Underestimation of Flow Velocity in 2-D Super-Resolution Ultrasound Imaging.

Velocity estimation in ultrasound imaging is a technique to measure the speed and direction of blood flow. The flow velocity in small blood vessels, i.e., arterioles, venules, and capillaries, can be estimated using super-resolution ultrasound imaging (SRUS). However, the vessel width in SRUS is relatively small compared with the full-width-half-maximum of the ultrasound beam in the elevation direction (FWHMy), which directly impacts the velocity estimation. By taking into consideration the small vessel widths in SRUS, it is hypothesized that the velocity is underestimated in 2-D super-resolution ultrasound imaging when the vessel diameter is smaller than the FWHMy. A theoretical model is introduced to show that the velocity of a 3-D parabolic velocity profile is underestimated by up to 33% in 2-D SRUS, if the width of the vessel is smaller than the FWHMy. This model was tested using Field II simulations and 3-D printed micro-flow hydrogel phantom measurements. A Verasonics Vantage 256™ scanner and a GE L8-18i-D linear array transducer with FWHMy of approximately 770 µm at the elevation focus were used in the simulations and measurements. Simulations of different parabolic velocity profiles showed that the velocity underestimation was 36.8%±1.5% (mean±standard deviation). The measurements showed that the velocity was underestimated by 30%±6.9%. Moreover, the results of vessel diameters, ranging from 0.125×FWHMy to 3×FWHMy, indicate that velocities are estimated according to the theoretical model. The theoretical model can, therefore, be used for the compensation of velocity estimates under these circumstances.

Super Resolution Ultrasound Imaging Using the Erythrocytes: II: Velocity Images.

Super resolution ultrasound imaging using the erythrocytes (SURE) has recently been introduced. The method uses erythrocytes as targets instead of fragile microbubbles (MBs). The abundance of erythrocyte scatterers makes it possible to acquire SURE data in just a few seconds compared to several minutes in ultrasound localization microscopy (ULM) using MBs. A high number of scatterers can reduce the acquisition time, however, the tracking of uncorrelated and high-density scatterers is quite challenging. This paper hypothesizes that it is possible to detect and track erythrocytes as targets to obtain vascular flow images. A SURE tracking pipeline is used with modules for beamforming, recursive synthetic aperture imaging, motion estimation, echo canceling, peak detection, and recursive nearest neighbor tracker. The SURE tracking pipeline is capable of distinguishing the flow direction and separating tubes of a simulated Field II phantom with 125 to 25 µm wall-to-wall tube distances, as well as a 3D-printed hydrogel micro-flow phantom with 100 to 60 µm wall-to-wall channel distances. The comparison of an in-vivo SURE scan of a Sprague-Dawley rat kidney with ULM and micro-CT scans with voxel sizes of 26.5µm and 5µm demonstrated consistent findings. A microvascular structure composed of 16 vessels exhibited similarities across all imaging modalities. The flow direction and velocity profiles in the SURE scan were found to be concordant with those from ULM.

Super Resolution Ultrasound Imaging using the Erythrocytes: I: Density Images.

A new approach for vascular super resolution imaging using the erythrocytes as targets (SURE imaging) is described and investigated. SURE imaging does not require fragile contrast agent bubbles, making it possible to use the maximum allowable mechanical index for ultrasound scanning for an increased penetration depth. A synthetic aperture ultrasound sequence was employed with 12 virtual sources using a 10 MHz GE L8-18i-D linear array hockey stick probe. The axial resolution was 1.20λ,(185.0µm) and the lateral resolution was 1.50λ,(231.3µm). Field IIpro simulations were conducted on 12.5 µm radius vessel pairs with varying separations. A vessel pair with a separation of 70 µm could be resolved, indicating a SURE image resolution below half a wavelength. A Verasonics research scanner was used for the in vivo experiments to scan the kidneys of Sprague-Dawley rats for up to 46 s to visualize their microvasculature by processing from 0.1 up to 45 s of data for SURE imaging, and for 46.8 s for super resolution (SR) imaging with a SonoVue contrast agent. Afterward, the renal vasculature was filled with the ex vivo micro-CT contrast agent Microfil, excised, and scanned in a micro-CT scanner at both a 22.6 µm voxel size for 11 hours, and for 20 hours in a 5 µm voxel size for validating the SURE images. Comparing the SURE and micro-CT images revealed that vessels with a diameter of 28 µm, five times smaller than the ultrasound wavelength, could be detected, and the dense grid of microvessels in the full kidney was shown for scan times between 1 to 10 s. The vessel structure in the cortex was also similar for the SURE and SR images. Fourier ring correlation indicated a resolution capability of 29 µm. SURE images are acquired in seconds rather than minutes without any patient preparation or contrast injection, making the method translatable to clinical use.

