Bentonite as a water-insoluble amorphous solid dispersion matrix for enhancing oral bioavailability of poorly water-soluble drugs.

Bentonite (BT), an orally administrable natural clay, is widely used for medical and pharmaceutical purposes due to its unique properties, including swelling, adsorption and ion-exchange. However, its application as a matrix of amorphous solid dispersion (ASD) formulations is rarely reported, despite the fact that drugs can adsorb to BT in an amorphous state. The objective of this study was to explore the feasibility of BT as a water-insoluble ASD matrix for enhancing the oral bioavailability of poorly water-soluble drugs, including sorafenib (SF). We prepared a novel BT-based ASD of an SF-BT composite (SFBTC) by adsorbing SF onto BT under acidic conditions using the ionic interaction between cationic SF and negatively charged BT. Scanning electron microscopy (SEM), powder X-ray diffractometry (pXRD), and differential scanning calorimetry (DSC) analyses revealed that SF adsorbed to BT in an amorphous state at SF:BT ratios from 1:3 to 1:10. In pharmacokinetic studies in rats, SFBTC (1:3) significantly improved the oral bioavailability of SF, and the AUClast of SFBTC (1:3) was 3.3-fold higher than that of NEXAVAR®, a commercial product of SF. An in vitro release study under sink conditions revealed that SFBTC (1:3) completely released SF in a pH-dependent manner, while a nonsink condition study indicated the generation of supersaturation under intestinal pH conditions. A kinetic solubility study showed that the release of SFBTC (1:3) followed the diffusion-controlled mechanism, which is a typical characteristic of water-insoluble matrix-based ASDs. The pharmacokinetic studies of drug-BT composites of various drugs belonging to BCS class II indicated that the pKa value of the adsorbed drugs is one of the most important factors determining their dissolution and oral bioavailability. These results suggest that BT could be a promising water-insoluble ASD matrix for improving the oral bioavailability of poorly water-soluble drugs, including SF.

Volumetric T2 -weighted spin echo imaging with improved SNR using localized quadratic encoding and a spiral readout trajectory.

PURPOSE: To demonstrate T2 -weighted (single-echo) spin-echo (SE) imaging with near-optimal acquisition efficiency by applying SNR-efficient RF slice encoding and spiral readout. METHODS: A quadratic-phase (frequency swept) excitation RF pulse replaced the conventional excitation in T2 -weighted SE sequence to excite a thick slab that is internally spatially encoded by a variable phase along the slice direction. Highly overlapping slabs centered at every desired slice location were acquired in multiple passes, such that the entire imaging volume was excited by contiguous slabs in any given pass. Following 90° excitation, each slab was refocused with a conventional 180° RF to produce a SE signal, followed by a spiral in-out readout. A noise-insensitive reconstruction removed the quadratic phase in the spatial frequency domain, yielding desired slice resolution and improved SNR. RESULTS: Increasing the RF frequency sweep (hence, excitation width) allowed more frequent encoding of each slice over the multiple passes, improving final image SNR, until crosstalk ensued at excessive slab widths compared to their center-to-center spacing. With an optimized slab width, the proposed technique used all passes to acquire every prescribed slice, with substantially improved SNR over conventional SE or 2D-turbo-spin-echo (TSE) scans. Quantitative SNR measurements indicated similar SNR as 3D-TSE, but radiologist scoring favored 3D-TSE, mainly because of spiral-related artifacts and possibly because of regularized reconstructions in 3D-TSE. CONCLUSION: Using SNR-efficient slice excitation scheme and spiral readout helped eliminate SNR and temporal inefficiencies in conventional T2 -weighted imaging, yielding SNR independent of TR or number of passes.

Tailoring renal-clearable zwitterionic cyclodextrin for colorectal cancer-selective drug delivery.

