Lung Epithelial Cell Membrane-Camouflaged ROS-Activatable Berberine Nanoparticles for Targeted Treatment in Acute Lung Injury

Abstract

Introduction: Acute lung injury (ALI) seriously threatens human health and is induced by multiple factors. When ALI occurs, lung lesions affect gas exchange and may trigger respiratory failure. Current clinical treatments are limited, and traditional drug delivery has drawbacks. Berberine, a natural drug with anti-inflammatory effects, has difficulty in effectively exerting its efficacy.

Methods: The study designed a nano-micelle. Hydrophobic berberine was encapsulated with diselenide bonds as the linker. Then, lung epithelial cell membranes were extracted to encapsulate and disguise the nano-micelle. These nanoparticles were injected intravenously. Thanks to the cell membrane's specificity, they could bind to lung tissue, achieving targeted lung delivery. In the inflamed area of acute lung injury, the significantly increased reactive oxygen species level was used to break the diselenide bonds, enabling precise berberine release at the lung injury site.

Results: The nano-drug (MM-NPs) was successfully prepared, with the encapsulation efficiency of berberine in the micelles reaching 68.2%. In a ROS environment, the nano-micelles could quickly release over 80% of berberine. In inflammatory MLE-12 cells, MM-NPs responded well to ROS, and cellular inflammatory factor levels were significantly improved after treatment. In a lipopolysaccharide (LPS)-induced pneumonia mouse model, MM-NPs achieved lung targeting. Further studies showed that MM-NPs administration significantly alleviated LPS-induced lung injury in mice. Additionally, evaluation indicated MM-NPs had good in-vivo safety with no obvious adverse reactions.

Conclusion: This study successfully developed a novel delivery system, MM-NPs, overcoming berberine's low bioavailability problem in treating acute lung injury. The system has excellent physicochemical properties, biocompatibility, and metabolic safety. In vitro and animal experiments verified it can significantly enhance the therapeutic effect, offering new ideas and hopes for acute lung injury treatment. In the future, clinical trials can be advanced, and new lung targeting strategies explored for more therapeutic breakthroughs.

Keywords: acute lung injury; berberine; lung-targeted delivery; nano-micelles; reactive oxygen species.

Figure 1

Schematic diagram describing the important role of MM-NPs in acute lung injury by inhibiting the release of inflammatory factors and reducing oxidative stress damage, and the design principle of how to construct a new delivery system (MM-NPs) in which ROS-responsive micelles carry berberine and wrap lung epithelial cell membranes.

Figure 2

Preparation and characterization of nanomedicines. (A) Schematic illustration of the preparation of RBPSe-NPs. (B) TEM images of RBPSe-NPs. Scale bar = 100 nm. (C) RBPSe-NPs could maintain stability at 37 °C for 2 weeks. (D) RBPSe micelles can rapidly accumulate and release more than 80% of berberine because the diselenide bond in the structure of RBPSe is sensitive to reactive oxygen species. (E) Schematic illustration of preparation of MM-NPs through an extrusion method. (F) TEM images of MM-NPs. Scale bar = 200 nm. (G) Zeta potentials of NPs analyzed by DLS. (H) Particle sizes of NPs analyzed by DLS.

Figure 3

ROS response of MM-NPs in inflammatory MLE-12 cells and improvement of inflammatory factors. (A) Confocal images on detecting and comparing the levels of reactive oxygen species in LPS-induced inflammatory and normal lung epithelial cells. DCFH (green, a probe for detecting ROS), DAPI (blue, a nuclear stain). Scale bar, 10 μm. (B) Flow cytometry plots on detecting and comparing the levels of reactive oxygen species in LPS-induced inflammatory and normal lung epithelial cells. (C) Quantitative statistics of flow cytometry results in (B). (D) Detect the uptake of MM-NPs in lipopolysaccharide (LPS)-induced inflammatory lung epithelial cells and normal lung epithelial cells through confocal images, and detect the uptake efficiency of MM-NPs compared with free berberine, RBPSe-NPs and MMC-NPs. Scale bar, 10 μm. (E) Perform fluorescence quantification of the uptake in parallel images in (D). (n=3). (F) The intracellular location of berberine and mitochondria in MM-NPs was determined by confocal laser scanning microscopy (CLSM) imaging. Mito-red was used to mark intracellular mitochondria. Scale bar, 10 μm. (G) Plot profile for co-localization analysis of berberine and mitochondria in MM-NPs. (H) qPCR of the indicated cytokines in MLE-12 cells. (n=3). Data are from three experiments and presented as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

In vivo fluorescence imaging of MM-NPs in LPS-induced pneumonia mice. (A) Representative whole-body imaging of LPS-induced pneumonia mice at different time points after injection of MM-NPs, MMC-NPs and RBPSe-NPs. Combine DID with the outer cell membranes of MM-NPs and MMC-NPs for binding staining, and synthesize RBPSe-NPs by substituting DID for berberine. (DID, ex=535 nm, em=640 - 660 nm). (B) Quantitative analysis of the fluorescence imaging of mice from parallel images (A). (n=3). (C) Representative ex vivo imaging images of five major organs (heart, liver, spleen, lung and kidney) obtained from LPS-induced pneumonia mice at 72 hours after injection of MM-NPs, MMC-NPs and RBPSe-NPs. (D) Quantitative analysis of the fluorescence imaging of major organs from parallel images (C). (n=3). Data are presented as means ± SE. *P < 0.05, **P < 0.01.

Figure 5

Administration of MM-NPs alleviates lipopolysaccharide-induced lung injury in mice. (A) Simplified experimental protocol. C57BL/6 mice (n=5) were intratracheally administered LPS (4 mg/kg) for 4 hours and then treated with MM-NPs (2 mg/kg of berberine, intravenously), etc. The mice were sacrificed after 24 hours. (B) Protein concentration in BALF. (n=3). (C) Total cell counts in BALF. (n=3). (D) H&E staining of lung tissues. Scale bar, 100 μm. (E) Flow cytometry of the ratios of CD45+CD11b+Ly6G+ neutrophils in BALF. (n=3). (F) Flow cytometry of the ratios of CD45+CD11b+F4/80+ MMs in BALF. (n=3). (G) qPCR of the indicated cytokines in lungs. (n=3). Data are presented as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Transcriptome sequencing analysis of lipopolysaccharide-induced lung injury mice treated with MM-NPs. (A) GSEA results of the statistically significant gene set: Altemeier Response To Lps With Mechanical Ventilation. (B) GSEA results of the statistically significant gene set: Hallmark Inflammatory Response. (C) KEGG analysis of DEGs. (D) GO analysis of DEGs. (E) Volcano plot analysis of DEGs between PBS control or MM-NPs treated. (F) Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) of some important genes. (G) Heat map of expression of inflammatory genes of PBS control or MM-NPs treated. (H) Heat map of expression of oxidative stress genes of PBS control or MM-NPs treated.

Figure 7

The safety of MM-NPs in vivo. (A) H&E staining of heart, liver, spleen and kidney tissues from the mice in different groups. Scale bar, 100 μm. (B) The levels of Hemoglobin (HGB). (C) The numbers of platelets (PLT). (D) The numbers of red blood cells (RBC). (E) The numbers of white blood cells (WBC). (F) The levels of alanine transaminase (ALT). (G) The levels of aspartate transaminase (AST). (H) The levels of creatinine (CR). (B–H, n=5). Data are presented as means ± SE.