Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor

Abstract

Research on cell‐penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans‐activator of transcription (TAT)‐derived peptides and, to a lesser extent, on proline‐rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen‐sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15–24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH‐SY5Y and HeLa, via cell‐type‐dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time‐resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen‐sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O2 levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action

“…Even more striking results were obtained with eukaryotic cells, into which Bac7 fragments were capable of delivering a noncovalently linked protein [103]. More recently, a functionalized derivative of Pt(II) coproporphyrin I (PEPP0) was conjugated to the 15-24 fragment of Bac7 to make an oxygen-sensitive phosphorescent probe for intracellular use [104].…”

Optical probes and techniques for O2 measurement in live cells and tissue

“…Another group of supramolecular probes are conjugates of the phosphorescent dyes with cell-penetrating peptides (CPP) for sensing intracellular O 2 [42, 60–62]. In this case, the luminescent moiety [a Ru(II) dye or Pt-coproporphyrin (PtCP)] is linked to a targeted delivery vector, such as polyarginine or proline-rich peptides which escort the whole structure into the cell by passive means and retain it inside [63].…”

Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

“…These probes exhibit a selective and reversible response to O 2 within the full physiological oxygen range (0 to 250 μM) combined with optimal photophysical properties (for example, high fluorescence brightness). Nevertheless, in comparison to genetically encoded sensors intracellular loading of these dyes appears to be relatively inefficient, slow and, furthermore, they cannot be generally targeted to specific cells or intracellular compartments [19,44,45]. Consequently, intracellular visualization of O 2 with high spatio-temporal resolution is limited.…”