Although cyclodextrin-based renal-clearable nanocarriers have a high potential for clinical translation in targeted cancer therapy, their designs remain to be optimized for tumour retention. Here we report on the design of a tailored structure for renal-clearable zwitterionic cyclodextrin for colorectal cancer-selective drug delivery. Twenty cyclodextrin derivatives with different charged moieties and spacers are synthesized and screened for colloidal stability. The resulting five candidates are evaluated for biodistribution and an optimized structure is identified. The optimized cyclodextrin shows a high tumour accumulation and is used for delivery of doxorubicin and ulixertinib. Higher tumour accumulation and tumour penetration facilitates tumour elimination. The improved antitumour efficacy is demonstrated in heterotopic and orthotopic colorectal cancer models.

Bone tumor-homing nanotherapeutics for prolonged retention in tumor microenvironment and facilitated apoptotic process via mevalonate pathway inhibition.

Bone malignancy features a mineralized extracellular matrix primarily composed of hydroxyapatite, which interferes with the distribution and activity of antineoplastic agents. Herein, we report bone tumor-homing polymeric nanotherapeutics consisting of alendronate-decorated chondroitin sulfate A-graft-poly(lactide-co-glycolide) and doxorubicin (DOX), named PLCSA-AD, which displayed a prolonged retention profile in the tumor microenvironment and augmented therapeutic efficacy via inhibition of the mevalonate pathway. PLCSA-AD exhibited a 1.72-fold lower IC50 value than free DOX and a higher affinity for hydroxyapatite than PLCSA in HOS/MNNG cell-based 2D bone tumor-mimicking models. The inhibition of the mevalonate pathway by PLCSA-AD in tumor cells was verified by investigating the cytosolic fraction of unprenylated proteins, where blank PLCSA-AD significantly increased the expression of cytosolic Ras and RhoA without changing their total cellular amounts. In a bone tumor-mimicking xenografted mouse model, AD-decorated nanotherapeutics significantly increased tumor accumulation (1.73-fold) compared with PLCSA, and higher adsorption to hydroxyapatites was observed in the histological analysis of the tumor. As a result, inhibition of the mevalonate pathway and improvement in tumor accumulation led to markedly enhanced therapeutic efficacy in vivo, suggesting that PLCSA-AD could be promising nanotherapeutics for bone tumor treatment.

Accelerated 4D-flow MRI with 3-point encoding enabled by machine learning.

PURPOSE: To investigate the acceleration of 4D-flow MRI using a convolutional neural network (CNN) that produces three directional velocities from three flow encodings, without requiring a fourth reference scan measuring background phase. METHODS: A fully 3D CNN using a U-net architecture was trained in a block-wise fashion to take complex images from three flow encodings and to produce three real-valued images for each velocity component. Using neurovascular 4D-flow scans (n = 144), the CNN was trained to predict velocities computed from four flow encodings by standard reconstruction including correction for residual background phase offsets. Methods to optimize loss functions were investigated, including magnitude, complex difference, and uniform velocity weightings. Subsequently, 3-point encoding was evaluated using cross validation of pixelwise correlation, flow measurements in major arteries, and in experiments with data at differing acceleration rates than the training data. RESULTS: The CNN-produced 3-point velocities showed excellent agreements with the 4-point velocities, both qualitatively in velocity images, in flow rate measures, and quantitatively in regression analysis (slope = 0.96, R2  = 0.992). Optimizing the training to focus on vessel velocities rather than the global velocity error and improved the correlation of velocity within vessels themselves. The lowest error was observed when the loss function used uniform velocity weighting, in which the magnitude-weighted inverse of the velocity frequency uniformly distributed weighting across all velocity ranges. When applied to highly accelerated data, the 3-point network maintained a high correlation with ground truth data and demonstrated a denoising effect. CONCLUSION: The 4D-flow MRI can be accelerated using machine learning requiring only three flow encodings to produce three-directional velocity maps with small errors.

Simultaneous 3D-TOF angiography and 4D-flow MRI with enhanced flow signal using multiple overlapping thin slab acquisition and magnetization transfer.

