Elaborate construction of pH-sensitive polymyxin B loaded nanoparticles for safe and effective treatment of carbapenem-resistant <i>Klebsiella pneumoniae</i>

Elaborate construction of pH-sensitive polymyxin B loaded nanoparticles for safe and effective treatment of carbapenem-resistant Klebsiella pneumoniae

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Wen Zhong^a^,¹, Dan Zheng^a^,¹, Xukun Liao^a, Yadi Zhou^b, Yan Jiang^c, Ting Gao^d, Ming Li^b^,^*, Chengli Yang^a^,^b^,^*

Chinese Chemical Letters | 2025, 36(3) : 110448

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Chinese Chemical Letters | 2025, 36(3): 110448

• Communications •

Elaborate construction of pH-sensitive polymyxin B loaded nanoparticles for safe and effective treatment of carbapenem-resistant Klebsiella pneumoniae

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Wen Zhong^a^,¹, Dan Zheng^a^,¹, Xukun Liao^a, Yadi Zhou^b, Yan Jiang^c, Ting Gao^d, Ming Li^b^,^*, Chengli Yang^a^,^b^,^*

Affiliations

^aDepartment of Clinical Pharmacy, School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang 550025, China

^bDepartment of Pharmacy, The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

^cDepartment of Clinical Microbiology and Immunology, School of Clinical Laboratory Science, Guizhou Medical University, Guiyang 550004, China

^dDepartment of Clinical Laboratory, The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

Published: 2025-03-15 doi: 10.1016/j.cclet.2024.110448

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Abstract

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The escalation in the incidence of multidrug-resistant Gram-negative bacteria is becoming a pressing global concern. Polymyxin B (PMB), a conventional antibiotic with notable therapeutic efficacy against Gram-negative bacterial infections, serves as a crucial final recourse against carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Nevertheless, the clinical usage of PMB is impeded by its pronounced nephrotoxicity and poor infection site targeting. This investigation is geared to construct a nanoparticle formulation (named HA-PMB@H) comprising hyaluronic acid (HA) and PMB via a simple Schiff base reaction and further coating HA by electrostatic action. HA-PMB@H shows an average size of (153.8 ± 24.3) nm, and a mean zeta potential of (−25.6 ± 5.2) mV. Additionally, PMB can be released from HA-PMB@H more thoroughly and efficiently at pH 5.5 compared to pH 7.4, which demonstrates the Schiff base modification of PMB paves the way for its release at focus of infection. The uptake ratio of HA-PMB@H by alveolar epithelial cells (RLE-6TN) surpassed that of free PMB devoid of HA, which facilitates to the intracellular sterilization of PMB. Furthermore, the employment of HA-PMB@H ameliorated the toxicity of PMB towards human embryonic kidney cells (HEK 293) and pulmonary microvascular endothelial cells (HULEC-5a). What is more, HA-PMB@H effectively managed severe pneumonia induced by CRKP samples from clinical patients diagnosed with CRKP infection in vivo, substantially enhancing the survival rate of mice. Consequently, this nano-delivery system holds promising clinical significance in the combat against drug-resistant bacterial infections.

Key words

Polymyxin B / pH-sensitive modification / Carbapenem-resistant Klebsiella pneumoniae / Antibacterial / Biological security

Cite this Article

Wen Zhong, Dan Zheng, Xukun Liao, Yadi Zhou, Yan Jiang, Ting Gao, Ming Li, Chengli Yang. Elaborate construction of pH-sensitive polymyxin B loaded nanoparticles for safe and effective treatment of carbapenem-resistant Klebsiella pneumoniae[J]. Chinese Chemical Letters, 2025 , 36 (3) : 110448 - . DOI: 10.1016/j.cclet.2024.110448

