Isolation of NDs bound to NPCs.

<p>(a–f) Unprocessed TEM images of anti-Nup98–conjugated NDs bound to NPCs in 90 nm epoxy resin sections from HeLa cells fixed 12 h after transfection with the ND conjugates. NPCs are highlighted in green. (g–i) Gradient morphometry filter applied to (d–f). (j–l) Images processed with a color threshold applied to isolate NDs. All scale bars are 50 nm.</p

Gradient enhancement alone allows detection of quasi-spherical NDs.

<p>Cropped sections from (a) central ROI and (b) upper ROI of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179295#pone.0179295.g004" target="_blank">Fig 4</a>. From left to right are the original images (left), gradient morphometry enhanced images (center), and contrast thresholds applied to the gradient images (right).</p

Nup98 and the NPC as a target for NDs.

<p>(a) <i>In situ</i> structure of human nuclear pore from isolated intact HeLa nuclei. Reproduced from Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179295#pone.0179295.ref027" target="_blank">27</a>] with permission. (b) Location of Nup98 within the NPC showing that Nup98 can localize in the central channel on both sides of the nuclear pore and outside, where it helps anchor the NPC to the nuclear envelope. Reproduced from Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179295#pone.0179295.ref015" target="_blank">15</a>] with permission. (c) High magnification (3.8 Å/pixel) TEM image of human nuclear pore in 90 nm epoxy resin slice from HeLa cells showing structural details including the cytoplasmic ring, internal ring, nuclear ring and basket. Scale bar, 20 nm, rotated. (d) Wide area view of the NPC in (c), scale bar 100 nm, no rotation. C and N designate cytoplasmic and nuclear regions.</p

Nanodiamonds can be detected individually from low magnification (5,000×) images.

<p>(a) Original image. (b) Zoom of ROI from (a). (c) Gradient morphometry enhancement of (b). (d) Contrast inverted image of (b). (e) Inverted and gradient images summed with threshold applied to isolate diamond. Red and blue indicate saturated maximum and minimum intensities, respectively, resulting in a binary colored image.</p

Successful targeting of NDs to the NPC.

<p>(a–f) Representative images of anti-Nup98–conjugated NDs (a, b) bound to or (c) located near an NPC, and (d–f) unconjugated NDs. C and N designate cytoplasmic and nuclear regions. Scale bar, 125 nm. Black arrows point to NDs. (g,i) Histograms of edge-to-edge distances from each ND or cluster to the nearest nuclear pore for anti-Nup98–conjugated (g) and unconjugated NDs (i). Central bin counts as a percentage of the total NDs counted are 31.0% and 1.26% for (g) and (i), respectively. (h,j) Rescaled data from (g) and (i). Negative and positive values refer to distances into the nucleus and cytoplasm, respectively. We analyzed twenty-five (25) representative images from both the conjugated and unconjugated TEM samples and identified 158 and 239 NDs or ND clusters, respectively, including approximately 1,000 intracellular NDs for both sets. Minimum distance (bin 0) was defined as ± 7.5 nm to account for antibody length. The center 3 bin edges are: -200, -7.5, 7.5, 200, and all others increment by 200 nm. The blue curve is a normal distribution based on the mean and standard deviation of the targeted NDs not bound to the NPC and the untargeted NDs, respectively. The distances measured are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179295#pone.0179295.s005" target="_blank">S2 Table</a>.</p

Aqueous Ligand-Stabilized Palladium Nanoparticle Catalysts for Parahydrogen-Induced ¹³C Hyperpolarization

