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

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5442
Author(s):  
Sergey S. Ovcherenko ◽  
Olga A. Chinak ◽  
Anton V. Chechushkov ◽  
Sergey A. Dobrynin ◽  
Igor A. Kirilyuk ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Laser Scanning ◽  
Human Lung ◽  
A549 Cells ◽  
Laser Scanning Microscopy ◽  
Human Lung Cancer ◽  
Confocal Laser ◽  
Paramagnetic Resonance ◽  
Cell Penetrating ◽  
Electron Paramagnetic

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.

scholarly journals Free Radical-Mediated Protein Radical Formation in Differentiating Monocytes

2021 ◽  
Vol 22 (18) ◽  
pp. 9963
Author(s):  
Ankush Prasad ◽  
Renuka Ramalingam Manoharan ◽  
Michaela Sedlářová ◽  
Pavel Pospíšil
Keyword(s):  
Free Radical ◽  
Laser Scanning ◽  
Model System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Paramagnetic Resonance ◽  
Nadph Oxidase 4 ◽  
Ros Formation ◽  
Inflammatory Macrophages ◽  
Electron Paramagnetic

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

scholarly journals pH and Reduction Dual-Responsive Bi-Drugs Conjugated Dextran Assemblies for Combination Chemotherapy and In Vitro Evaluation

Polymers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1515
Author(s):  
Xiukun Xue ◽  
Yanjuan Wu ◽  
Xiao Xu ◽  
Ben Xu ◽  
Zhaowei Chen ◽  
...  
Keyword(s):  
Combination Chemotherapy ◽  
Laser Scanning ◽  
Rational Design ◽  
A549 Cells ◽  
Chemotherapeutic Agents ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tumor Resistance ◽  
Oxidized Dextran

Polymeric prodrugs, synthesized by conjugating chemotherapeutic agents to functional polymers, have been extensively investigated and employed for safer and more efficacious cancer therapy. By rational design, a pH and reduction dual-sensitive dextran-di-drugs conjugate (oDex-g-Pt+DOX) was synthesized by the covalent conjugation of Pt (IV) prodrug and doxorubicin (DOX) to an oxidized dextran (oDex). Pt (IV) prodrug and DOX were linked by the versatile efficient esterification reactions and Schiff base reaction, respectively. oDex-g-Pt+DOX could self-assemble into nanoparticles with an average diameter at around 180 nm. The acidic and reductive (GSH) environment induced degradation and drug release behavior of the resulting nanoparticles (oDex-g-Pt+DOX NPs) were systematically investigated by optical experiment, DLS analysis, TEM measurement, and in vitro drugs release experiment. Effective cellular uptake of the oDex-g-Pt+DOX NPs was identified by the human cervical carcinoma HeLa cells via confocal laser scanning microscopy. Furthermore, oDex-g-Pt+DOX NPs displayed a comparable antiproliferative activity than the simple combination of free cisplatin and DOX (Cis+DOX) as the extension of time. More importantly, oDex-g-Pt+DOX NPs exhibited remarkable reversal ability of tumor resistance compared to the cisplatin in cisplatin-resistant lung carcinoma A549 cells. Take advantage of the acidic and reductive microenvironment of tumors, this smart polymer-dual-drugs conjugate could serve as a promising and effective nanomedicine for combination chemotherapy.

scholarly journals Cell-Penetrating Peptide and Transferrin Co-Modified Liposomes for Targeted Therapy of Glioma

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3540 ◽  
Author(s):  
Xi Wang ◽  
Yarong Zhao ◽  
Shiyan Dong ◽  
Robert J. Lee ◽  
Dongsheng Yang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Therapeutic Agent ◽  
Spherical Shape ◽  
Cell Penetrating Peptides ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Great Promise ◽  
Uniform Size ◽  
Cell Penetrating ◽  
Low Toxicity

