Effects of NPs on swelling kinetics and diffusivity of secretory granule matrix.

<p>(A) Data points of swelling kinetics of secretory granule matrices of A549 cells were fit with equation (1). Exocytosis was triggered by ionomycin. Two representative lines are displayed here. The control (NP free; open circles) shows a higher rate of swelling of newly released mucin network than the swelling rate of mucin matrix when exposed to positively-charged NP (160 nm, 1 mg/L, solid circle). The black arrows indicate the swelling of exocytosed mucin matrix at different time points. (B) Positively-charged NPs hinder the rate of mucin diffusivity (hydration). Mucin post-exocytotic swelling from A549 cells was significantly reduced by positively- charged NPs (160 and 57 nm). This protocol to estimate mucin diffusivity of newly released mucin gels from A549 cells was adapted from our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015434#pone.0015434-Chin2" target="_blank">[65]</a>. Data are shown as mean±SD (n≥10). NP treated groups are significantly different from the untreated control at p<0.005 as indicated by *.</p

SEM images of mucin-NP complexes.

<p>SEM micrographs of mucin-NPs complexes with 160 nm (A, C) and 57 nm (B, D) positively-charged NPs after 5 hrs (A, B) and 72 hrs (C, D). There SEM images clearly demonstrate that NPs and mucins aggregate together forming large mucin-NPs gels. The black holes in the images are the pores in Isopore membranes. The scale bar is 1 µm.</p

Interaction of NPs and mucin aggregation.

<p>(A) Positively-charged NPs (160 nm) (n≥6) induce significant mucin aggregation while (B) negatively-charged (120 nm) (n≥6) and (C) non-functionalized NPs (99 nm) (n≥6) fail to induce mucin aggregation since the aggregate size remained below 300 nm throughout 72 hrs incubation. (D) Positively-charged NPs (160 nm) (n≥5) alone can not generate large aggregates in the same Hanks' solution. Smaller size positively-charged (57 nm) (n≥6) promote larger mucin aggregates (E). At the same time, negative controls show that (F) positively-charged NPs (57 nm) (n≥5) or (G) mucin alone (1 mg/L, n≥5) can not generate large aggregates. Various concentrations (solid circles: 100 µg/L, solid triangles: 1 mg/L, solid squares: 10 mg/L) of positively-charged, negatively-charged and non-functionalized NPs (160, 57, 120 and 99 nm) were added to mucin solution (1 mg/L). Significant mucin aggregates were found at 10 mg/L and 1 mg/L of positive NPs. The size of mucin-NPs aggregates were determined with DLS as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015434#pone.0015434-Bhaskar1" target="_blank">[37]</a>.</p

Inability of EGTA (Ca²⁺ chelator) to disperse NPs-induced mucin aggregates.

<p>Positively-charged NPs with sizes of 160 nm (A) and 57 nm (B) at 10 mg/L induced mucin (1 mg/L) to aggregate forming large size mucin-NPs aggregates (∼6 µm) in 72 hrs (n≥6). EGTA (2 mM) was added to chelate Ca²⁺ ions that can crosslink mucins forming gels. However, EGTA can not disperse mucin-NPs aggregates after 72 hrs incubation.</p

Decreased microgel equilibrium size (black circles) and bound Ca²⁺ (blue triangles) with concomitant increase in hydrophobicity (red squares).

<p>The non-linear rate of declining microgel size with increased temperature indicates potential cooperativity; around 32˚C all three parameters experienced the most pronounced associative effect—a major drop in microgel size and bound Ca²⁺, with a concomitant rise in hydrophobicity.</p

DOM assembly monitored with temperature and pH reveals that, as either pH decreases or temperature increases, microgel equilibrium size and assembly rates decrease at a non-linear rate.

<p>a. DOM assembly at three temperatures—22˚C (black circles), 30˚C (blue triangles), 32˚C (red squares)—over time at three pH units. Each data point represents (mean ± SD) of six measurements made in each of six replicate samples. b. DOM assembly at three pHs—8.0 (black circles), 7.7 (blue triangles), 7.5 (red squares)—over time at three constant temperature incubations. Microgels assembled in identical pH conditions showed equilibrium size reduction and decelerated non-linear assembly rates when exposed to increased temperature. Each data point represents the mean (+/− SD) of six measurements made in each of six replicate samples. Shaded windows represent an average microgel equilibrium size range (4–6 μm) at 22˚C and pH 8.</p

Measurement of the [Ca²⁺]_C and calcein dye leakage after TiO₂ NP treatment.

<p>Cells were treated with TiO₂ NPs with concentrations ranging from 0.05 mg/ml–1 mg/ml, in A) Ca²⁺-free Hanks' solution, B) in the presence of CdCl₂ (200 µM), C) nifedipine (10 µM), D) calcein (50 µM) (n = 12, **P<0.005), and E) NAC (250 µM) (colors are as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016198#pone-0016198-g001" target="_blank">Figure 1B</a>).</p

Microgel assembly / dispersion are temperature dependent.

<p>1-a. Microgel assembly rate and equilibrium size decreases with increased temperature. Samples were incubated at 22˚C (black circles), 32˚C (blue triangles) and 35˚C (red squares) for 24 hours, then stored in the dark at 22˚C for the remainder of the experiment. Assembly was measured using dynamic laser scattering at 22˚C. Each data point represents (mean ± SD) of six measurements made in each of six replicate samples. Data highlight that short-term temperature exposure above 35˚C confers significant DOM assembly loss with no obvious recovery. 1-b. Microgel dispersion depends on temperature variation. Self-assembled microgels (size ~ 6 μm) were incubated at various temperatures (from 22˚C to 40 ˚C) for 24 hours. The equilibrium microgel sizes were monitored with dynamic laser scattering spectroscopy. Each data point represents six replicate samples. Non-linear temperature responses of microgels were observed—particularly for microgels incubated at temperatures above 32˚C, which showed a marked size decrease.</p

Measurement of [Ca²⁺]_C after stimulation by TiO₂ NPs.

<p>Cells were treated with TiO₂ NPs with concentrations ranging from 0.1 mg/ml–1 mg/ml, in the presence of A) thapsigargin (100 nM), and B) ryanodine (100 µM) (colors used are consistent with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016198#pone-0016198-g001" target="_blank">Figure 1</a>).</p

Mucin secretion in response to ionomycin application (positive control, n≥3).

<p>Concentration of ionomycin used was 1 µM.</p