Effects of As₂O₃ on A431 cell EGFR expression.

<p>Cells were exposed to different concentrations of As₂O₃. At 48 h post-treatment, cells were assessed by fluorescence microscopy for visualization of the intake of EGF-Cy5.5 and cell immunohistochemistry for assay of EGFR expression. A–D, representative fluorescence images of different groups (Scale bar = 100 µm), Cy5.5 was pseudo-colored red, DAPI was pseudo-colored blue; E-H, representative images of cellular EGFR Immunohistochemistry assay (Scale bar = 100 µm), diaminobenzidine (DAB) showed as brown color represented EGFR expression and hematoxylin showed as blue color indicated the cellular nuclear. Interestingly, cell numbers decreased as the arsenic trioxide concentration increased from 0 µM to 5 µM in both fluorescent and immunostained images, indicating that As₂O₃ induced a dose-dependent inhibition on tumor cell proliferation as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046255#pone.0046255-Kumar1" target="_blank">[31]</a>.</p

Monitoring tumor growth and body weight of tumor-bearing mice during treatment.

<p>Tumor growth (A) and body weight (B) of tumor-bearing mice treated with saline (as untreated control group), arsenic trioxide (ATO) at 0.5 mg/kg, 2.5 mg/kg or 5.0 mg/kg daily for 12 days. Six mice were used in each group. The tumor volume and body weight of all four groups were also measured every two days. Tumor volume was calculated according to the formula V = (a×b²)/2 where a and b represent the length and width of the tumor. Measurements were continued to 12th day. <i>p</i><0.05 is a significant difference between control and treatment groups.</p

<i>In vivo</i> dynamic near-infrared fluorescent imaging of A-431 tumor models.

<p>A: The representative fluorescence images of the tumor regions in mice were acquired at 4 h post injection of EGF-Cy5.5. Fluorescence signal from Cy5.5 was pseudo-colored red. B: The dynamic measurement comparison of fluorescence intensity of tumor in different groups. It was demonstrated that the fluorescence intensity in the tumor regions were changed with time (<i>p</i><0.05). On day 0 (before As₂O₃ treatment), there was no significant difference of signal intensity of tumors between treatment and control groups (<i>p</i>>0.05). On day 4, 8, 12 (after As₂O₃ treatment), the signal intensity of EGF-Cy5.5 uptake by control group (0 mg/kg/day As₂O₃) gradually increased, while the intensities in other three groups with different concentrations (0.5 mg/kg/day, 2.5 mg/kg/day, 5.0 mg/kg/day) of As₂O₃ treatment gradually decreased (<i>p</i><0.05). C: The <i>in vivo</i> fluorescence intensity was compared between post-treatment (on day 12) in four different groups compared with respective pre-treatment (on day 0). All plots are representative of results from groups of mice treated under the same experimental conditions. Each point represents the mean values (n = 6/group, *<i>p</i><0.05, **<i>p</i><0.01).</p

Physicochemical properties and stability of blank NPs and mel-NPs.

<p>(<b>A</b> and <b>B</b>) Size and zeta potential of blank NPs and mel-NPs immediately following preparation. (<b>C</b> and <b>D</b>) Size and zeta potential of blank NPs and mel-NPs following incubation for up to 3 days at pH 7.4 or pH 4.4. Error bars represent S.D. of n = 3 replicates, NS p>0.05, ** p<0.01, *** p<0.001.</p

The cellular EGFR expression percentage of different treatment groups <i>in vitro</i> assessed by flow cytometry.

<p>The dynamic EGFR cellular expression percentage after treatment of different concentrations of As₂O₃ (0 µM, 0.5 µM, 2.5 µM, 5.0 µM) varied with time (*<i>p</i><0.05, **<i>p</i><0.01). All experiments were carried out in triplicate; each point represents the mean ± standard error values.</p

Characterization of SPAM1-targeted NPs and their interactions with sperm.

<p>(<b>A</b> and <b>B</b>) Size and zeta potential of anti-SPAM1 NPs and anti-SPAM1-mel-NPs immediately following preparation. (<b>C</b>) Fluorescence of sperm-bound blank NPs and anti-SPAM1 NPs following incubation with sperm for 30 minutes and removal of unbound NPs by density gradient centrifugation. Control samples did not contain sperm and were used to determine background fluorescence due to remaining unbound NPs. (<b>D</b>) Standard curve of blank NP and anti-SPAM1 NP fluorescence used for quantification of sperm binding. Error bars represent S.D. of n = 3 replicates, NS p>0.05, *** p<0.001. (<b>E</b>, <b>F</b>, <b>G</b> and <b>H</b>) Brightfield and fluorescence images of semen samples following addition of 3×10¹⁰ blank NPs or anti-SPAM1 NPs. Scale bar = 50 µm.</p

EGFR immunohistochemical assay of the tumor sections from different groups.

<p>A: 0 mg/kg As₂O₃ (control). The strongest red-brownish membrane-bound immunostaining on the A431 tumor tissue slice reflected the abundant over expression of EGFR (+++). B: 0.5 mg/kg As₂O₃ treatment group. Strong brownish membrane staining indicated plentiful of EGFR expression (++∼+++). C: 2.5 mg/kg As₂O₃ treatment group demonstrated moderate to low EGFR expression (+∼++). D: 5.0 mg/kg As₂O₃ treatment group showed weak EGFR expression (+). (Scale bar = 100 µm).</p

Effect of free melittin, blank NPs and mel-NPs on vaginal epithelial cell viability.

<p>(<b>A</b>) VK2 vaginal cell viability following treatment with a single dose of free melittin, blank NPs or mel-NPs for 12 hours. (<b>B</b>) VK2 vaginal cell viability following repeated treatment with fresh media (control) or a 10 µM equivalent melittin dose of blank NPs or mel-NPs once per day for 3 days. VK2 viability was determined using MTT assay. Error bars represent S.D. of n = 6 replicates. Labels indicate level of statistical significance compared to untreated control, NS p>0.05, *** p<0.001.</p

Effect of free melittin, blank NPs, mel-NPs, anti-SPAM1 NPs, and anti-SPAM1-mel-NPs on sperm motility and viability.

<p>(A and B) Sperm motility and viability following treatment with the appropriate agent for 30 minutes. Motility and viability were determined by IVOS Computer Assisted Sperm Analysis and normalized to the motility and viability of untreated sperm. Error bars represent S.D. of n = 3 replicates using sperm samples from distinct donors. Labels indicate level of statistical significance compared to untreated control, NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.</p

The effect of PFC plus carbogen and carbogen alone on survival of mice with intracranial tumours who received a single dose of whole brain radiation.

<p>Kaplan Meier curves were compiled from 3 separate experiments. Mice were randomly assigned to groups of 5 mice. Mice were left untreated (black line), given 4.5Gy of radiation (red line), breathing carbogen (95%O₂/5%CO₂) for 1h immediately prior to irradiation (green line) or injected IV with 1.5cc/kg of a 40% PFC emulsion in addition to breathing carbogen for one hour immediately prior to irradiation (blue line). Only data from animals that died from tumour progression were used. Data from animals that died from other causes were excluded.</p