Interactions of oral permeation enhancers with lipid membranes in simulated intestinal environments.

The oral bioavailability of therapeutic peptides is generally low. To increase peptide transport across the gastrointestinal barrier, permeation enhancers are often used. Despite their widespread use, mechanistic knowledge of permeation enhancers is limited. To address this, we here investigate the interactions of six commonly used permeation enhancers with lipid membranes in simulated intestinal environments. Specifically, we study the interactions of the permeation enhancers sodium caprate, dodecyl maltoside, sodium cholate, sodium dodecyl sulfate, melittin, and penetratin with epithelial cell-like model membranes. To mimic the molecular composition of the real intestinal environment, the experiments are performed with two peptide drugs, salmon calcitonin and desB30 insulin, in fasted-state simulated intestinal fluid. Besides providing a comparison of the membrane interactions of the studied permeation enhancers, our results demonstrate that peptide drugs as well as intestinal-fluid components may substantially change the membrane activity of permeation enhancers. This highlights the importance of testing permeation enhancement in realistic physiological environments and carefully choosing a permeation enhancer for each individual peptide drug.

Contrast-enhanced ultrasound imaging using capacitive micromachined ultrasonic transducers.

Capacitive micromachined ultrasonic transducers (CMUTs) have a nonlinear relationship between the applied voltage and the emitted signal, which is detrimental to conventional contrast enhanced ultrasound (CEUS) techniques. Instead, a three-pulse amplitude modulation (AM) sequence has been proposed, which is not adversely affected by the nonlinearly emitted harmonics. In this paper, this is shown theoretically, and the performance of the sequence is verified using a 4.8 MHz linear capacitive micromachined ultrasonic transducer (CMUT) array, and a comparable lead zirconate titanate (PZT) array, across 6-60 V applied alternating current (AC) voltage. CEUS images of the contrast agent SonoVue flowing through a 3D printed hydrogel phantom showed an average enhancement in contrast-to-tissue ratio (CTR) between B-mode and CEUS images of 49.9 and 37.4 dB for the PZT array and CMUT, respectively. Furthermore, hydrophone recordings of the emitted signals showed that the nonlinear emissions from the CMUT did not significantly degrade the cancellation in the compounded AM signal, leaving an average of 2% of the emitted power between 26 and 60 V of AC. Thus, it is demonstrated that CMUTs are capable of CEUS imaging independent of the applied excitation voltage when using a three-pulse AM sequence.

Imaging therapeutic peptide transport across intestinal barriers.

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro, ex vivo, and in vivo platforms that currently are being developed to accelerate this emerging field of research.

3D printed calibration micro-phantoms for super-resolution ultrasound imaging validation.

This study evaluates the use of 3D printed phantoms for 3D super-resolution ultrasound imaging (SRI) algorithm calibration. The main benefit of the presented method is the ability to do absolute 3D micro-positioning of sub-wavelength sized ultrasound scatterers in a material having a speed of sound comparable to that of tissue. Stereolithography is used for 3D printing soft material calibration micro-phantoms containing eight randomly placed scatterers of nominal size 205 µm × 205 µm × 200 µm. The backscattered pressure spatial distribution is evaluated to show similar distributions from micro-bubbles as the 3D printed scatterers. The printed structures are found through optical validation to expand linearly in all three dimensions by 2.6% after printing. SRI algorithm calibration is demonstrated by imaging a phantom using a λ/2 pitch 3 MHz 62+62 row-column addressed (RCA) ultrasound probe. The printed scatterers will act as point targets, as their dimensions are below the diffraction limit of the ultrasound system used. Two sets of 640 volumes containing the phantom features are imaged, with an intervolume uni-axial movement of the phantom of 12.5 µm, to emulate a flow velocity of 2 mm/s at a frame rate of 160 Hz. The ultrasound signal is passed to a super-resolution pipeline to localise the positions of the scatterers and track them across the 640 volumes. After compensating for the phantom expansion, a scaling of 0.989 is found between the distance between the eight scatterers calculated from the ultrasound data and the designed distances. The standard deviation of the variation in the scatterer positions along each track is used as an estimate of the precision of the super-resolution algorithm, and is expected to be between the two limiting estimates of (σ̃x,σ̃y,σ̃z) = (22.7 µm, 27.6 µm, 9.7 µm) and (σ̃x,σ̃y,σ̃z) = (18.7 µm, 19.3 µm, 8.9 µm). In conclusion, this study demonstrates the use of 3D printed phantoms for determining the accuracy and precision of volumetric super-resolution algorithms.

