skip to main content

This content will become publicly available on January 1, 2024

Title: Scanning ion conductance microscopy reveals differential effect of PM2.5 exposure on A549 lung epithelial and SH-SY5Y neuroblastoma cell membranes
Numerous studies have linked a wide range of diseases including respiratory illnesses to harmful particulate matter (PM) emissions indoors and outdoors, such as incense PM and industrial PM. Because of their ability to penetrate the lower respiratory tract and the circulatory system, fine particles with diameters of 2.5 µm or less (PM2.5) are believed to be more hazardous than larger PMs. Despite the enormous number of studies focusing on the intracellular processes associated with PM2.5 exposure, there have been limited reports studying the biophysical properties of cell membranes, such as nanoscale morphological changes induced by PM2.5. Our study assesses the membrane topographical and structural effects of PM2.5 from incense PM2.5 exposure in real time on A549 lung carcinoma epithelial cells and SH-SY5Y neuroblastoma cells that had been fixed to preclude adaptive cell responses. The size distribution and mechanical properties of the PM2.5 sample were characterized with atomic force microscopy (AFM). Nanoscale morphological monitoring of the cell membranes utilizing scanning ion conductance microscopy (SICM) indicated statistically significant increasing membrane roughness at A549 cells at half an hour of exposure and visible damage at 4 h of exposure. In contrast, no significant increase in roughness was observed on SH-SY5Y cells after half an hour of PM2.5 exposure, although continued exposure to PM2.5 for up to 4 h affected an expansion of lesions already present before exposure commenced. These findings suggest that A549 cell membranes are more susceptible to structural damage by PM2.5 compared to SH-SY5Y cell membranes, corroborating more enhanced susceptibility of airway epithelial cells to exposure to PM2.5 than neuronal cells. SICM · Particulate matter · Membrane topography · Single-cell imaging  more » « less
Award ID(s):
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Analytical and Bioanalytical Chemistry
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Scanning ion conductance microscopy (SICM) offers the ability to obtain nanoscale resolution images of the membranes of living cells. Here, we show that a dual-barrel nanopipette probe based potentiometric SICM (P-SICM) can simultaneously map the topography and surface potential of soft, rough and heterogeneously charged surfaces under physiological conditions. This technique was validated and tested by systematic studies on model samples, and the finite element method (FEM) based simulations confirmed its surface potential sensing capability. Using the P-SICM method, we compared both the topography and extracellular potential distributions of the membranes of normal (Mela-A) and cancerous (B16) skin cells. We further monitored the structural and electrical changes of the membranes of both types of cells after exposing them to the elevated potassium ion concentration in extracellular solution, known to depolarize and damage the cell. From surface potential imaging, we revealed the dynamic appearance of heterogeneity of the surface potential of the individual cell membrane. This P-SICM method provides new opportunities to study the structural and electrical properties of cell membrane at the nanoscale. 
    more » « less
  2. Torrecilhas, Ana Claudia (Ed.)
    Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s). 
    more » « less
  3. ABSTRACT Physical agents, such as low electric voltage and current, have recently gained attention for antimicrobial treatment due to their bactericidal capability. Although microampere electric current was shown to suppress the growth of bacteria, it remains unclear to what extent the microampere current damaged the bacterial membrane. Here, we investigated the membrane damage and two-way leakage caused by microampere electric current (≤100 μA) with a short exposure time (30 min). Based on MitoTracker staining, propidium iodide staining, filtration assays, and quantitative single-molecule localization microscopy, we observed significant membrane damage, which allowed two-way leakage of ions, small molecules, and proteins. This study paves the way to new development of antimicrobial applications for ultralow electric voltage and current. IMPORTANCE Although electric voltage and current have been studied for a long time in terms of their ability to suppress the growth of bacteria and to kill bacteria, increasing interest has been aroused more recently due to the prevalence of antibiotic resistance of microbes in past decades. Toward understanding the antimicrobial mechanism of low electric voltage and current, previous studies showed that treating bacteria with milliampere electric currents (≥5 mA) for ≥72 h led to significant damage of the bacterial membrane, which likely resulted in leakage of cellular contents and influx of toxic substances through the damaged membrane. However, it remains unclear to what extent membrane damage and two-way (i.e., inward and outward) leakage are caused by lower (i.e., microampere) electric current in a shorter time frame. In this work, we set out to answer this question. We observed that the membrane damage was caused by microampere electric current in half an hour, which allowed two-way leakage of ions, small molecules, and proteins. 
