The mice in the GEM treatment group only experienced slight fluctuations in body weight at the beginning of treatment, followed by a rapid recovery and gradual return to near-normal levels. This indicates that GEM microspheres can effectively control infection, reduce lung damage, and promote the recovery of the body's condition.

[0067] Figure 7 Immunohistochemical analysis of mouse lung tissue pathology and inflammatory factors: (A) HE staining; (B) IFN-γ immunohistochemistry; (C) IL-6 immunohistochemistry; (D) TNF-α immunohistochemistry. Results showed that in the blank control group, the alveolar structure was intact, and the blue cell nuclei (lung parenchymal cells and normal immune cells) were evenly distributed and morphologically intact, with no obvious inflammatory cell infiltration, indicating that the lung tissue had no pathological damage and was in a normal physiological state. In contrast, in the model group, the number and disordered distribution of blue cell nuclei (numerously infiltrated inflammatory cells and damaged lung parenchymal cells) were abnormally increased, and some cell nuclei were broken. Alveolar structure destruction and interstitial congestion and edema were also observed, indicating that NDM-1 infection caused severe lung inflammation and damage, resulting in significant pathological changes in the lung tissue. In the lung tissue of the treatment group containing GEM microspheres loaded with EDTA and meropenem, the number of blue cell nuclei was significantly reduced compared with the model group, and the distribution was more uniform. The cell nuclei were basically intact, the alveolar structure gradually recovered, and the inflammatory infiltration was significantly reduced. This indicates that the prepared GEM microspheres can effectively inhibit lung inflammation, reduce lung tissue damage, and promote lung tissue repair, proving that they have a significant therapeutic effect on NDM-1 pneumonia.

[0068] Figure 8 HE staining results of GEM microspheres on major organs of healthy mice, including heart, liver, spleen, lung, and kidney tissue sections. The results showed that the structures of all major organs remained intact, with no obvious pathological damage, inflammatory infiltration, or necrotic foci, indicating that GEM microspheres at therapeutic doses showed no significant organ toxicity in mice and possessed good biosafety.

[0069] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A process for the preparation of respirable microspheres loaded with meropenem and EDTA, characterized in that, Includes the following steps: Meropenem, EDTA, gelatin, and deionized water were mixed and stirred until completely dissolved to obtain an aqueous solution. Liquid paraffin was mixed with Span-80 and homogenized at high speed until the system was homogeneous to obtain an oil solution. The aqueous solution was slowly added dropwise to the oil solution, and high-speed homogenization was continued to emulsify the mixture. Subsequently, a crosslinking agent solution dissolved in dimethyl sulfoxide was added, and the mixture was stirred at low speed to carry out a crosslinking reaction. After the reaction was completed, unencapsulated free drug was removed by washing with n-hexane, and then the mixture was freeze-dried to obtain inhalable microspheres loaded with meropenem and EDTA.

2. The process for the preparation of inhalable microspheres loaded with meropenem and EDTA according to claim 1, characterized by the fact that: In the aqueous solution, the mass ratio of gelatin, EDTA, and meropenem is 200:10-20:10-20.

3. The process for the preparation of inhalable microspheres loaded with meropenem and EDTA according to claim 1, characterized by the fact that: In the oil phase solution, the volume ratio of liquid paraffin to Span-80 is 40-50:8-10.

4. The process for the preparation of inhalable microspheres loaded with meropenem and EDTA according to claim 1, characterized by the fact that: The high-speed homogenizing stirring speed is 10,000 to 15,000 rpm, and the emulsification treatment time is 20 to 40 min; the stirring speed of the crosslinking reaction is 500 to 1,000 rpm, the reaction time is 12 to 24 h, and the reaction temperature is room temperature.

5. The process for the preparation of inhalable microspheres loaded with meropenem and EDTA according to claim 1, characterized by the fact that: The crosslinking agent is any one of genipin, glutaraldehyde, and calcium chloride.

6. An inhalable microsphere loaded with meropenem and EDTA, prepared by the method according to any one of claims 1-5, characterized in that: The particle size of the inhalable microspheres is 2–5 μm.

7. The inhalable microspheres loaded with meropenem and EDTA according to claim 6, characterized in that: The inhalable microspheres possess drug release characteristics that respond to the infection microenvironment.

8. The inhalable microspheres loaded with meropenem and EDTA according to claim 6, characterized in that: The inhalable microspheres can inactivate NDM-1 metallo-β-lactamase and disrupt bacterial biofilms; the inhalable microspheres can reshape the lung immune microenvironment, promote M1 macrophage polarization, and enhance IFN-γ secretion.

9. The use of the inhalable microspheres loaded with meropenem and EDTA according to any one of claims 6-8 in the preparation of a medicament for treating pneumonia and / or pulmonary biofilm infections caused by NDM-1 enzyme-resistant bacteria.

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