Sonophoresis with ultrasound-responsive liquid-core nuclei for transdermal drug delivery.

BACKGROUND: Sonophoresis can increase the delivery efficiency of various drugs into the skin. A recent advance in sonophoresis is the use of ultrasound-responsive liquid-core nuclei (URLN) to increase the probability of cavitation. In this study, we developed a URLN and ultrasound device, and demonstrated its effectiveness through in vitro and clinical tests. MATERIALS AND METHODS: Three types of experiments were designed to evaluate the efficiency of sonophoresis with URLN. First, a Franz diffusion cell with cosmetic ingredients was used to analyze quantitatively the amount of drug delivered to the porcine skin. Second, after the application of sonophoresis with URLN, the porcine skin surface was examined using scanning electron microscopy (SEM) to see the changes in morphology. Finally, a clinical test was performed to verify the utility of sonophoresis with URLN. RESULTS: The results indicate that sonophoresis with URLN can increase the amount of compound delivered by approximately 11.9-fold over 6 h for niacinamide and by 7.33-fold over 6 h for adenosine. In addition, we observed approximately 20-30 µm sized pores on porcine skin in SEM images. In clinical testing, the application of sonophoresis with cosmetics containing URLN for 3 min improved the efficiency of transdermal drug delivery by 1.9-fold, the depth of absorption by 2.0-fold, and the speed of absorption by 2.0-fold at 30 min after application. CONCLUSION: We expect that sonophoresis with specialized URLN in transdermal drug delivery could be used widely for various skin-related applications.

Multi-Focus Image Fusion Using Focal Area Extraction in a Large Quantity of Microscopic Images.

The non-invasive examination of conjunctival goblet cells using a microscope is a novel procedure for the diagnosis of ocular surface diseases. However, it is difficult to generate an all-in-focus image due to the curvature of the eyes and the limited focal depth of the microscope. The microscope acquires multiple images with the axial translation of focus, and the image stack must be processed. Thus, we propose a multi-focus image fusion method to generate an all-in-focus image from multiple microscopic images. First, a bandpass filter is applied to the source images and the focus areas are extracted using Laplacian transformation and thresholding with a morphological operation. Next, a self-adjusting guided filter is applied for the natural connections between local focus images. A window-size-updating method is adopted in the guided filter to reduce the number of parameters. This paper presents a novel algorithm that can operate for a large quantity of images (10 or more) and obtain an all-in-focus image. To quantitatively evaluate the proposed method, two different types of evaluation metrics are used: "full-reference" and "no-reference". The experimental results demonstrate that this algorithm is robust to noise and capable of preserving local focus information through focal area extraction. Additionally, the proposed method outperforms state-of-the-art approaches in terms of both visual effects and image quality assessments.

Parametric response mapping of dynamic CT for predicting intrahepatic recurrence of hepatocellular carcinoma after conventional transcatheter arterial chemoembolization.

OBJECTIVES: The aim of our study was to determine the diagnostic value of a novel image analysis method called parametric response mapping (PRM) for prediction of intrahepatic recurrence of hepatocellular carcinoma (HCC) treated with conventional transcatheter arterial chemoembolization (TACE). METHODS: This retrospective study was approved by the IRB. We recruited 55 HCC patients who achieved complete remission (CR) after TACE and received longitudinal multiphasic liver computed tomography (CT). The patients fell into two groups: the recurrent tumour group (n = 29) and the non-recurrent tumour group (n = 26). We applied the PRM analysis to see if this technique could distinguish between the two groups. The results of the PRM analysis were incorporated into a prediction algorithm. We retrospectively removed data from the last time point and attempted to predict the response to therapy of the removed data. RESULTS: The PRM analysis was able to distinguish between the non-recurrent and recurrent groups successfully. The prediction algorithm detected response to therapy with an area under the curve (AUC) of 0.76, while the manual approach had AUC 0.64. CONCLUSIONS: Adopting PRM analysis can potentially distinguish between recurrent and non-recurrent HCCs and allow for prediction of response to therapy after TACE. KEY POINTS: Parametric response mapping (PRM) could help assess patients with recurrent HCCs after TACE. Parametric response mapping could direct patients to individualized therapy. Longitudinal CT images were analyzed with advanced image analysis method.

