In vivo targeting of hydrogen peroxide by activatable cell-penetrating peptides

Abstract

A hydrogen peroxide (H2O2)-activated cell-penetrating peptide was developed through incorporation of a boronic acid-containing cleavable linker between polycationic cell-penetrating peptide and polyanionic fragments. Fluorescence labeling of the two ends of the molecule enabled monitoring its reaction with H2O2 through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H2O2 sensor selectively reacts with endogenous H2O2 in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H2O2 has potential applications in imaging and therapy of diseases related to oxidative stress.

Figure 1

Schematic illustration of H₂O₂-ACPP structure and its H₂O₂-triggered fragmentation process. (A) Fluorescence labeling of H₂O₂-ACPP peptide domains enables visualization of its cleavage through FRET disruption. Shown are the fluorescence emissions of (B) ACPP 1 and (C) ACPP 2 (1 μM each) before (purple) and 20 min after (green) reaction with H₂O₂ (2 mM).

Figure 2

Selective and concentration-dependent cleavage of ACPP 1 by H₂O₂. (A) Fold increase in fluorescein/Cy5 emission ratio (524/672 nm) after 20 min incubation of ACPP 1 (1 μM) with indicated concentrations of H₂O₂. Error bars represent ± standard deviation. *p < 0.05. (B) Time course fluorescence emission spectra of ACPP 1 (1 μM) in the presence of H₂O₂ (1 mM). (C) H₂O₂-dependent cleavage of ACPP 1 (1 μM) after 30 min incubation with 1 mM H₂O₂. (D) Fold increase in fluorescein/Cy5 emission ratio at indicated times of ACPP 1 (1 μM) with indicated ROS or their donors (100 μM, catalase 0.5 mg/mL).

Figure 3

Detection of H₂O₂ by ACPP 1 in cellular environment. (A) Fold increase in fluorescein/Cy5 emission ratio (524/672 nm) after 30 min of ACPP 1 (1 μM) upon exogenous addition of H₂O₂ at indicated concentration in the presence of HL-60 cells. Error bars represent ± standard deviation. (B) Time course of fold increase in fluorescein/Cy5 emission ratio (524/672 nm) of ACPP 1 (1 μM) incubated with HL-60 cells at the indicated conditions (catalase 0.5 mg/mL, PMA 0.5 μM). Error bars represent ± standard deviation. *p < 1 × 10^–10, **p < 1 × 10^–4.

Figure 4

In vivo targeting of H₂O₂ by ACPPs 1 and 2. (A) Representative ratiometric fluorescent images of fluorescein/Cy5 (ACPP 1) or Alexa488/594 (ACPP 2) emission ratios of mouse lungs in the indicated conditions treated with ACPP 1 (10 nmol) or ACPP 2 (5 nmol) for 6 h. Right: scales of appropriate emission ratios. (B) Mean change in ratios of images presented in (A) with additional animals (n = 5). Error bars represent ± standard deviation. *p < 0.05, **p < 0.005. (C) SDS-PAGE analysis of lung extracts from LPS-mice treated with ACPPs 1–3 for 6 h. Bands were pseudocolored according to their emission spectra (Figure S9 (SI)): intact ACPP (purple) or cleaved ACPP (green).