Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy

Abstract

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

3,4-Unsubstituted 2-tert-Butyl-pyrrolidine-1-oxyls with Hydrophilic Functional Groups in the Side Chains

“…Recently, nitroxides with methyl groups replaced with bulkier alkyl substituents have attracted much attention. These so-called “sterically shielded” nitroxides demonstrated much higher stability against chemical reduction to diamagnetic compounds with components of biological systems then their tetramethyl analogs did [ 4 , 5 , 6 ]. The advantage of these reduction-resistant radicals over conventional tetramethyl-substituted nitroxides is especially obvious when they are used for EPR measurements inside living cells [ 7 , 8 ], or in vivo for functional imaging using MRI or EPRI techniques [ 9 , 10 ].…”

An EPR Study on Highly Stable Nitroxyl-Nitroxyl Biradicals for Dynamic Nuclear Polarization Applications at High Magnetic Fields

“…The structure of our tetraethyl nitroxides 3 and 4 obtained by X-ray [ 42 , 43 ] clearly shows that this “shielding” arises due to tetraethyl substituents, which form a dense shell at a distance of ~3 Å around the nitroxide moiety. On the other hand, it is this shielding that ensures tetraethyl nitroxide’s high stability against reduction by ascorbate or any other reducing agent, and allows their use in living systems and in the cells [ 44 ]. To underline this hypothesis, for our biradicals 3 and 4 , the resistance to reduction by ascorbic acid was inspected (for details see Section 2.3 ) showing that 3 and 4 are stable for several hours, in contrast to spiro-substituted PAs, which exist for only ~10 min in the presence of ascorbate [ 36 ].…”

Dance with spins: site-directed spin labeling coupled to electron paramagnetic resonance spectroscopy directly inside cells

“…This approach allowed tracking dynamics and accessibility to reducing agents with a time resolution of maximum 10 minutes. 52,86 The development of a commercially available Rapid-scan setup coupled with in-cell EPR will improve the time resolution up to ms ms À1 according to the T 2 of the sample, increasing up to 17 times the signal-to-noise ratio. 87 The first proof-of-concept in this matter has been recently published, demonstrating that is possible to follow the interaction of alphasynuclein and lipids in X. Laevis oocytes.…”