Connectivity Analysis and Feature Classification in Attention Deficit Hyperactivity Disorder Sub-Types: A Task Functional Magnetic Resonance Imaging Study.

Attention deficit hyperactivity disorder (ADHD) is a pervasive neuropsychiatric disorder. Patients with different ADHD subtypes show different behaviors under different stimuli and thus might require differential approaches to treatment. This study explores connectivity differences between ADHD subtypes and attempts to classify these subtypes based on neuroimaging features. A total of 34 patients (13 ADHD-IA and 21 ADHD-C subtypes) underwent functional magnetic resonance imaging (fMRI) with six task paradigms. Connectivity differences between ADHD subtypes were assessed for the whole brain in each task paradigm. Connectivity measures of the identified regions were used as features for the support vector machine classifier to distinguish between ADHD subtypes. The effectiveness of connectivity measures of the regions were tested by predicting ADHD-related Diagnostic and Statistical Manual of Mental Disorders (DSM) scores. Significant connectivity differences between ADHD subtypes were identified mainly in the frontal, cingulate, and parietal cortices and partially in the temporal, occipital cortices and cerebellum. Classifier accuracy for distinguishing between ADHD subtypes was 91.18 % for both gambling punishment and emotion task paradigms. Linear prediction under the two task paradigms showed significant correlation with DSM hyperactive/impulsive score. Our study identified important brain regions from connectivity analysis based on an fMRI paradigm using gambling punishment and emotion task paradigms. The regions and associated connectivity measures could serve as features to distinguish between ADHD subtypes.

Real-Time Analysis of Cellular Response to Small-Molecule Drugs within a Microfluidic Dielectrophoresis Device.

Quantitative detection of the biological properties of living cells is essential for a wide range of purposes, from the understanding of cellular characteristics to the development of novel drugs in nanomedicine. Here, we demonstrate that analysis of cell biological properties within a microfluidic dielectrophoresis device enables quantitative detection of cellular biological properties and simultaneously allows large-scale measurement in a noise-robust and probeless manner. Applying this technique, the static and dynamic biological responses of live B16F10 melanoma cells to the small-molecule drugs such as N-ethylmaleimide (NEM) and [(dihydronindenyl)oxy]alkanoic acid (DIOA) were quantitatively and statistically examined by investigating changes in movement of the cells. Measurement was achieved using subtle variations in dielectrophoresis (DEP) properties of the cells, which were attributed to activation or deactivation of K(+)/Cl(-) cotransporter channels on the cell membrane by the small-molecule drugs, in a microfluidic device. On the basis of quantitative analysis data, we also provide the first report of the shift of the complex permittivity of a cell induced by the small-molecule drugs. In addition, we demonstrate interesting quantifiable parameters including the drug effectiveness coefficient, antagonistic interaction coefficient, kinetic rate, and full width at half-maximum, which corresponded to changes in biological properties of B16F10 cells over time when NEM and DIOA were introduced alone or in combination. Those demonstrated parameters represent very useful tools for evaluating the effect of small-molecule drugs on the biological properties of cells.

Parametric response mapping of dynamic CT as an imaging biomarker to distinguish viability of hepatocellular carcinoma treated with transcatheter arterial chemoembolization.

PURPOSE: Accurate assessment of viability of hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE) is important for therapy planning. The purpose of this study is to determine the diagnostic value of a novel image analysis method called parametric response mapping (PRM) in predicting viability of tumor in HCC treated with TACE for dynamic CT images. METHODS: 35 patients who had 35 iodized-oil defect areas (IODAs) in HCCs treated with TACE were included in our study. These patients were divided into two groups, one group with viable tumors (n = 22) and the other group with non-viable tumors (n = 13) in the IODA. All patients were followed up using triple-phase dynamic CT after the treatment. We compared (a) manual analysis, (b) using PRM results, and (c) using PRM results with automatic classifier to distinguish between two tumor groups based on dynamic CT images from two longitudinal exams. Two radiologists performed the manual analysis. The PRM approach was implemented using prototype software. We adopted an off-the-shelf k nearest neighbor (kNN) classifier and leave-one-out cross-validation for the third approach. The area under the curve (AUC) values were compared for three approaches. RESULTS: Manual analysis yielded AUC of 0.74, using PRM results yielded AUC of 0.84, and using PRM results with an automatic classifier yielded AUC of 0.87. CONCLUSIONS: We improved upon the standard manual analysis approach by adopting a novel image analysis method of PRM combined with an automatic classifier.