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The global prevalence of multidrug-resistant gram-negative bacteria is an escalating concern in the field of healthcare [1]. Particularly, infection with strains resistant to carbapenems significantly increases the risk of mortality for patients in intensive care units and other healthcare facilities [2]. The excessive and prolonged usage of antibiotics has triggered the emergence of antibiotic resistance, which has become a major public health challenge [3]. This is exemplified by the widespread proliferation of multidrug-resistant Gram-negative bacteria, posing a substantial threat to the global population [4]. Among these bacteria, Klebsiella pneumoniae (KP), as a common pathogen, plays a significant role in causing severe infections, particularly in individuals with compromised immune systems [5]. KP is responsible for various infections, including pneumonia, urinary tract infections, and sepsis [6,7]. Previously, carbapenems were the preferred treatment option for multidrug-resistant Klebsiella pneumoniae (MDR-KP), but the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has presented new challenges in the healthcare setting [8]. Recent survey findings indicated a notable surge in cases of CRKP, escalating from 2.5% to around 16% over the last ten years [9]. The rising morbidity and mortality rates associated with CRKP infections, sometimes exceeding 40%, highlight the urgent need for more effective treatment options [10,11].

One potential strategy to address this situation is the reevaluation of "old-fashioned" antibiotics [12]. Polymyxin B (PMB), a member of the polymyxin antibiotic family, enhances the permeability of the outer membrane of Gram-negative bacteria by strongly interacting with the lipid A fragment of lipopolysaccharide (LPS), thereby exhibiting antibiotic properties [13]. PMB was previously widely used in the treatment of pneumonia, bacteremia, and tissue infections [14]. However, intravenous administration of PMB was limited in clinical practice during the 1960s due to its toxic side effects, such as anaphylaxis, nephrotoxicity, and neurotoxicity [15,16]. The cationic property of PMB, particularly the α and γ amino groups on the aminobutyric acid residue, presents a double-edged sword in infection therapy [17]. On one hand, it binds and disrupts the negatively charged LPS in the outer membrane of Gram-negative bacteria to achieve effective treatment of bacterial infections. On the other hand, under physiological conditions, it becomes protonated and interacts with phosphate anions in the cell membrane, especially in the kidney where PMB accumulates, thereby resulting in cell membrane damage of healthy cells [18]. This renal accumulation is the critical mechanism leading to PMB-induced nephrotoxicity because it increases the permeability of tubular epithelial cell membranes [19]. Therefore, if PMB is to be extensively used in clinical practice, it must undergo modifications to mitigate its toxicity and elevate its targeting effects.

The application of biomaterials is becoming increasingly widespread [20], among which nano-drug delivery systems are currently garnering significant interest in the territories of anti-tumor, antibacterial, and anti-inflammatory [21-25] research due to their ability to target drug delivery precisely and regulate the release of conventional medications. This enhances drug effectiveness and minimizes harm to healthy tissues. It has come to our attention that, hyaluronic acid (HA) is a natural linear mucopolysaccharide consisting of alternating units of d-glucuronic acid and naphthyl-d-glucosamine, serving as the primary component of the extracellular matrix [26]. Its notable hydrophilicity plays a crucial role in reducing protein adsorption and permeation, thereby enhancing the circulation and stability of drug delivery systems (DDS) containing HA in vivo [27,28]. Furthermore, HA is recognized as the key ligand for CD44 [29,30], a cell adhesion molecule prevalent in white blood cells, endothelial cells, and tumor cells [31]. Studies have indicated that the interaction between CD44 and its HA is highly active in inflammatory diseases, suggesting HA's enormous potential as a drug carrier for PMB to target infections effectively [32-34]. Furthermore, researchers have indicated that the site of infection is acidic, with a pH of around 5.5, and the pH-sensitive modification of antibodies can improve their targeted antibacterial effect [35,36]. As a consequence, the study introduces a nanoparticle named HA-PMB@H (Scheme 1), which is constructed by chemically bonding of HA and PMB molecules via Schiff base and further coated with HA to enhance its bacterial targeting. This formulation exhibits the potential for safe and effective pneumonia treatment by concealing the cationic properties of PMB, thereby enhancing biosafety. Furthermore, the PMB release from HA-PMB@H with the characteristic of being pH-responsive, which facilitates to its targeting of the infection site. Upon intravenous administration, HA-PMB@H is anticipated to effectively eliminate infections, targeting the CRKP-infected area while safeguarding healthy tissues. Overall, the study underscores the clinical promise of HA-PMB@H and presents a viable strategy for the secure and efficient management of severe CRKP pneumonia.

