Surface Chemistry of Quantum Dots Determines Their Behavior in Postischemic Tissue

The behavior of quantum dots (QDs) in the microvasculature and their impact on inflammatory reactions under pathophysiological conditions are still largely unknown. Therefore, we designed this study to investigate the fate and effects of surface-modified QDs in postischemic skeletal and heart muscle. Under these pathophysiological conditions, amine-modified QDs, but not carboxyl-QDs, were strongly associated with the vessel wall of postcapillary venules and amplified ischemia-reperfusion-elicited leukocyte transmigration. Importantly, strong association of amine-QDs with microvessel walls was also present in the postischemic myocardium. As shown by electron microscopy and verified by FACS analyses, amine-modified QDs, but not carboxyl-QDs, were associated with endogenous microparticles. At microvessel walls, these aggregates were attached to endothelial cells. Taken together, we found that both the surface chemistry of QDs and the underlying tissue conditions (<i>i.e.</i>, ischemia-reperfusion) strongly determine their uptake by endothelial cells in microvessels, their association to endogenous microparticles, as well as their potential to modify inflammatory processes. Thus, this study strongly corroborates the view that the surface chemistry of nanomaterials and the physiological state of the tissue are crucial for the behavior of nanomaterials <i>in vivo.</i

Surface Chemistry of Quantum Dots Determines Their Behavior in Postischemic Tissue

The behavior of quantum dots (QDs) in the microvasculature and their impact on inflammatory reactions under pathophysiological conditions are still largely unknown. Therefore, we designed this study to investigate the fate and effects of surface-modified QDs in postischemic skeletal and heart muscle. Under these pathophysiological conditions, amine-modified QDs, but not carboxyl-QDs, were strongly associated with the vessel wall of postcapillary venules and amplified ischemia-reperfusion-elicited leukocyte transmigration. Importantly, strong association of amine-QDs with microvessel walls was also present in the postischemic myocardium. As shown by electron microscopy and verified by FACS analyses, amine-modified QDs, but not carboxyl-QDs, were associated with endogenous microparticles. At microvessel walls, these aggregates were attached to endothelial cells. Taken together, we found that both the surface chemistry of QDs and the underlying tissue conditions (<i>i.e.</i>, ischemia-reperfusion) strongly determine their uptake by endothelial cells in microvessels, their association to endogenous microparticles, as well as their potential to modify inflammatory processes. Thus, this study strongly corroborates the view that the surface chemistry of nanomaterials and the physiological state of the tissue are crucial for the behavior of nanomaterials <i>in vivo.</i

The Endothelial Glycocalyx Controls Interactions of Quantum Dots with the Endothelium and Their Translocation across the Blood–Tissue Border

Advances in the engineering of nanoparticles (NPs), which represent particles of less than 100 nm in one external dimension, led to an increasing utilization of nanomaterials for biomedical purposes. A prerequisite for their use in diagnostic and therapeutic applications, however, is the targeted delivery to the site of injury. Interactions between blood-borne NPs and the vascular endothelium represent a critical step for nanoparticle delivery into diseased tissue. Here, we show that the endothelial glycocalyx, which constitutes a glycoprotein–polysaccharide meshwork coating the luminal surface of vessels, effectively controls interactions of carboxyl-functionalized quantum dots with the microvascular endothelium. Glycosaminoglycans of the endothelial glycocalyx were found to physically cover endothelial adhesion and signaling molecules, thereby preventing endothelial attachment, uptake, and translocation of these nanoparticles through different layers of the vessel wall. Conversely, degradation of the endothelial glycocalyx promoted interactions of these nanoparticles with microvascular endothelial cells under the pathologic condition of ischemia–reperfusion, thus identifying the injured endothelial glycocalyx as an essential element of the blood–tissue border facilitating the targeted delivery of nanomaterials to diseased tissue

Model of post-transcriptional regulation after SCNT.