Synthesis of laboratory Ultrasound Contrast Agents.

Ultrasound Contrast Agents (UCAs) were developed to maximize reflection contrast so that organs can be seen clearly in ultrasound imaging. UCAs increase the signal to noise ratio (SNR) by linear and non-linear mechanisms and thus help more accurately visualize the internal organs and blood vessels. However, the UCAs on the market are not only expensive, but are also not optimized for use in various therapeutic research applications such as ultrasound-aided drug delivery. The UCAs fabricated in this study utilize conventional lipid and albumin for shell formation and perfluorobutane as the internal gas. The shape and density of the UCA bubbles were verified by optical microscopy and Cryo SEM, and compared to those of the commercially available UCAs, Definity® and Sonovue®. The size distribution and characteristics of the reflected signal were also analyzed using a particle size analyzer and ultrasound imaging equipment. Our experiments indicate that UCAs composed of spherical microbubbles, the majority of which were smaller than 1 um, were successfully synthesized. Microbubbles 10 um or larger were also identified when different shell characteristics and filters were used. These laboratory UCAs can be used for research in both diagnoses and therapies.

A phased array ultrasound system with a robotic arm for neuromodulation.

BACKGROUND: Ultrasonic neuromodulation (UNMOD) provides a non-invasive brain stimulation. However, the high-resolution region-specificity of UNMOD with a single element transducer combined with a mechanical positioning system could have limits due to the intrinsic positioning error from mechanical systems. OBJECTIVE/HYPOTHESIS: A phased array system could lead to highly selective neuromodulation with electronic control. METHODS: A specialized phased-array system with a robotic arm is implemented for a rhesus monkey model. Various primary motor cortex areas related to tail, hand, and mouth were stimulated with a 200 µm step size. The ultrasonic parameters were ISPTA of 840 mW/cm2, pulse repetition frequency of 100 Hz, and a 5% duty factor at 600 kHz. The induced movement were recorded and analyzed. RESULTS: Separate digits, mouth, and tongue motions were successfully induced by electronically controlling the focus. The identical body part movement could be induced when the focus was moved back to the identical primary motor cortex with electronic control. Accordingly, the reproducibility of UNMOD could be partially validated with rhesus monkey model. CONCLUSION: A phased-array system appears to have a potential for the non-invasive and region-selective neuromodulation method.

Electrochemical detection of high-sensitivity CRP inside a microfluidic device by numerical and experimental studies.

The concentration of C-reactive protein (CRP), a classic acute phase plasma protein, increases rapidly in response to tissue infection or inflammation, especially in cases of cardiovascular disease and stroke. Thus, highly sensitive monitoring of the CRP concentration plays a pivotal role in detecting these diseases. Many researchers have studied methods for the detection of CRP concentrations such as optical, mechanical, and electrochemical techniques inside microfluidic devices. While significant progress has been made towards improving the resolution and sensitivity of detection, only a few studies have systematically analyzed the CRP concentration using both numerical and experimental approaches. Specifically, systematic analyses of the electrochemical detection of high-sensitivity CRP (hsCRP) using an enzyme-linked immunosorbant assay (ELISA) inside a microfluidic device have never been conducted. In this paper, we systematically analyzed the electrochemical detection of CRP modified through the attachment of an alkaline phosphatase (ALP-labeled CRP) using ELISA inside a chip. For this analysis, we developed a model based on antigen-antibody binding kinetics theory for the numerical quantification of the CRP concentration. We also experimentally measured the current value corresponding to the ALP-labeled CRP concentration inside the microfluidic chip. The measured value closely matched the calculated value obtained by numerical simulation using the developed model. Through this comparison, we validated the numerical simulation methods, and the calculated and measured values. Lastly, we examined the effects of various microfluidic parameters on electrochemical detection of the ALP-labeled CRP concentration using numerical simulations. The results of these simulations provide insight into the microfluidic electrochemical reactions used for protein detection. Furthermore, the results described in this study should be useful for the design and optimization of electrochemical immunoassay chips for the detection of target proteins.