The development of HA-PMB@H aims to broaden the utility of PMB in biological systems. In order to enhance the infection site targeting and decrease the cytotoxicity of PMB, the authors modified PMB with HA through a pH-sensitive Schiff base (named HA-PMB). Furthermore, HA molecules could effectively neutralize and cloak the positive charge of PMB. The Schiff base bond typically involves an interaction between nitrogen atoms and a protonated carbon atom [37]. In this part, the chemical structure of the Schiff base in HA-PMB was confirmed by hydrogen-nuclear magnetic resonance (¹H NMR) spectrum. As depicted in Fig. 1a, a characteristic peak at approximately 6.3 ppm was observed for synthesized HA-PMB, indicating the successful synthesis of the surface through the Schiff base reaction.

To enhance antibacterial efficacy, the HA-PMB was further coated with HA to reduce the cationic toxicity of PMB, and various molar ratios of HA-PMB and HA were explored, as shown in the Figs. 1b and c, culminating in a ratio of 17:1 found to optimize antibacterial properties while fully neutralizing PMB's positive charge. The encapsulation efficiency of HA-PMB@H nanoparticles was quantified at 66.2% ± 0.56% using ultraviolet and visible spectrophotometer (UV–vis) spectroscopy at 215 nm with a standard curve. The particle size of HA-PMB@H was approximately (153.8 ± 24.3) nm (Fig. 1d). In addition, the transmission electron microscopy (TEM) and high angle toroidal dark field image-scanning transmission electron microscope (HAADF-STEM) image showed that formulations had a uniform and approximately spherical morphology as displayed in Fig. 1e. These results indicated that HA-PMB@H can obtain a formulation with good in vitro antibacterial properties and uniform stability, laying a solid foundation for subsequent experiments.

Schiff bases demonstrate pH responsiveness due to their chemical structure. Imine bonds, along with hydrazones and oximes, are formed through condensation reactions between aldehyde groups and various nucleophilic amine groups [38]. Consequently, the pH sensitive release of PMB from HA-PMB@H nanoparticle was observed and relevant results were demonstrated in Fig. 1f. The HA-PMB@H nanoparticles demonstrated a fast cumulative release of PMB (approximately 70%) within the first 0.5 h at pH 5.5, whereas it was only about 25% at pH 7.4. In addition, the cumulative release of PMB in HA-PMB@H group reached about 85% within 12 h in pH 5.5 condition, but it was only about 35% at pH 7.4. These results exhibited that the delivery system of HA-PMB@H can achieve highly effective sustained release in acidic focuses that are infected with bacteria.

The efficient uptake of antibiotics by bacteria is very critical to the improvement of antibacterial efficiency. Consequently, we compared the bacterial uptake efficiency of free PMB and HA-PMB@H nanoparticles. In this work, the PMB was fluorescently labeled by Cy5, and relevant results were demonstrated in Fig. 2a. The fluorescence intensity in free PMB group was significantly weaker than that in HA-PMB@H group. These results indicated that the nanocrystallization of PMB and HA coated of HA-PMB nanoparticles was facilitated by the uptake ratio of PMB.