<p>Interlinked events at the metabolic level, disturbed after SCNT, are proposed to impact on reprogramming and embryo development outcome. SCNT manipulation alters mitochondrial membrane potential (Δψ), triggers cytosolic Ca²⁺ concentration ([Ca²⁺]_c) increase, ATP decrease and reactive oxygen species (ROS) increase. Increased cytosolic Ca²⁺-induced retrograde response, and ROS-induced redox imbalance, could induce transcriptional regulation. Oxidative stress and decreased ATP could induce cellular responses driving post-transcriptional regulation. Proposed effects could be direct (bold arrows), or follow after a cascade of events (indirect; dashed arrows).</p

Metabolic disturbances after SCNT.

<p>(A) Energy (ATP and ADP) content decreases in SCNT at the 4-cell stage, compared to ICSI embryos (significant difference for ATP; t-test, <i>p</i> = 0.008). (B) Levels of reactive oxygen species (ROS) are increased in 4-cell SCNT (t-test, <i>p</i> = 1.0E-06), as measured by DCHFDA. At the same stage, cellular antioxidant capacity (measured as reduced glutathione levels, with MCB) is similar for SCNT and ICSI embryos. (C) Increased TMRM signal indicates higher mitochondrial membrane potential after SCNT (t-test, <i>p</i> = 0.007), while (D) cytosolic calcium (measured with Fura-2 AM) is significantly increased in 2-cell SCNT (t-test, <i>p</i> = 1.4E-06).</p

Mitochondria ultrastructure in pre-implantation embryos.

<p>Representative micrographs show mitochondria ultrastructure in mouse wild-type SCNT and ICSI embryos, throughout pre-implantation development. nucleus: n; mitochondria: d, developed; md, medium developed; ud, undeveloped. Mitochondria morphology and analysis are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036850#s4" target="_blank">Materials and Methods</a>. Magnification: 12200X.</p

Transcription and translation in mouse blastocysts.

<p>mRNA levels were determined for pools of six ICSI and SCNT blastocysts (in duplicate), by quantitative RT-PCR, over two independent experiments. Technical replicates were determined three times for each sample, and results were normalized to Flag-Oct4 mRNA. (A) dCT values for transcripts in mtGFP-Tg SCNT, compared to ICSI. (B) dCT values for Gapdh and Actb mRNA, as well as protein levels (by immunocytochemistry), in wild-type SCNT relative to ICSI. Gapdh and Actb levels were significantly different, both for mRNA (t-test, <i>p</i> = 0,001 and <i>p</i> = 1,29E-08, respectively) and protein (t-test, <i>p</i> = 0.006 and <i>p</i> = 0.04, respectively). Box plot: fluorescence intensities distribution in the blastocyst; top and bottom lines: inter-quartile range; middle line: median; whiskers: range of variation limited to 1.5 times inter-quartile range.</p

Gene Ontology (GO) categories enriched among the genes that are differentially expressed (2-fold) in SCNT and ICSI embryos.

<p>130 upregulated and 112 downregulated genes in SCNT compared to ICSI. <i>p</i>-values were truncated at 0.00001.</p

EGFP localization in mtGFP-Tg cells.

<p>(A) In the mtGFP-Tg oocyte, EGFP is mainly localized in mitochondria (A’, insert in A), as shown by cryo-immuno electron microscopy. (A”) In cryosections of B6C3F1 wild-type ooplast (SCNT recipient), only low unspecific background labeling in the cytoplasm is found (arrow), while there is no detectable mitochondrial signal. Scale bar, A: 1 µm; A’ and A”: 200 nm. (B) Embryonic stem cells (here feeder-free) derived from fertilized blastocysts express EGFP (B”, green), co-localizing with mitochondria (stained with TMRM; B’, red); B’”, merge B’ and B”. Scale bar, 25 µm.</p

Primer sequences for SYBR green PCR.

<p>Flag-Oct4 mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036850#pone.0036850-Pfeiffer1" target="_blank">[45]</a> was used as external reference. n.a., not applicable.</p