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. 2022 Jun 16:13:901979.
doi: 10.3389/fmicb.2022.901979. eCollection 2022.

Functionalized Erythrocyte Membrane-Coated Nanoparticles for the Treatment of Klebsiella pneumoniae-Induced Sepsis

Jun Liu  1 , Hui Ding  1 , Mingjie Zhao  2 , Fan Tu  1 , Tian He  1 , Lizhu Zhang  3 , Yanfei Jing  4 , Xiaohong Rui  1 , Shiliang Zhang  1
Affiliations

Functionalized Erythrocyte Membrane-Coated Nanoparticles for the Treatment of Klebsiella pneumoniae-Induced Sepsis

Jun Liu et al. Front Microbiol. .

Abstract

Sepsis is a systemic inflammatory response syndrome caused by infection, with high incidence and mortality. Therefore, it is necessary to carry out an effective anti-infection treatment. In this work, we designed and synthesized red blood cell (RBC) membrane-coated PLGA nanoparticles named γ3-RBCNPs, which target the highly expressed intercellular adhesion molecule-1 (ICAM-1) at the site of infection through the γ3 peptide on its surface and kill the Klebsiella pneumoniae through ciprofloxacin encapsulated in its core. In addition, the homogenous RBC membrane coated on the surface of the nanoparticles helps them avoid immune surveillance and prolong the circulation time of the drug in the body. We found that the γ3-RBCNPs target human umbilical vein endothelial cells (HUVECs) activated by TNF-α in vitro and the infected lung of mice in the sepsis model very well. In vitro evaluation suggested that γ3-RBCNPs have a low risk of acute hemolysis and are less likely to be engulfed by macrophages. In vivo evaluation showed that γ3-RBCNPs has a long half-life and good bio-safety. More importantly, we confirmed that γ3-RBCNPs have the good antibacterial and anti-infection ability in vivo and in vitro. Our research provides a new strategy for the nano-drug treatment of Klebsiella pneumoniae-induced sepsis.

Keywords: Klebsiella pneumoniae; red blood cell membrane; sepsis; targeted therapy; γ3 peptide.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Characterization of γ3-RBCNPs. (A,B) DLS analysis of the size intensity (A) and mean diameter (B) of the prepared NPs. (C) Zeta potential of the prepared NPs. (D) TEM images of the NPs. Scale bar = 100 nm. (E) In vitro drug release curves of RBCNPs and γ3-RBCNPs. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NP cores group.
FIGURE 2
FIGURE 2
Biological characteristics of γ3-RBCNPs. (A) The appearance of the prepared NP-cores, RBC vesicles, RBCNPs, and γ3-RBCNPs. (B) SDS-PAGE analysis of the membrane protein of RBC on the surface of NP-cores, RBC vesicles, RBCNPs, and γ3-RBCNPs. (C) The targeting ability of NP-cores, RBCNPs, and γ3-RBCNPs on TNF-α-stimulated HUVECs was evaluated with LSCM. Scale bar = 50 μm. (D) The mean fluorescence intensity in HUVECs stimulated by LPS was assessed by flow cytometry. ***P < 0.001 vs. NP cores group.
FIGURE 3
FIGURE 3
Evaluation of the therapeutic effect of γ3-RBCNPs in vitro. (A) Quantification of the number of bacteria in HUVECs treated with NP-cores, RBCNPs, and γ3-RBCNPs after K.P infection. ***P < 0.001 vs. PBS group. Flow cytometry (B) and IF staining (C) detect the apoptosis of HUVECs treated with γ3-RBCNP after K.P infection. Scale bar = 50 μm. ***P < 0.001 vs. unstimulated groups. ###P < 0.001 vs. K.P stimulated PBS group.
FIGURE 4
FIGURE 4
The targeting capacity of γ3-RBCNPs in vivo. Drug distribution in major organs of mice with acute pulmonary infection treated with RBCNPs (A) and γ3-RBCNPs (B) at 4, 16, and 24 h. Quantification of the fluorescence intensity in the main organs of mice in RBCNPs (C) and γ3-RBCNPs (D) groups. **P < 0.01 vs. 4 h group. ***P < 0.001 vs. 4 h group.
FIGURE 5
FIGURE 5
In vitro evaluation of γ3-RBCNPs. (A,B) Assessment of the hemolytic activity of NP-cores, RBCNPs, and γ3-RBCNPs, PBS was used as the negative control, and Triton-X100 was used as a positive control. (C,D) The phagocytosis of RBCNPs and γ3-RBCNPs by macrophages in vitro was observed by LSCM. Scale bar = 50 μm. ***P < 0.001 vs. PBS group.
FIGURE 6
FIGURE 6
Assessment of the bio-safety of γ3-RBCNPs in vivo. (A) Evaluation of half-life of the drug in NP-cores, RBCNPs, and γ3-RBCNPs in vivo. The changes of body weight (B), liver function (C) and blood routine (D) indexes of mice with acute pulmonary infection treated with NP-cores, RBCNPs, and γ3-RBCNPs. (E) H&E staining of the main tissues in mice. Scale bar = 100 μm.
FIGURE 7
FIGURE 7
Evaluation of the therapeutic effect of γ3-RBCNPs in vivo. (A) Survival curves of mice with an acute lung infection in each group after being treated with NP-cores, RBCNPs, and γ3-RBCNPs. (B) Quantification of bacterial counts in main tissues of mice after treatment. (C) HE staining was performed to evaluate the degree of lung infection in mice after treatment. Scale bar = 100 μm. (D) ELISA assay was conducted to detect the level of TNF-α, IL-1β, and IL-6 in the serum of mice. **P < 0.01 vs. PBS group, ***P < 0.001 vs. PBS group.
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