Research has found that HA has certain antibacterial activity against biofilms and respiratory bacteria [39]. In addition, HA could be hydrolyzed by hyaluronidase secreted by bacteria in the infectious microenvironment and further exposed to PMB to achieve its antibacterial effect [40,41]. This research assessed the antimicrobial properties of nanoparticles on KP and CRKP using an in vitro micro broth dilution technique. Various concentrations of diluted PBS, HA, PMB, and HA-PMB@H ranging from 128 µg/mL were added to a microplate containing bacterial cultures. The inhibitory effects were determined by measuring the optical density at OD600 nm of the supernatant in each well. As evident in Figs. 2b and c, the minimum inhibitory concentration (MIC) values for KP, PMB, and HA-PMB@H were 2 µg/mL, while for CRKP, the MIC values were 2 and 1 µg/mL, respectively. These findings suggest that the HA-based nanoparticle delivery system demonstrates enhanced bactericidal efficacy against CRKP. Furthermore, the impact of the formulation on the growth patterns of KP and CRKP was investigated (Figs. 2d and e). Monitoring over a 24-h incubation period revealed that HA-PMB@H (PMB: 1 µg/mL) completely halted visible bacterial growth of CRKP and maintained a steady OD₆₀₀ value throughout the incubation period.

The visual evidence of bacteria presented in the SEM images further substantiates the previously discussed anti-bacterial results. As depicted in Fig. 2f, the group of PBS and HA bacteria displays a characteristic rod-shaped structure with a sleek and intact surface. In contrast, following exposure to PMB and HA-PMB@H, the bacterial membrane was compromised, leading to notable alterations in its morphology. In conclusion, HA-PMB@H displays superior antibacterial activity against CRKP compared to PMB, effectively suppressing drug-resistant bacteria. The presence of HA molecules enhances the anti-bacterial effectiveness of PMB against CRKP. We further studied its mechanism of action (Fig. 2g). We have learned that HA-PMB@H can significantly flip the bacterial membrane potential, destroy the bacterial membrane, and achieve a good bactericidal effect.

The precise treatment of pneumonia requires nanoparticles with specific targeting abilities. In our design concept, the HA on the outer surface of HA-PMB@H binds to the CD44 receptor on cell membranes, providing the nanoparticles with lung-targeting specificity. The potential lung targeting ability of HA-PMB@H was evaluated using HULEC-5a and RLE-6TN cells, demonstrating significant CD44 expression on their membranes in Figs. 3a and b. This supports the inference that HA-PMB@H can effectively target the lungs due to the HA-CD44 binding affinity.

Specific targeting properties of nanoparticles are vital for treating CRKP infection effectively, and antibodies getting inside the cells and killing the bacteria inside them are very critical to anti-infective therapy [42]. At first, our research suggests that the developed nanoparticles can reach infected lungs through post-intravenous administration and be internalized by alveolar cells. Confocal microscopy analysis of HA-PMB@H—Cy5 and PMB-Cy5 in HULEC-5a (Fig. 3c) and RLE-6TN (Fig. 3e) revealed enhanced intracellular fluorescence with HA-PMB@H—Cy5, indicating superior internalization and rapid entry into target cells for antibacterial efficacy, as shown in Figs. 3d and f.

The potent inhibitory activity of HA-PMB@H against CRKP in vitro, coupled with its favorable biosafety profile, prompted the establishment of a pneumonia model in mice (Fig. 4a). All animal experiments were approved by the Ethics Committee of the Animal Experimental Center of Guizhou Medical University (approval No. 12304086) and were carried out in compliance with all relevant ethical regulations. This model involved inducing acute lung injury along with CRKP infection to assess the in vivo anti-infective efficacy of HA-PMB@H. Healthy ICR mice were randomly divided into five groups (n = 5), and a detailed in vivo treatment protocol was implemented. Post-modeling, mice were administered intravenous injections of PBS, HA, PMB, and HA-PMB@H. Over the course of 5 days, the survival rates of mice under various treatments were monitored. The results illustrated a stark contrast, with a 0% survival rate observed in pneumonia mice infected with CRKP by day 5. Conversely, mice infected with CRKP and treated with PMB and HA-PMB@H (PMB dose: 5 mg/kg) exhibited survival rates of 80% and 100% (Fig. 4b). This outcome underscores the enhanced antibacterial activity achieved by encapsulating PMB in nanoparticles and highlights the effectiveness of HA-based nanodrug delivery systems in combating CRKP infection. The superior therapeutic effect of the HA-PMB@H group can be attributed to its targeted distribution and reduced toxicity. Animals administered with HA-PMB@H initially experienced a decrease in weight, followed by a prompt return to their original state (Fig. 4c), and expedited recovery from hypothermia (Fig. 4d). Conversely, mice treated with HA-PMB@H exhibited weight reduction not exceeding 12%, with all subjects fully recuperating within a span of 24 h.