On-Demand Reversible UV-Triggered Interpenetrating Polymer Network-Based Drug Delivery System Using the Spiropyran-Merocyanine Hydrophobicity Switch.

A reversible switchable on-demand UV-triggered drug delivery system (DDS) based on interpenetrating polymer networks (IPNs) with silicone as the host polymer and spiropyran (SP)-functionalized guest polymer is designed and demonstrated. The photo-responsive IPNs provide a new triggered drug delivery concept as they exploit the change in intermolecular interactions (work of adhesion) among the drug, matrix, and solvent when the incorporated hydrophobic SP moieties transform into the hydrophilic merocyanine form upon light irradiation without degradation and disruption of the DDS. The change in how the copolymer composition (hydrophilicity and content) and the lipophilicity of the drug (log P) affect the release profile was investigated. A thermodynamic model, based on Hansen solubility parameters, was developed to design and optimize the polymer composition of the IPNs to obtain the most efficient light-triggered drug release and suppression of the premature release. The developed IPNs showed excellent result for dopamine, l-dopa, and prednisone with around 90-95% light-triggered release. The model was applied to study the release behavior of drugs with different log P and to estimate if the light-induced hydrophobic-to-hydrophilic switch can overcome the work of adhesion between polymers and drugs and hence the desorption and release of the drugs. To the best of our knowledge, this is the first time that work of adhesion is used for this aim. Comparing the result obtained from the model and experiment shows that the model is useful for evaluating and estimating the release behavior of specific drugs merocyanine, IPN, DDS, and spiropyran.

Detection and Localization of Ultrasound Scatterers Using Convolutional Neural Networks.

Delay-and-sum (DAS) beamforming is unable to identify individual scatterers when their density is so high that their point spread functions overlap. This paper proposes a convolutional neural network (CNN)-based method to detect and localize high-density scatterers, some of which are closer than the resolution limit of delay-and-sum (DAS) beamforming. A CNN was designed to take radio frequency channel data and return non-overlapping Gaussian confidence maps. The scatterer positions were estimated from the confidence maps by identifying local maxima. On simulated test sets, the CNN method with three plane waves achieved a precision of 1.00 and a recall of 0.91. Localization uncertainties after excluding outliers were ±46 [Formula: see text] (outlier ratio: 4%) laterally and ±26 [Formula: see text] (outlier ratio: 1%) axially. To evaluate the proposed method on measured data, two phantoms containing cavities were 3-D printed and imaged. For the phantom study, the training data were modified according to the physical properties of the phantoms and a new CNN was trained. On an uniformly spaced scatterer phantom, a precision of 0.98 and a recall of 1.00 were achieved with the localization uncertainties of ±101 [Formula: see text] (outlier ratio: 1%) laterally and ±37 [Formula: see text] (outlier ratio: 1%) axially. On a randomly spaced scatterer phantom, a precision of 0.59 and a recall of 0.63 were achieved. The localization uncertainties were ±132 [Formula: see text] (outlier ratio: 0%) laterally and ±44 [Formula: see text] with a bias of 22 [Formula: see text] (outlier ratio: 0%) axially. This method can potentially be extended to detect highly concentrated microbubbles in order to shorten data acquisition times of super-resolution ultrasound imaging.

3D Printed Hydrogel Multiassay Platforms for Robust Generation of Engineered Contractile Tissues.

We present a method for reproducible manufacture of multiassay platforms with tunable mechanical properties for muscle tissue strip analysis. The platforms result from stereolithographic 3D printing of low protein-binding poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Contractile microtissues have previously been engineered by immobilizing suspended cells in a confined hydrogel matrix with embedded anchoring cantilevers to facilitate muscle tissue strip formation. The 3D shape and mechanical properties of the confinement and the embedded cantilevers are critical for the tissue robustness. High-resolution 3D printing of PEGDA hydrogels offers full design freedom to engineer cantilever stiffness, while minimizing unwanted cell attachment. We demonstrate the applicability by generating suspended muscle tissue strips from C2C12 mouse myoblasts in a compliant fibrin-based hydrogel matrix. The full design freedom allows for new platform geometries that reduce local stress in the matrix and tissue, thus, reducing the risk of tissue fracture.

Autocrine CCL19 blocks dendritic cell migration toward weak gradients of CCL21.

