Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Methods
2.2.1. RL2 Expression and Chromatographic Separation of RL2 Dimers and Monomers
2.2.2. The Synthesis of the Conjugate of Spin Label 1 with the Recombinant Lactaptin Analogue (sRL2)
2.2.3. Analysis of the Internalization of Spin Label 1 and sRL2 into Cells
2.2.4. EPR Measurements
2.2.5. Confocal Microscopy
3. Results
3.1. The Synthesis of a Conjugate of Spin Label 1 with the Recombinant Lactaptin Analogue (sRL2)
3.2. Penetration of Nitroxide 2 into A549 Cells
3.3. Penetration of sRL2 into A549 Cells
3.4. Simulation of EPR Spectra
3.5. Localization of the Fluorescent RL22 Conjugate in A549 Cells
3.6. The Influence of Sodium Azide on the Penetration of sRL2 into A549 Cells
3.7. Verification of Trypsin Effectivity to Remove sRL2 from Membrane Surface
3.8. Viability of the Cells during the EPR Measurements
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Ovcherenko, S.S.; Chinak, O.A.; Chechushkov, A.V.; Dobrynin, S.A.; Kirilyuk, I.A.; Krumkacheva, O.A.; Richter, V.A.; Bagryanskaya, E.G. Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy. Molecules 2021, 26, 5442. https://doi.org/10.3390/molecules26185442
Ovcherenko SS, Chinak OA, Chechushkov AV, Dobrynin SA, Kirilyuk IA, Krumkacheva OA, Richter VA, Bagryanskaya EG. Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy. Molecules. 2021; 26(18):5442. https://doi.org/10.3390/molecules26185442
Chicago/Turabian StyleOvcherenko, Sergey S., Olga A. Chinak, Anton V. Chechushkov, Sergey A. Dobrynin, Igor A. Kirilyuk, Olesya A. Krumkacheva, Vladimir A. Richter, and Elena G. Bagryanskaya. 2021. "Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy" Molecules 26, no. 18: 5442. https://doi.org/10.3390/molecules26185442