Probing Slow Protein Dynamics by Adiabatic <i>R</i>_1ρ and <i>R</i>_2ρ NMR Experiments

Slow μs/ms dynamics involved in protein folding, binding, catalysis, and allostery are currently detected using NMR dispersion experiments such as CPMG (Carr−Purcell−Meiboom−Gill) or spin-lock R1ρ. In these methods, protein dynamics are obtained by analyzing relaxation dispersion curves obtained from either changing the time spacing between 180° pulses or by changing the effective spin-locking field strength. In this Communication, we introduce a new method to induce a dispersion of relaxation rates. Our approach relies on altering the shape of the adiabatic full passage pulse and is conceptually different from existing approaches. By changing the nature of the adiabatic radiofrequency irradiation, we are able to obtain rotating frame R1ρ and R2ρ dispersion curves that are sensitive to slow μs/ms protein dynamics (demonstrated with ubiquitin). The strengths of this method are to (a) extend the dynamic range of the relaxation dispersion analysis, (b) avoid the need for multiple magnetic field strengths to extract dynamic parameters, (c) measure accurate relaxation rates that are independent of frequency offset, and (d) reduce the stress to NMR hardware (e.g., cryoprobes)

Ferrozine Assay for Simple and Cheap Iron Analysis of Silica-Coated Iron Oxide Nanoparticles

The Ferrozinen assay is applied as an accurate and rapid method to quantify the iron content of iron oxide nanoparticles (IONPs) and can be used in biological matrices. The addition of ascorbic aqcid accelerates the digestion process and can penetrate an IONP core within a mesoporous and solid silica shell. This new digestion protocol avoids the need for hydrofluoric acid to digest the surrounding silica shell and provides and accessible alternative to inductively coupled plasma methods. With the updated digestion protocol, the quantitative range of the Ferrozine assay is 1 - 14 ppm. <br

A Low-Cost, Tabletop LOD-EPR System for Nondestructive Quantification of Iron Oxide Nanoparticles in Tissues

Iron oxide nanoparticles (IONPs) have wide utility in applications from drug delivery to the rewarming of cryopreserved tissues. Due to the complex behavior of IONPs (e.g., uneven particle distribution and aggregation), further developments and clinical translation can be accelerated by having access to a noninvasive method for tissue IONP quantification. Currently, there is no low-cost method to nondestructively track IONPs in tissues across a wide range of concentrations. This work describes the performance of a low-cost, tabletop, longitudinally detected electron paramagnetic resonance (LOD-EPR) system to address this issue in the field of cryopreservation, which utilizes IONPs for rewarming of rat kidneys. A low-cost LOD-EPR system is realized via simultaneous transmit and receive using MHz continuous-wave transverse excitation with kHz modulation, which is longitudinally detected at the modulation frequency to provide both geometric and frequency isolation. The accuracy of LOD-EPR for IONP quantification is compared with NMR relaxometry. Solution measurements show excellent linearity (R2 > 0.99) versus Fe concentration for both measurements on EMG308 (a commercial nanoparticle), silica-coated EMG308, and PEG-coated EMG308 in water. The LOD-EPR signal intensity and NMR longitudinal relaxation rate constant (R1) of water are affected by particle coating, solution viscosity, and particle aggregation. R1 remains linear but with a reduced slope when in cryoprotective agent (CPA) solution, whereas the LOD-EPR signal is relatively insensitive to this. R1 does not correlate well with Fe concentration in rat kidney sections (R2 = 0.3487), while LOD-EPR does (R2 = 0.8276), with a linear regression closely matching that observed in solution and CPA

Injectable and Repeatable Inductive Heating of Iron Oxide Nanoparticle-Enhanced “PHIL” Embolic toward Tumor Treatment