Furthermore, surviving mice from different treatment groups underwent euthanasia, and lung tissue homogenates were subjected to colony analysis, revealing that the PMB and HA-PMB@H treatment groups exhibited significantly lower colony counts and inhibition rates exceeding 90% in CRKP-infected pneumonia mice (Figs. 4e and f). In summary, HA-PMB@H exhibited more effective therapeutic outcomes on mice with CRKP-infected pneumonia compared to the other treatment groups.

PMB encounters obstacles in its clinical utilization due to the emergence of severe adverse reactions [43]. An optimal antibacterial agent should possess the capacity to target bacteria selectively while causing minimal harm to regular human cells. One strategy to achieve this objective involves enveloping PMB with HA molecules, which act as a protective barrier against the positive charge of PMB. This protective mechanism is anticipated to notably diminish the cytotoxic effects of PMB. To assess the impact of HA encapsulation on PMB-induced cellular toxicity, we conducted cell counting kit-8 (CCK-8) cell proliferation assays utilizing HEK 293 and HULEC-5a cell lines. The outcomes depicted in the figure reveal that HA-PMB@H nanoparticles demonstrate favorable biosafety when compared to PMB. Even at a concentration of 200 µg/mL, the cell viability rates of both cell lines treated with HA-PMB@H surpassed 80%, while PMB exhibited noticeable cytotoxicity towards HEK 293 cells with escalating concentrations (Figs. S1–S4 in Supporting information). These results validate the considerable reduction in PMB toxicity following HA encapsulation, laying a robust groundwork for subsequent in vivo investigations. As shown in Figs. S5 and S6 (Supporting information), the hemolysis rate of HA-PMB@H is < 1%, which is lower than that of PMB. The hemolysis percentages of HA, PMB, and HA-PMB@H were 1.54% ± 0.14%, 1.81% ± 0.09%, and 0.74% ± 0.15%, respectively, indicating that the self-assembly of PMB and HA improved their biological safety. To delve deeper into the histopathological alterations in the lungs of mice subjected to treatment and those left untreated, the remaining lung specimens from each group were sectioned and stained with H & E for histological scrutiny (Fig. S7 in Supporting information). The illustration manifests that, in contrast to the other treatment cohorts, mice treated with HA-PMB@H displayed marked enhancements in pathological conditions, characterized by diminished inflammatory infiltration in lung tissue (blue arrows) and reduced renal impairment (green arrows). These observations substantiate the superior efficacy and reduced toxicity of HA-PMB@H in comparison to PMB, thereby establishing a fundamental framework for future clinical applications.

In brief, a specialized nano-delivery system named HA-PMB@H nanoparticles has been successfully developed to target and effectively treat CRKP infection. These nanoparticles are formed through the electrostatic self-assembly of Schiff-base-modified PMB and HA molecules. In comparison to PMB, HA-PMB@H nanoparticles demonstrate superior biosafety both in vitro and in vivo by concealing the cationic properties of PMB. Extensive research indicates that upon intravenous administration, HA-PMB@H nanoparticles precisely target the infected site and disrupt the bacterial membrane through the affinity between PMB and the membrane of bacterium. This mechanism proves pivotal in combating severe pneumonia by inhibiting bacterial growth and minimizing organ damage. HA-PMB@H not only enhances treatment efficacy but also boosts biosafety. Consequently, HA-PMB@H sets a promising path for the future clinical management of bacterial inflammatory conditions.

