10 research outputs found
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Toxicological Responses of α-Pinene-Derived Secondary Organic Aerosol and Its Molecular Tracers in Human Lung Cell Lines
Secondary organic aerosol (SOA) is a major component of airborne fine particulate matter (PM2.5) that contributes to adverse human health effects upon inhalation. Atmospheric ozonolysis of α-pinene, an abundantly emitted monoterpene from terrestrial vegetation, leads to significant global SOA formation; however, its impact on pulmonary pathophysiology remains uncertain. In this study, we quantified an increasing concentration response of three well-established α-pinene SOA tracers (pinic, pinonic, and 3-methyl-1,2,3-butanetricarboxylic acids) and a full mixture of α-pinene SOA in A549 (alveolar epithelial carcinoma) and BEAS-2B (bronchial epithelial normal) lung cell lines. The three aforementioned tracers contributed ∼57% of the α-pinene SOA mass under our experimental conditions. Cellular proliferation, cell viability, and oxidative stress were assessed as toxicological end points. The three α-pinene SOA molecular tracers had insignificant responses in both cell types when compared with the α-pinene SOA (up to 200 μg mL-1). BEAS-2B cells exposed to 200 μg mL-1 of α-pinene SOA decreased cellular proliferation to ∼70% and 44% at 24- and 48-h post exposure, respectively; no changes in A549 cells were observed. The inhibitory concentration-50 (IC50) in BEAS-2B cells was found to be 912 and 230 μg mL-1 at 24 and 48 h, respectively. An approximate 4-fold increase in cellular oxidative stress was observed in BEAS-2B cells when compared with untreated cells, suggesting that reactive oxygen species (ROS) buildup resulted in the downstream cytotoxicity following 24 h of exposure to α-pinene SOA. Organic hydroperoxides that were identified in the α-pinene SOA samples likely contributed to the ROS and cytotoxicity. This study identifies the potential components of α-pinene SOA that likely modulate the oxidative stress response within lung cells and highlights the need to carry out chronic exposure studies on α-pinene SOA to elucidate its long-term inhalation exposure effects. © 2021 American Chemical Society
Double casting prototyping with a thermal aging step for fabrication of 3D microstructures in poly(dimethylsiloxane)
The paper describes a cheap and accessible technique of a poly(dimethylsiloxane) (PDMS) master treatment by thermal aging as a step of double casting microfabrication process. Three-dimensional PDMS microstructures could have been achieved using this technique. It was proved, that thermal aging changes nanotopology of a PDMS surface and thus enhances efficiency of double casting prototyping. The thermally aged PDMS master could have been used for multiple and correct replication of over 98% of the fabricated microstructures. Moreover, lack of chemical modification preserved the biocompatibility of PDMS devices. The fabricated microstructures were successfully utilized for 3D cell culture
Three-layer poly(methyl methacrylate) microsystem for analysis of lysosomal enzymes for diagnostic purposes
Cellular Uptake of Bevacizumab in Cervical and Breast Cancer Cells Revealed by Single-Molecule Spectroscopy
Bevacizumab is a biological drug that is now extensively
studied
in clinics against various types of cancer. Although bevacizumab’s
action is preferably extracellular, there are reports suggesting its
internalization into cancer cells, consequently decreasing its therapeutic
potential. Here we are solving this issue by applying fluorescence
correlation spectroscopy in living cells. We tracked single molecules
of fluorescent bevacizumab in MDA-MB-231 and HeLa cells and proved
that mobility measurements bring significant added value to standard
imaging techniques. We confirmed the presence of the drug in intracellular
vesicles. Additionally, we explicitly excluded the presence of a free
cytosolic fraction of bevacizumab in both studied cell types. Extracellular
and intracellular concentrations of the drug were measured, giving
a partition coefficient on the order of 10–5, comparable
with the spontaneous uptake of biologically inert nanoparticles. Our
work presents how techniques and models developed for physics can
answer biological questions
Quantitative analysis of biochemical processes in living cells at a single-molecule level:a case of olaparib–PARP1 (DNA repair protein) interactions
Quantitative description of biochemical processes inside living cells and at single-molecule levels remains a challenge at the forefront of modern instrumentation and spectroscopy. This paper demonstrates such single-cell, single-molecule analyses performed to study the mechanism of action of olaparib – an up-to-date, FDA-approved drug for germline-BRCA mutated metastatic breast cancer. We characterized complexes formed with PARPi-FL – fluorescent analog of olaparib in vitro and in cancer cells using the advanced fluorescent-based method: Fluorescence Correlation Spectroscopy (FCS) combined with a length-scale dependent cytoplasmic/nucleoplasmic viscosity model. We determined in vitro olaparib–PARP1 equilibrium constant (6.06 × 108 mol L−1). In the cell nucleus, we distinguished three states of olaparib: freely diffusing drug (24%), olaparib–PARP1 complex (50%), and olaparib–PARP1–RNA complex (26%). We show olaparib accumulation in 3D spheroids, where intracellular concentration is twofold higher than in 2D cells. Moreover, olaparib concentration was tenfold higher (506 nmol L−1 vs. 57 nmol L−1) in cervical cancer (BRCA1 high abundance) than in breast cancer cells (BRCA1 low abundance) but with a lower toxic effect. Thus we confirmed that the amount of BRCA1 protein in the cells is a better predictor of the therapeutic effect of olaparib than its penetration into cancer tissue. Our single-molecule and single-cell approach give a new perspective of drug action in living cells. FCS provides a detailed in vivo insight, valuable in drug development and targeting
Apparent Anomalous Diffusion in the Cytoplasm of Human Cells: The Effect of Probes’ Polydispersity
This
work, based on <i>in vivo</i> and <i>in vitro</i> measurements, as well as <i>in silico</i> simulations,
provides a consistent analysis of diffusion of polydisperse nanoparticles
in the cytoplasm of living cells. Using the example of fluorescence
correlation spectroscopy (FCS), we show the effect of polydispersity
of probes on the experimental results. Although individual probes
undergo normal diffusion, in the ensemble of probes, an effective
broadening of the distribution of diffusion times occursî—¸similar
to anomalous diffusion. We introduced fluorescently labeled dextrans
into the cytoplasm of HeLa cells and found that cytoplasmic hydrodynamic
drag, exponentially dependent on probe size, extraordinarily broadens
the distribution of diffusion times across the focal volume. As a
result, the <i>in vivo</i> FCS data were effectively fitted
with the anomalous subdiffusion model while for a monodisperse probe
the normal diffusion model was most suitable. Diffusion time obtained
from the anomalous diffusion model corresponds to a probe whose size
is determined by the weight-average molecular weight of the polymer.
The apparent anomaly exponent decreases with increasing polydispersity
of the probes. Our results and methodology can be applied in intracellular
studies of the mobility of nanoparticles, polymers, or oligomerizing
proteins