Deep-seated tumors of the liver, brain, and other organ systems often recur after initial surgical, chemotherapeutic, radiation, or focal treatments. Repeating these treatments is often invasive and traumatic. We propose an iron oxide nanoparticle (IONP)-enhanced precipitating hydrophobic injectable liquid (PHIL, MicroVention inc.) embolic as a localized dual treatment implant for nutrient deprivation and multiple repeatable thermal ablation. Following a single injection, multiple thermal treatments can be repeated as needed, based on monitoring of tumor growth/recurrence. Herein we show the ability to create an injectable stable PHIL-IONP solution, monitor deposition of the PHIL-IONP precipitate dispersion by μCT, and gauge the IONP distribution within the embolic by magnetic resonance imaging. Once precipitated, the implant could be heated to reach therapeutic temperatures >8 °C for thermal ablation (clinical temperature of ∼45 °C), in a model disk and a 3D tumor bed model. Heat output was not affected by physiological conditions, multiple heating sessions, or heating at intervals over a 1 month duration. Further, in ex vivo mice hind-limb tumors, we could noninvasively heat the embolic to an “ablative” temperature elevation of 17 °C (clinically 54 °C) in the first 5 min and maintain the temperature rise over +8 °C (clinically a temperature of 45 °C) for longer than 15 min

MRI relaxation constants of the articular and sub-articular viable and necrotic epiphyseal cartilage.

<p>Large lesion (5 × 5 mm incision): 3, 5, 9, weeks post-surgery</p><p>Small lesion (3 × 4 mm incision): 4, 6, 10 weeks post-surgery</p><p>Due to technical problems, the T₂ measurement was invalid for the 3-week specimen and was not included.</p><p>MRI relaxation constants of the articular and sub-articular viable and necrotic epiphyseal cartilage.</p

Regression analysis of the percent differences of all parametric MRI relaxation constants and of the light absorption between the viable and necrotic epiphyseal cartilage.

<p>The solid line shows the linear fitting (R² = 0.39) between the percent differences of all MR relaxation constants (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140400#pone.0140400.t004" target="_blank">Table 4</a>) and the percent difference of the light absorption (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140400#pone.0140400.t002" target="_blank">Table 2</a>, column 4), resulting in the slope equal to 0.21.</p

Percent difference in relaxation times between viable and necrotic epiphyseal cartilage using various MRI sequences.

<p>Large lesion (5 × 5 mm incision): 3, 5, 9, weeks post surgery</p><p>Small lesion (3 × 4 mm incision): 4, 6, 10 weeks post surgery</p><p>Due to technical problems, the T₂ measurement was invalid for the 3-week specimen and was not included.</p><p>Percent difference in relaxation times between viable and necrotic epiphyseal cartilage using various MRI sequences.</p

Protocols for MR relaxometry.

<p>Protocols for MR relaxometry.</p

Safranin O-stained sections of femoral condyle.

<p>Top row: Surgically induced large lesions (3, 5, and 9 weeks post induction). Bottom row: Surgically induced small lesions (4, 6 and 10 weeks post surgical induction). Decreased staining in the chondronecrosis shows a variable degree of pallor. The optical density experiment of the safranin O-stained sections of the femoral condyle was conducted and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140400#pone.0140400.g002" target="_blank">Fig 2</a> to estimate the PG loss in the chondronecrosis.</p

Light absorption (arbitrary units [A.U.]) in safranin O stained sections of femoral condyle.

<p>Top row: Surgically induced large lesions (3, 5, and 9 weeks post induction). Bottom row: Surgically induced small lesions (4, 6 and 10 weeks post surgical induction). The areas of chondronecrosis are outlined in black (the area in (E) is selected based on the PG loss, in which the color shows yellow or light blue). Intralesional color spectrum ranges from yellow to dark blue, as proteoglycans are progressively lost from the cartilage matrix. The late-stage lesions in (C) and (F) similarly demonstrated very low proteoglycan content and either resulted in a marked delay in endochondral ossification (C) or became completely surrounded by bone (F).</p