Glioma is one of the most aggressive and common malignant brain tumors. Due to the presence of the blood-brain barrier (BBB), the effectiveness of therapeutics is greatly affected. In this work, to develop an efficient anti-glioma drug with targeting and which was able to cross the BBB, cell-penetrating peptides (R8) and transferrin co-modified doxorubicin (DOX)-loaded liposomes (Tf-LPs) were prepared. Tf-LPs possessed a spherical shape and uniform size with 128.64 nm and their ξ-potential was 6.81 mV. Tf-LPs were found to be stable in serum within 48 h. Uptake of Tf-LPs in both U87 and GL261 cells was analyzed by confocal laser scanning microscopy and by flow cytometry. Tf-LPs were efficiently taken up by both U87 and GL261 cells. Moreover, Tf-LPs exhibited sustained-release. The cumulative release of DOX from Tf-LPs reached ~50.0% and showed excellent anti-glioma efficacy. Histology of major organs, including brain, heart, liver, spleen, lungs and kidney, and the bodyweight of mice, all indicated low toxicity of Tf-LPs. In conclusion, Tf-LPs showed great promise as an anti-glioma therapeutic agent.

scholarly journals Quantifying Cytosolic Cytochrome c Concentration Using Carbon Quantum Dots as a Powerful Method for Apoptosis Detection

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1556
Author(s):  
Cristian Silviu Moldovan ◽  
Anca Onaciu ◽  
Valentin Toma ◽  
Radu Marginean ◽  
Alin Moldovan ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Cytochrome C ◽  
Laser Scanning ◽  
A549 Cells ◽  
Carbon Quantum Dots ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Fluorescent Nanoparticles ◽  
Cyt C

Background: Cytochrome c (Cyt c) is a key biomarker for early apoptosis, and many methods were designed to detect its release from mitochondria. For a proper evaluation of these programed cell death mechanisms, fluorescent nanoparticles are excellent candidates due to their valuable optical properties. Among all classes of nanoparticles developed thus far, carbon-based quantum dots bring qualitative and efficient imaging strategies for biomedical applications as a consequence of their biocompatibility and low cytotoxicity. Methods: In this study, we synthesized carbon quantum dots smaller than 5 nm from sodium citrate and polyethylene imine. These nanoparticles were rigorously characterized, and their quenching capacity in apoptotic events was assessed in A549 cells treated with staurosporine and etoposide. For the evaluation of Cyt c release, a phenomenon directly correlated with apoptotic events, we ran a semiquantitative analysis using confocal laser scanning microscopy. Results: Carbon quantum dots were synthesized and were successfully employed for Cyt c detection by means of fluorescence microscopy. Significant drops in fluorescence intensity were observed in the case of cells treated with apoptosis-inducing therapeutic compounds compared to untreated cells, confirming Cyt c release from mitochondria to cytosol. Conclusion: Considering these results, we strongly believe this method can contribute to an indirect in vitro evaluation of apoptosis.

scholarly journals Design of new acid-activated cell-penetrating peptides for tumor drug delivery

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3429 ◽  
Author(s):  
Jia Yao ◽  
Yinyun Ma ◽  
Wei Zhang ◽  
Li Li ◽  
Yun Zhang ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Targeted Delivery ◽  
Laser Scanning ◽  
Critical Role ◽  
Cell Penetrating Peptides ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ph Response ◽  
Cell Penetrating ◽  
Ph Dependent

TH(AGYLLGHINLHHLAHL(Aib)HHIL-NH2), a histidine-rich, cell-penetrating peptide with acid-activated pH response, designed and synthesized by our group, can effectively target tumor tissues with an acidic extracellular environment. Since the protonating effect of histidine plays a critical role in the acid-activated, cell-penetrating ability of TH, we designed a series of new histidine substituents by introducing electron donating groups (Ethyl, Isopropyl, Butyl) to the C-2 position of histidine. This resulted in an enhanced pH-response and improved the application of TH in tumor-targeted delivery systems. The substituents were further utilized to form the corresponding TH analogs (Ethyl-TH, Isopropyl-TH and Butyl-TH), making them easier to protonate for positive charge in acidic tumor microenvironments. The pH-dependent cellular uptake efficiencies of new TH analogs were further evaluated using flow cytometry and confocal laser scanning microscopy, demonstrating that ethyl-TH and butyl-TH had an optimal pH-response in an acidic environment. Importantly, the new TH analogs exhibited relatively lower toxicity than TH. In addition, these new TH analogs were linked to the antitumor drug camptothecin (CPT), while butyl-TH modified conjugate presented a remarkably stronger pH-dependent cytotoxicity to cancer cells than TH and the other conjugates. In short, our work opens a new avenue for the development of improved acid-activated, cell-penetrating peptides as efficient anticancer drug delivery vectors.

all-D proline-rich cell-penetrating peptides: a preliminary in vivo internalization study