Parahydrogen-induced polarization (PHIP) is a method for enhancing NMR sensitivity. The pairwise addition of parahydrogen in aqueous media by heterogeneous catalysts can lead to applications in chemical and biological systems. Polarization enhancement can be transferred from ¹H to ¹³C for longer lifetimes by using zero field cycling. In this work, water-dispersible <i>N</i>-acetylcysteine- and l-cysteine-stabilized palladium nanoparticles are introduced, and carbon polarizations up to 2 orders of magnitude higher than in previous aqueous heterogeneous PHIP systems are presented. <i>P</i>_¹³C values of 1.2 and 0.2% are achieved for the formation of hydroxyethyl propionate from hydroxyethyl acrylate and ethyl acetate from vinyl acetate, respectively. Both nanoparticle systems are easily synthesized in open air, and TEM indicates an average size of 2.4 ± 0.6 nm for NAC@Pd and 2.5 ± 0.8 nm for LCys@Pd nanoparticles with 40 and 25% ligand coverage determined by thermogravimetric analysis, respectively. As a step toward biological relevance, results are presented for the unprotected amino acid allylglycine upon aqueous hydrogenation of propargylglycine

Biothiol Xenon MRI Sensor Based on Thiol-Addition Reaction

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play an important role in regulating the vital functions of living organisms. Knowledge of their biodistribution in real-time could help diagnose a variety of conditions. However, existing methods of biothiol detection are invasive and require assays. Herein we report a molecular biosensor for biothiol detection using the nuclear spin resonance of ¹²⁹Xe. The ¹²⁹Xe biosensor consists of a cryptophane cage encapsulating a xenon atom and an acrylate group. The latter serves as a reactive site to covalently bond biothiols through a thiol-addition reaction. The biosensor enables discrimination of Cys from Hcy and GSH through the chemical shift and average reaction rate. This biosensor can be detected at a concentration of 10 μM in a single scan and it has been applied to detect biothiols in bovine serum solution. Our results indicate that this biosensor is a promising tool for the real-time imaging of biothiol distributions

Mitochondria Targeted and Intracellular Biothiol Triggered Hyperpolarized ¹²⁹Xe Magnetofluorescent Biosensor

Biothiols such as gluthathione (GSH), cysteine (Cys), homocysteine (Hcy), and thioredoxin (Trx) play vital roles in cellular metabolism. Various diseases are associated with abnormal cellular biothiol levels. Thus, the intracellular detection of biothiol levels could be a useful diagnostic tool. A number of methods have been developed to detect intracellular thiols, but sensitivity and specificity problems have limited their applications. To address these limitations, we have designed a new biosensor based on hyperpolarized xenon magnetic resonance detection, which can be used to detect biothiol levels noninvasively. The biosensor is a multimodal probe that incorporates a cryptophane-A cage as ¹²⁹Xe NMR reporter, a naphthalimide moiety as fluorescence reporter, a disulfide bond as thiol-specific cleavable group, and a triphenylphosphonium moiety as mitochondria targeting unit. When the biosensor interacts with biothiols, disulfide bond cleavage leads to enhancements in the fluorescence intensity and changes in the ¹²⁹Xe chemical shift. Using Hyper-CEST (chemical exchange saturation transfer) NMR, our biosensor shows a low detection limit at picomolar (10^–10 M) concentration, which makes a promise to detect thiols in cells. The biosensor can detect biothiol effectively in live cells and shows good targeting ability to the mitochondria. This new approach not only offers a practical technique to detect thiols in live cells, but may also present an excellent in vivo test platform for xenon biosensors

Increasing Cancer Therapy Efficiency through Targeting and Localized Light Activation

Currently, the potential of cancer therapy is compromised by a variety of problems related to tumor specificity, drug access, and limited efficacy. We report a novel approach to improve the effectiveness of cancer treatment utilizing a light-responsive nanoconstruct. Effectiveness is increased by enhancing drug absorption through heating and the production of free radicals. Treatment specificity is increased through chemical targeting of the nanoconstruct and localization of light delivery to the tumor. When reaching the tumor, magnetic resonance imaging is enhanced and near-infrared fluorescence is activated upon drug release, making it possible to visualize the localized treatment at both the tissue and cellular levels. This dual-modality imaging nanoconstruct enables the synergistic treatment and observable evaluation of solid tumors with dramatically improved efficacy, giving rise to a promising new approach for cancer therapy and evaluation