BACKGROUND AIMS: Maturation of dendritic cells (DCs) induces their homing from peripheral to lymphatic tissues guided by CCL21. However, in vitro matured human monocyte-derived DC cancer vaccines injected intradermally migrate poorly to lymph nodes (LNs). In vitro maturation protocols generate DCs with high (type 1 DCs) or low (prostaglandin E2 [PGE2]-DCs) autocrine CCL19 levels, which may potentially interfere with LN homing of DCs. METHODS: Employing a three-dimensional (3D) chemotaxis assay, chemokine competition/desensitization studies and short interfering RNA (siRNA) against CCL19, we analyzed the effect of autocrine CCL19 on in vitro migration of human DCs toward CCL21. RESULTS: Using human monocyte-derived DCs in a 3D chemotaxis assay, we are the first to demonstrate that CCL19 more potently induces directed migration of human DCs compared with CCL21. When comparing migration of type 1 DCs and PGE2-DCs, migration of type 1 DCs was strikingly impaired compared with PGE2-DCs, but only toward low concentrations of CCL21. When type 1 DCs were cultured overnight in fresh culture medium (reducing autocrine CCL19 levels), a rescuing effect was observed on migration toward low concentrations of CCL21 in a 3D chemotaxis assay. Finally pre-incubation with CCL19 negatively affected PGE2-DC migration, whereas silencing of CCL19 by siRNA improved type 1 DC migration. Importantly, in both cases, the effect was observed only at low concentrations of CCL21. CONCLUSIONS: Our results demonstrate that autocrine CCL19 negatively affects DC migratory potential toward CCL21, the potency difference between CCL19 and CCL21 being the underlying cause. CCL19 secretion level of in vitro matured DCs is an important indicator of DC vaccine homing potential.

Designing CAF-adjuvanted dry powder vaccines: spray drying preserves the adjuvant activity of CAF01.

Dry powder vaccine formulations are highly attractive due to improved storage stability and the possibility for particle engineering, as compared to liquid formulations. However, a prerequisite for formulating vaccines into dry formulations is that their physicochemical and adjuvant properties remain unchanged upon rehydration. Thus, we have identified and optimized the parameters of importance for the design of a spray dried powder formulation of the cationic liposomal adjuvant formulation 01 (CAF01) composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) via spray drying. The optimal excipient to stabilize CAF01 during spray drying and for the design of nanocomposite microparticles was identified among mannitol, lactose and trehalose. Trehalose and lactose were promising stabilizers with respect to preserving liposome size, as compared to mannitol. Trehalose and lactose were in the glassy state upon co-spray drying with the liposomes, whereas mannitol appeared crystalline, suggesting that the ability of the stabilizer to form a glassy matrix around the liposomes is one of the prerequisites for stabilization. Systematic studies on the effect of process parameters suggested that a fast drying rate is essential to avoid phase separation and lipid accumulation at the surface of the microparticles during spray drying. Finally, immunization studies in mice with CAF01 in combination with the tuberculosis antigen Ag85B-ESAT6-Rv2660c (H56) demonstrated that spray drying of CAF01 with trehalose under optimal processing conditions resulted in the preservation of the adjuvant activity in vivo. These data demonstrate the importance of liposome stabilization via optimization of formulation and processing conditions in the engineering of dry powder liposome formulations.

Comparison of clinical grade type 1 polarized and standard matured dendritic cells for cancer immunotherapy.

Monocyte-derived dendritic cells (DCs) used for immunotherapy e.g. against cancer are commonly matured by pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) and prostaglandin E(2) although the absence of Toll-like receptor mediated activation prevents secretion of IL-12 from DCs and subsequent efficient induction of type 1 effector T cells. Standard matured clinical grade DCs "sDCs" were compared with DCs matured with either of two type 1 polarizing maturation cocktails; the alpha-type-1 DCs "αDC1s" (TNF-α, IL-1ß, IFN-γ, IFN-α, Poly(I:C)) and "mDCs" (monophosphoryl lipid A (MPL), IFN-γ) or a mixed cocktail - "mpDCs", containing MPL, IFN-γ and PGE(2). αDC1s and mDCs secreted IL-12 directly and following re-stimulation with CD40L-expressing cells and they mainly secreted the T effector cell attracting chemokines CXCL10 and CCL5 as opposed to sDCs that mainly secreted CCL22, known to attract regulatory T cells. αDC1s and mDCs were functionally superior to sDCs as they polarized naïve CD4(+) T cells most efficiently into T helper type 1 effector cells and primed more functional MART-1 specific CD8(+) T cells although with variation between donors. αDC1s and mDCs were transiently less capable of CCL21-directed transwell migration than standard matured DCs, likely due to their increased secretion of CCL19, which mediate internalization of CCR7. mpDCs were intermediate between standard and polarized DCs both in terms of IL-12 secretion and transwell migratory ability but functionally they resembled sDCs and strikingly had the highest expression of the inhibitory molecules PD-L1 and CD25. Thus, further studies with type 1 polarized DCs are warranted for use in immunotherapy, but when combined with PGE(2) as in mpDCs, they seems to be less optimal for maturation of DCs.