    more » « less
  4. Abstract. In mid-August through mid-September of 2017 a major wildfire smoke and hazeepisode strongly impacted most of the NW US and SW Canada. During this periodour ground-based site in Missoula, Montana, experienced heavy smoke impactsfor ∼ 500 h (up to 471 µg m−3 hourly averagePM2.5). We measured wildfire trace gases, PM2.5 (particulate matter≤2.5 µm in diameter), and black carbon and submicron aerosolscattering and absorption at 870 and 401 nm. This may be the most extensivereal-time data for these wildfire smoke properties to date. Our range oftrace gas ratios for ΔNH3∕ΔCO and ΔC2H4∕ΔCO confirmed that the smoke from mixed, multiple sourcesvaried in age from ∼ 2–3 h to ∼ 1–2 days. Our study-averageΔCH4∕ΔCO ratio (0.166±0.088) indicated a largecontribution to the regional burden from inefficient smoldering combustion.Our ΔBC∕ΔCO ratio (0.0012±0.0005) for our groundsite was moderately lower than observed in aircraft studies (∼ 0.0015)to date, also consistent with a relatively larger contribution fromsmoldering combustion. Our ΔBC∕ΔPM2.5 ratio (0.0095±0.0003) was consistent with the overwhelmingly non-BC (black carbon),mostly organic nature of the smoke observed in airborne studies of wildfiresmoke to date. Smoldering combustion is usually associated with enhanced PMemissions, but our ΔPM2.5∕ΔCO ratio (0.126±0.002)was about half the ΔPM1.0∕ΔCO measured in freshwildfire smoke from aircraft (∼ 0.266). Assuming PM2.5 isdominated by PM1, this suggests that aerosol evaporation, at least nearthe surface, can often reduce PM loading and its atmospheric/air-qualityimpacts on the timescale of several days. Much of the smoke was emitted latein the day, suggesting that nighttime processing would be important in theearly evolution of smoke. The diurnal trends show brown carbon (BrC),PM2.5, and CO peaking in the early morning and BC peaking in the earlyevening. Over the course of 1 month, the average single scattering albedo forindividual smoke peaks at 870 nm increased from ∼ 0.9 to ∼ 0.96.Bscat401∕Bscat870 was used as a proxy for the size and“photochemical age” of the smoke particles, with this interpretation beingsupported by the simultaneously observed ratios of reactive trace gases toCO. The size and age proxy implied that the Ångström absorptionexponent decreased significantly after about 10 h of daytime smoke aging,consistent with the only airborne measurement of the BrC lifetime in anisolated plume. However, our results clearly show that non-BC absorption canbe important in “typical” regional haze and moderately aged smoke, with BrCostensibly accounting for about half the absorption at 401 nm on average forour entire data set. 
    more » « less
  5. Abstract Particulate matter (PM) concentration levels in the London Underground (LU) are higher than London background levels and beyond World Health Organization (WHO) defined limits. Wheel, track, and brake abrasion are the primary sources of particulate matter, producing predominantly Fe-rich particles that make the LU microenvironment particularly well suited to study using environmental magnetism. Here we combine magnetic properties, high-resolution electron microscopy, and electron tomography to characterize the structure, chemistry, and morphometric properties of LU particles in three dimensions with nanoscale resolution. Our findings show that LU PM is dominated by 5–500 nm particles of maghemite, occurring as 0.1–2 μm aggregated clusters, skewing the size-fractioned concentration of PM artificially to larger sizes when measured with traditional monitors. Magnetic properties are largely independent of the PM filter size (PM 10 , PM 4 , and PM 2.5 ), and demonstrate the presence of superparamagnetic (< 30 nm), single-domain (30–70 nm), and vortex/pseudo-single domain (70–700 nm) signals only (i.e., no multi-domain particles > 1 µm). The oxidized nature of the particles suggests that PM exposure in the LU is dominated by resuspension of aged dust particles relative to freshly abraded, metallic particles from the wheel/track/brake system, suggesting that periodic removal of accumulated dust from underground tunnels might provide a cost-effective strategy for reducing exposure. The abundance of ultrafine particles identified here could have particularly adverse health impacts as their smaller size makes it possible to pass from lungs to the blood stream. Magnetic methods are shown to provide an accurate assessment of ultrafine PM characteristics, providing a robust route to monitoring, and potentially mitigating this hazard. 
    more » « less