S-thanatin functionalized liposome potentially targeting on Klebsiella pneumoniae and its application in sepsis mouse model

Abstract

S-thanatin (Ts) was a short antimicrobial peptide with selective antibacterial activity. In this study, we aimed to design a drug carrier with specific bacterial targeting potential. The positively charged Ts was modified onto the liposome surface by linking Ts to the constituent lipids via a PEG linker. The benefits of this design were evaluated by preparing a series of liposomes and comparing their biological effects in vitro and in vivo. The particle size and Zeta potential of the constructed liposomes were measured with a Zetasizer Nano ZS system and a confocal laser scanning microscope. The in vitro drug delivery potential was evaluated by measuring the cellular uptake of encapsulated levofloxacin using HPLC. Ts-linked liposome or its conjugates with quantum dots favored bacterial cells, and increased the bacterial uptake of levofloxacin. In antimicrobial assays, the Ts and levofloxacin combination showed a synergistic effect, and Ts-LPs-LEV exhibited excellent activity against the quality control stain Klebsiella pneumoniae ATCC 700603 and restored the susceptibility of multidrug-resistant K. pneumoniae clinical isolates to levofloxacin in vitro. Furthermore, Ts-LPs-LEV markedly reduced the lethality rate of the septic shock and resulted in rapid bacterial clearance in mouse models receiving clinical multidrug resistant (MDR) isolates. These results suggest that the Ts-functionalized liposome may be a promising antibiotic delivery system for clinical infectious disorders caused by MDR bacteria, in particular the sepsis related diseases.

Keywords: antimicrobial peptide; liposome; multidrug resistance; sepsis; targeting delivery.

FIGURE 1

Schematic representation of the preparation of Ts-LPs-LEV.

FIGURE 2

Factors affecting the encapsulation efficiency (EE). The drug-loading experiments were performed using the ammonium sulfate gradient method. (A) Influence of ammonium sulfate on EE (settings: 60°C, 30 min). (B) Influence of incubation temperature (settings: 0.2 mol/L ammonium sulfate, 30 min incubation). (C) Influence of incubation time (settings: 0.2 mol/L ammonium sulfate, 60°C). Results indicated peak EE values at 0.2 mol/L ammonium sulfate, 60°C, and an incubation time of 20 min.

FIGURE 3

Size distribution and transmission electron microscopy (TEM) image of Ts-LPs-LEV.

FIGURE 4

Ts-QDs605 and Ts-LPs-CM selectively targeting Klebsiella pneumoniae cells. The cell-targeting of Ts to K. pneumoniae ATCC 700603 observed by confocal laser scanning microscopy (CLSM) using quantum dots (QD) or coumarin (CM)-loaded liposomes (LPs): (A) QDs605. (B) Ts-QDs605. (C) LPs-CM. (D) Ts-LPs-CM. K. pneumonia ATCC 700603 cells (approximately 1 × 10⁵ bacteria/ml) were first incubated with Ts-QDs605 (10 nM) for 2 h at 37°C followed by trice rinse with PBS, and then placed on slides. A solution containing 4% (wt/vol) paraformaldehyde was added for sample fixation for 30 min. Ts-LPs-CM were used at 20 nM but different from Ts-QDs605 assay, the cells receiving Ts-LPs-CM were sent to CLSM without washing with PBS. The bar indicated scale of 5 μm.

FIGURE 5

Ts and Ts-LPs affecting membrane permeability of K. pneumoniae ATCC 700603. Bacteria incubated with Ts or Ts-LPs-LEV following by staining with (A) PI or (B) DiBAC4(3). The error bars represent SD (n = 5).

FIGURE 6

Intra-bacterial levofloxacin accumulation of levofloxacin in K. pneumoniae ATCC 700603. Error bars represent the standard deviation of three measurements.

FIGURE 7

Bactericidal effect of Ts-LPs-LEV. Electron micrographs of K. pneumoniae ATCC 700603 incubated for 1 h after receiving (A) saline or (B) Ts-LPs-LEV. The length of the scale bar in the TEM image is 200 nm.

FIGURE 8

Mortality data reported as Kaplan–Meier curves for levofloxacin formulations in a septic shock model induced by a multidrug resistant (MDR) clinical isolate. After bacterial challenge by injection of 2.5x10⁷ CFU of the multidrug-resistant clinical isolate K. pneumoniae CI 130702215, the animals were immediately injected in their tail veins with sterile saline, free levofloxacin, LPs-LEV or Ts-LPs-LEV. The survival rate was monitored every 6 h for 3 days without any intervention except for the experimental drugs.

FIGURE 9

Schematic representation of Ts-LPs-LEV killing bacteria process. Briefly, when Ts-LPs-LEV co-incubated with the bacteria, Ts carried a large amount of levofloxacin-loaded liposomes to the bacterial surface and anchored in the outer membrane by combination with LPS and/or negatively charged components. Then, the liposomes fused with the bacterial cell outer membrane, which could enhance levofloxacin entry through hydrophobic and/or self-promoted pathway or contact release. Meanwhile, Ts perturbed the membrane lipid bilayers of the bacteria and removed the electrical potential of the membrane, affecting the activity of the drug efflux pumps and thus resulting in an intra-bacterial levofloxacin accumulation. The cells finally became dead and broken.