Single-step electropolymerization patterning of a polypyrrole nanowire by ultra-short pulses via an AFM cantilever.

Conducting polymers (CPs) have attracted a great deal of attention due to their unique properties; these properties are useful in implementing various functional devices, such as memory, and chemical and biological sensors. In particular, the nanopatterning of CPs is a key technology that will accelerate the adoption of CPs in fabricating nanoscaled multifunctional devices. This paper presents an innovative technique for forming polypyrrole nanowire (PPy-NW) patterns, without any additional pretreatment on the gold surface, using atomic force microscopy (AFM) and ultra-short pulse voltage. Applying the ultra-short pulse voltage to the AFM tip has the following advantage: since the electrochemical current is extremely localized around the tip, the successful formation of CP nanowires results. This is because the pulse width is much shorter than the resistor-capacitor (RC) time constant of the equivalent electrochemical circuit of our experimental set-up. This paper provides systematic results regarding the dimensional variation of the PPy-NW patterns produced by varying the electrical conditions of the ultra-short pulse, such as the pulse amplitude, width, and frequency. The results show that use of an ultra-short pulse is essential in fabricating PPy-NW patterns. Additionally, an ultra-short pulse offers excellent pattern controllability for both width (353 nm ∼ 3.37 µm) and height (2.0 ∼ 88.3 nm).

Experimental and numerical study of electrochemical nanomachining using an AFM cantilever tip.

We fabricated nanopatterns on Cu thin films via an electrochemical route using an atomic force microscope (AFM). Experimental results were compared with an equivalent electrochemical circuit model representing an electrochemical nanomachining (ECN) technique. In order to precisely construct the nanopatterns, an ultra-short pulse was applied onto the Cu film through the AFM cantilever tip. The line width of the nanopatterns (the lateral dimension) increased with increased pulse amplitude, on-time, and frequency. The tip velocity effect on the nanopattern line width was also investigated. The study described here provides important insight for fabricating nanopatterns precisely using electrochemical methods with an AFM cantilever tip.

A physical method to enhance transdermal delivery of a tissue optical clearing agent: combination of microneedling and sonophoresis.

BACKGROUND AND OBJECTIVES: Various physical methods, such as microneedling, laser ablation, sonophoresis, and sandpaper, have been widely studied to enhance the transdermal delivery of tissue optical clearing (TOC) agents. A previous study demonstrated that the microneedling method could effectively enhance the permeability of a TOC agent through the skin barrier. STUDY DESIGN/MATERIALS AND METHODS: In this study, we introduce a new physical combination method which utilizes both microneedling and sonophoresis to further enhance the transdermal delivery of a TOC agent, glycerol. Porcine skin samples were divided into a control group treated only with the microneedle roller and a test group treated with both the microneedle roller and sonophoresis. Glycerol was applied topically after microneedling. The optimal concentration and transdermal delivery efficacy of glycerol were quantitatively evaluated. RESULTS: A 70% glycerol solution was determined to be the optimal concentration for the combination method. The combination method resulted in approximately a 2.3-fold higher transdermal diffusion rate of glycerol when compared to the microneedling method alone. CONCLUSION: The combination method and optimal glycerol concentration effectively enhanced transdermal delivery of glycerol by accelerating the diffusion rate through the skin barrier.

Development of 64Cu-loaded Perfluoropentane Nanodroplet: A Potential Tumor Theragnostic Nano-carrier and Dual-Modality PET-Ultrasound Imaging Agents.