PURPOSE: To investigate the fusion of 3D time-of-flight principles into 4D-flow MRI to enhance vessel contrast and signal without an exogenous contrast agent, enabling simultaneous in-flow based angiograms. METHODS: A 4D-flow MRI technique was developed consisting of multiple overlapping slabs with intermittent magnetization transfer preparation. The scan time penalty associated with multiple slab acquisitions was mitigated by using undersampled distributed spiral trajectories and compressed sensing reconstruction. A flow phantom was used to characterize in-flow enhancement, velocity noise improvement, and flow rate measurements against the single-slab 4D-flow MRI. In a patient-volunteer cohort (n = 15), magnitude-based angiograms were radiologically evaluated against 3D time-of-flight, and velocity measurements were compared pixel-wise against single-slab and contrast-enhanced 4D-flow MRI. RESULTS: Multiple-slab acquisitions, together with magnetization transfer preparation, substantially improved vessel signal, contrast, and vessel conspicuity in magnitude angiograms. Both clinical 3D time-of-flight and the proposed technique produced equivalent vessel depictions with no statistically significant difference (p < .1). Both techniques also produced clear depictions of brain aneurysms in all patients; however, very small vessels tended to show reduced conspicuity in the proposed technique. Velocity measurements agreed with contrast-enhanced and single-slab scans with high correlations (R2 = 0.941-0.974) and agreements (slopes = 0.994-1.071). Slab boundary and magnetization transfer-related artifacts were not observed in velocity measurements, and velocity noise was reduced with in-flow enhancement over single-slab scans (phantom). CONCLUSION: The vessel signal and contrast can be improved in 4D-flow MRI without exogenous contrast agents by utilizing in-flow enhancement, efficient sampling, and compressed sensing. The in-flow enhancement also enables simultaneous 3D time-of-flight angiograms useful for flow quantification and diagnosis.

Composite MRA: statistical approach to generate an MR angiogram from multiple contrasts.

PURPOSE: To develop a method to use information from multiple MRI contrasts to produce a composite angiogram with reduced sequence-specific artifacts and improved vessel depiction. METHODS: Bayesian posterior vessel probability was determined as a function of black blood (BB), contrast enhanced angiography (CE-MRA), and phase-contrast MRA (PC-MRA) intensities from training subjects (N = 4). To generate composite angiogram in evaluation subjects (N = 12), the voxel-wise vessel probabilities were weighted with a confidence measure and combined as a weighted product to yield angiogram intensity. For 23 internal carotid artery (ICA) segments (N = 23) from evaluation subjects, segmentation accuracy of composite MRA was evaluated and compared against CE-MRA using dice similarity coefficient (DSC). RESULTS: The composite MRA suppressed venous contaminations in CE-MRA, reduced flow artifacts, and velocity aliasing seen in PC-MRA and removed signal ambiguities in BB images. For ICA segmentations, the composite MRA improved segmentation over CE-MRA per DSC (0.908 ± 0.037 vs. 0.765 ± 0.079). Compared with CE-MRA, the composite MRA showed conservative changes in vessel appearance to small threshold changes. However, small vessels that are sensitive to registration errors or visible only weakly in CE-MRA were susceptible to poor depiction in composite MRA. CONCLUSION: By dynamically weighting vessel information from multiple contrasts and extracting their complementary information, the composite MRA produces reduced sequence-specific artifacts and improved vessel contrast. It is a promising technique for semi-automatic segmentation of vessels that are hard to segment because of artifacts.

Nanoscale imaging of caveolin-1 membrane domains in vivo.

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200-250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.

Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin.

The influenza viral membrane protein hemagglutinin (HA) is required at high concentrations on virion and host-cell membranes for infectivity. Because the role of actin in membrane organization is not completely understood, we quantified the relationship between HA and host-cell actin at the nanoscale. Results obtained using superresolution fluorescence photoactivation localization microscopy (FPALM) in nonpolarized cells show that HA clusters colocalize with actin-rich membrane regions (ARMRs). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMRs, and actin disruption caused specific changes to HA clustering. Surprisingly, the actin-binding protein cofilin was excluded from some regions within several hundred nanometers of HA clusters, suggesting that HA clusters or adjacent proteins within the same clusters influence local actin structure. Thus, with the use of imaging, we demonstrate a dynamic relationship between glycoprotein membrane organization and the actin cytoskeleton at the nanoscale.

Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy.

Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined.

Simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.