12 research outputs found
Cholera toxin B conjugated quantum dots for live cell labeling.
Cholera toxin subunit B (CTB)--quantum dot conjugates were developed for labeling mammalian cells. The conjugates were internalized by all tested cell lines into small vesicles dispersed throughout the cytoplasm, while commercially available polyarginine conjugates rapidly accumulated in large perinuclear endosomes. Although a large proportion of CTB conjugates eventually also accumulated in perinuclear endosomes, this accumulation required several days, and even then many CTB conjugated quantum dots remained in small vesicles dispersed throughout the cytoplasm. Thus CTB conjugates are a practical alternative to polyarginine conjugates for the general labeling of mammalian cells.</p
Nanoparticle Transport from Mouse Vagina to Adjacent Lymph Nodes
<div><p>To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes. A low level of transport was evident at 4 hr after intravaginal instillation, and transport peaked at about 36 hr after instillation. Transport was greatly enhanced by prior vaginal instillation of Nonoxynol-9. Hundreds of micrograms of nanoparticles/kg tissue (ppb) were found in the lumbar lymph nodes at 36 hr post-instillation. Our results imply that targeted transport of microbicides or immunogens from the vagina to local lymph organs is feasible. They also offer an in vivo model for assessing the toxicity of compounds intended for intravaginal use.</p> </div
Identification of Qdots in draining lymph nodes.
<p>A. CD3 staining (green) and CD4 staining (red) in mouse lymph node. N-9 pretreatment and instillation were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051995#pone-0051995-g001" target="_blank">Figure 1</a>. Inset shows tiled panel of entire lymph node. B. Region in white circle from A, which contains a Qdot aggregate, false-colored blue, which is not associated with CD4 or CD8 cells (arrow). C. Qdots (red) associated with macrophage (CD11b, green). Blue, DAPI.</p
Comparison of Qdot content as assessed by integrated spot brightness with cadmium content by ICP-MS.
<p>Data were from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051995#pone-0051995-t002" target="_blank"><b>Table 2</b></a>.</p
Uptake of mixed quantum dots in lymph nodes.
<p>All mice were pretreated with N-9 by instillation of 30 µL 1% N-9 in PBS 12 h before instillation of Qdots. Qdots were instilled as described in Materials and Methods, except that a total of 15 µl of quantum dots in 30 µL total volume (15 pmoles) were instilled singly, or 15 µLeach mixed to give a total volume of 30 µL(15 pmoles each). Mixtures were prepared so as to eliminate any effects due to Qdot size or emission wavelength; Mixture 1 contained 15 pmoles each 655-nm-emitting streptavidin-polyarg Qdots and 800-nm-emitting PEG Qdots, while the reciprocal Mixture 2 contained 15 pmoles each 655-nm-emitting PEG Qdots and 800-nm-emitting streptavidin-polyarg Qdots. Mice were harvested 36 hr after Qdot instillation, scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051995#pone-0051995-t001" target="_blank"><b>Table 1</b></a>, then integrated brightness was measured as described in Materials and Methods. <b>A</b>, Score; <b>B</b>, Integrated brightness (fluorescence was measured in arbitrary units, “a.u.”). Fluorescence due to 655-nm-emitting Qdots is shown in unfilled columns; fluorescence due to 800-nm-emitting Qdots is shown in filled columns. There are very few artifacts due to miscounts of 800-nm-emitters in nodes from mice exposed to 655 nm Qdots alone, or to miscounts of 655-nm-emitters in nodes from mice exposed to 800 nm Qdots alone (<b>A</b>); this is confirmed by measurements of integrated brightness (<b>B</b>). Standard errors are shown. Note that our scoring method collapses the differences in uptake between lymph nodes as compared to integrated brightness. This is why the standard errors shown in A appear much smaller than those in B. There are no significant differences in uptake between quantum dot having different coats or emission maxima.</p
Quantum dot uptake in lumbar lymph nodes.
<p>A–D. Right lumbar lymph node, visualized 36 hr after N-9 pretreatment and 24 hr after instillation of 30 µL 1 mM polyarg-streptavidin Qdots. A. GFP spectral window (autofluorescence), false-colored green. B. Qdot spectral window, false-colored red. C. Superposition of A and B. D. Superposition of A and B with B multiplied 5x. While detection sensitivity is low at 2.5x, this lens permits a view of a whole lymph node at once. Many more quantum dots are visible than are shown in B; exposure was set to avoid saturation. E–H. Control left lumbar lymph node (mouse sham-instilled with PBS only). E. GFP spectral window (autofluorescence), false-colored green. F. Qdot spectral window, false-colored red. G. Superposition of E and F. H. Superposition of E and F with F enhanced 5x. The relatively uniform color in H indicates that the background image in F is due to autofluorescence. Imaging times were adjusted to ensure that no pixels were saturated; then all images were adjusted to correspond to uniform exposure in both red channels and the green channel. Background (assessed by averaging 4 off-image fields, one in each quadrant) was subtracted from each image.</p
Qdots in vaginal wall close to cervix.
<p>Top to bottom, increasing magnification of circled areas. Red, Qdots; green, WGA (cell surfaces); blue, nuclei (DAPI). Note penetration of tissue in bottom figure. N-9 pretreatment and instillation were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051995#pone-0051995-g001" target="_blank">Figure 1</a>. Organs were harvested and flash-frozen without fixation 24 hr after Qdot instillation. Sectioning, staining and microscopy were as described in Materials and Methods.</p
Quantification by ICP-MS and Integrated z-Stacks.
<p>Samples 1–5 are from Qdot-instilled mice, 6–10 are from control (uninstilled) mice. All mice were pretreated using N-9. Mice were scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051995#pone-0051995-t001" target="_blank"><b>Table 1</b></a>. Molarity of quantum dots is computed using an average of 5,000 Cd atoms per QD. Scores were all based on single-blind counts. All numbers are rounded to two significant figures.</p