2007 ◽  
Vol 35 (4) ◽  
pp. 794-796 ◽  
Author(s):  
S. Pujals ◽  
E. Sabidó ◽  
T. Tarragó ◽  
E. Giralt
Keyword(s):  
Self Assembly ◽  
Laser Scanning ◽  
White Blood Cells ◽  
Cell Penetrating Peptides ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Penetrating ◽  
Peptide Derivatives ◽  
Degradation Problem

Proline-rich cell-penetrating peptides, particularly the SAP (sweet arrow peptide), (VRLPPP)3, have been proposed to be useful intracellular delivery vectors, as a result of their lack of cytotoxicity combined with their capacity to be internalized by cells. A common limitation of the therapeutic use of peptides is metabolic instability. In general, peptides are quickly degraded by proteases upon entry into the bloodstream. The use of all-D-peptide derivatives is emerging as a fruitful strategy to circumvent this degradation problem. In this context, we report on the internalization behaviour, protease-resistance enhancement and self-assembly properties of an all-D version of SAP [(vrlppp)3]. The cellular uptake of (vrlppp)3 was evaluated in an in vivo assay in mice. Both flow cytometry and confocal laser-scanning microscopy experiments showed that a carboxyfluoresceinated version of the molecule, carboxyfluorescein–(vrlppp)3, is internalized rapidly in white blood cells and kidney cells. Significant fluorescence was also detected in other organs such as the spleen and the liver. Finally, the toxicity of (vrlppp)3 was examined, and no significant differences in the main biochemical parameters nor in weight were detected compared with controls.

scholarly journals Casticin Induces DNA Damage and Affects DNA Repair Associated Protein Expression in Human Lung Cancer A549 Cells

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 341 ◽  
Author(s):  
Zheng-Yu Cheng ◽  
Yung-Ting Hsiao ◽  
Yi-Ping Huang ◽  
Shu-Fen Peng ◽  
Wen-Wen Huang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Damage ◽  
Human Lung ◽  
A549 Cells ◽  
Cell Number ◽  
Confocal Laser Microscopy ◽  
Human Lung Cancer ◽  
Confocal Laser ◽  
Dna Damage And Repair ◽  
Laser Microscopy

Casticin was obtained from natural plants, and it has been shown to exert biological functions; however, no report concerns the induction of DNA damage and repair in human lung cancer cells. The objective of this study was to investigate the effects and molecular mechanism of casticin on DNA damage and repair in human lung cancer A549 cells. Cell viability was determined by flow cytometric assay. The DNA damage was evaluated by 4’,6-diamidino-2-phenylindole (DAPI) staining and electrophoresis which included comet assay and DNA gel electrophoresis. The protein levels associated with DNA damage and repair were analyzed by western blotting. The expression and translocation of p-H2A.X were observed by confocal laser microscopy. Casticin reduced total viable cell number and induced DNA condensation, fragmentation, and damage in A549 cells. Furthermore, casticin increased p-ATM at 6 h and increased p-ATR and BRCA1 at 6–24 h treatment but decreased p-ATM at 24–48 h, as well as decreased p-ATR and BRCA1 at 48 h. Furthermore, casticin decreased p-p53 at 6–24 h but increased at 48 h. Casticin increased p-H2A.X and MDC1 at 6–48 h treatment. In addition, casticin increased PARP (cleavage) at 6, 24, and 48 h treatment, DNA-PKcs and MGMT at 48 h in A549 cells. Casticin induced the expressions and nuclear translocation of p-H2AX in A549 cells by confocal laser microscopy. Casticin reduced cell number through DNA damage and condensation in human lung cancer A549 cells.

scholarly journals Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells

2019 ◽  
Vol 20 (21) ◽  
pp. 5253 ◽  
Author(s):  
Arnold Sipos ◽  
Kwang-Jin Kim ◽  
Constantinos Sioutas ◽  
Edward D. Crandall
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Laser Scanning ◽  
A549 Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cancer Management ◽  
Lysosomal Dysfunction

Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

scholarly journals Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry

2005 ◽  
Vol 67 (2) ◽  
pp. 79-89 ◽  
Author(s):  
John L. Pauly ◽  
Erin M. Allison ◽  
Edward L. Hurley ◽  
Chukwumere E. Nwogu ◽  
Paul K. Wallace ◽  
...  
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Human Lung ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Lung Macrophages

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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