Declaration of competing interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

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Wen Zhong: Writing – original draft, Software, Methodology, Investigation, Formal analysis. Dan Zheng: Methodology, Investigation, Formal analysis. Xukun Liao: Methodology, Investigation, Formal analysis. Yadi Zhou: Writing – review & editing, Methodology. Yan Jiang: Validation, Methodology, Investigation. Ting Gao: Methodology, Data curation. Ming Li: Supervision, Investigation, Conceptualization. Chengli Yang: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Acknowledgments

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This work was supported by the National Natural Science Foundation of China (Nos. 81860543, 32360237), Guizhou Provincial Science and Technology Projects (Nos. ZK [2024] 235, ZK [2023] Key Project 041).

Supplementary materials

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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110448.

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Year 2025 volume 36 Issue 3

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doi: 10.1016/j.cclet.2024.110448

Receive Date：2024-06-26
Online Date：2025-11-07
Published：2025-03-15

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Received：2024-06-26
Revised：2024-09-10
Accepted：2024-09-11

Affiliations

^aDepartment of Clinical Pharmacy, School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang 550025, China

^bDepartment of Pharmacy, The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

^cDepartment of Clinical Microbiology and Immunology, School of Clinical Laboratory Science, Guizhou Medical University, Guiyang 550004, China

^dDepartment of Clinical Laboratory, The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Scheme 1 Schematic illustration of the construction of HA-PMB@H to kill the CRKP with excellent anti-inflammatory effectiveness and biological security.

Fig. 1 (a) The ¹H NMR of HA, PMB, and HA-PMB Schiff base. (b) The MICs of different HA to PMB ratios chemically bonded by Schiff bases through the microporous plate broth dilution method, and (c) the bacterial liquid coating results after HA-PMB@H treatment with their respective ratios. (d) The size distribution of HA-PMB@H. (e) The TEM and HAADF-STEM image of HA-PMB@H. Scale bar: 100 nm. (f) The cumulative release curve of PMB from HA-PMB@H at different pH. All data are presented as mean ± standard deviation (SD) (n = 3).

Fig. 2 (a) Fluorescence images of CRKP after incubation with cy5-modified PMB and HA-PMB@H. The bacteria were labeled with DAPI (blue), and the red fluorescence signal indicated the free PMB or HA-PMB@H. Scale bar: 20 µm. (b, c) MIC susceptibility profiles of HA-PMB@H via the microplate broth dilution method and corresponding quantification results after various treatments (n = 5 independent experiments). Growth curves of (d) KP and (e) CRKP co-incubated with various preparations (n = 3). (f) Representative SEM images of CRKP after 12 h incubated with PBS, HA, PMB, and HA-PMB@H. Scale bars: 2 µm and 1 µm. (g) Fluorescence image of the bacterial membrane potential of CRKP detected using DiOC₂(3) dyes. All data are presented as mean ± SD.

Fig. 3 (a, b) CD44 receptor expression in HULEC-5a and RLE-6TN cells detected by immuno-fluorescence assay. Scale bars: 2 µm and 20 µm. (c, d) The confocal laser scanning microscope (CLSM) images and relevant semi-quantitative of cellular uptake ratio of HULEC-5a cells. (e, f) The CLSM images and relevant semiquantitative of cellular uptake ratio of RLE-6TN cells. Scale bar: 20 µm. All data are presented as mean ± SD (n = 3).

Fig. 4 (a) Schematic design of ALI+CRKP model and experimental procedures. (b) Survival rates of acute CRKP-infected mice with pneumonia treated with PBS, HA, PMB, or HA-PMB@H for 5 days (n = 5 mice per group). Mice with pneumonia treated with PBS were used as a negative control. (c) Body weight and (d) body temperature changes of the mice. (e, f) Representative CRKP bacterial colonies formed on LB agar plates from the lung homogenization of mice with pneumonia receiving various treatments and bacteria colony forming unit (CFU) from the pulmonary tissue homogenization of mice in different treatment groups. All data are presented as mean ± SD (n = 5).

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