The purpose of this study was to develop and preliminarily evaluate phospholipid-shelled nanodroplets (NDs) encapsulating perfluoropentane (PFP) and radioactive 64Cu as a hybrid positron emission tomography (PET)-ultrasound (US) probe. PFP NDs were fabricated by mixing liquid-phase PFP with a phospholipid solution. The 64Cu was encapsulated into the NDs in a size-controlled manner by exploiting the hydrophobicity of 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) using a vial mixer and an extruder. The fabricated 64Cu-loaded PFP NDs (64Cu-PFP NDs) were evaluated using in vitro/in vivo PET-computed tomography (PET-CT), US imaging and transmission electron microscopy. In the in vitro PET images, the 64Cu-PFP NDs were observed as a hot spot in the lower section of the test tube. In the acquired US images, the mean region of interest brightness values of 64Cu-PFP NDs were revealed by their strong echo image. In a tumor-bearing mouse animal model, tumor uptake of the 64Cu-PFP NDs was low, that is, approximately 65%, compared with that of only free 64Cu, as determined by PET-delayed imaging analysis. The dual-function concept of the NDs is expected to contribute to the prognosis and effectiveness of therapy by fusing the science and technology of nuclear medicine and US.

Anti-foci for focused ultrasound.

An anti-focus is defined as the point where the pressure amplitude is zero. As an active protection means in focused ultrasound, the anti-focus was researched through simulations. A 513 element, 1 MHz, 15 cm focal depth, spherical shape transducer with a central hold was used for the simulation. An anti-focus was implemented with two different approaches and the simulation results were compared. First, the interference among foci was compared to the summation of the individual focus fields. Second, the interference among anti-foci was compared to the multiplication of the individual anti-focus fields. The minimum distance between a focus and an anti-focus was determined by the comparison between intended control values and the simulation results. The field simulation results indicate that the minimum distance to control the field is approximately 3 mm in the lateral direction and 15 mm in the axial direction. The results also demonstrate that a specific zone can be effectively protected with the addition of anti-foci and that the field can be effectively implemented by switching type amplifier systems. Results demonstrate the potential for anti-foci to significantly reduce the collateral damage in focused ultrasound through improved field control.

Transdermal Drug Delivery using a Specialized Cavitation Seed for Ultrasound.

GOAL: The Sonophoresis, which utilizes ultrasound for transdermal drug delivery (TDD), can improve the efficiency of drug delivery for a variety of drugs predominantly due to caviation effect. In order to increase the efficacy of sonophoresis, we propose an alternative cavitation seed specialized for sonophoresis, which can be concentrated on the skin surface by gravity adapting perfluorohexane as core. METHODS: An in vitro and in vivo experiments were conducted to assess the effect of the specialized cavitation seed. High performance liquid chromatography was used for in vitro experiments on porcine skin with ferulic acid and an optical imaging system was used for in vivo experiments on rat model with fluorescein isothiocyanate-dextran (FD, 150 kDa), respecitively. RESULTS: The amount of ferulic acid delivered by sonophoresis with the proposed cavitation seed was approximately 1,700 times greater than the amount delivered by diffusion. FD could be delivered to a depth of 500 »m under the skin, and the average total flux in the region of interest was increased 6.4-fold for the group using sonophoresis with the cavitation seed compared to the group using diffusion. CONCLUSION: Conclusively, sonophoresis with the proposed cavitation seed demonstrated significant improvement in TDD and the possibility of macromolecule delivery into the skin. SIGNIFICANCE: This approach has potential to be a main TDD method for variety of applications including medicine and cosmetics.

Functional brain networks associated with eating behaviors in obesity.

Obesity causes critical health problems including diabetes and hypertension that affect billions of people worldwide. Obesity and eating behaviors are believed to be closely linked but their relationship through brain networks has not been fully explored. We identified functional brain networks associated with obesity and examined how the networks were related to eating behaviors. Resting state functional magnetic resonance imaging (MRI) scans were obtained for 82 participants. Data were from an equal number of people of healthy weight (HW) and non-healthy weight (non-HW). Connectivity matrices were computed with spatial maps derived using a group independent component analysis approach. Brain networks and associated connectivity parameters with significant group-wise differences were identified and correlated with scores on a three-factor eating questionnaire (TFEQ) describing restraint, disinhibition, and hunger eating behaviors. Frontoparietal and cerebellum networks showed group-wise differences between HW and non-HW groups. Frontoparietal network showed a high correlation with TFEQ disinhibition scores. Both frontoparietal and cerebellum networks showed a high correlation with body mass index (BMI) scores. Brain networks with significant group-wise differences between HW and non-HW groups were identified. Parts of the identified networks showed a high correlation with eating behavior scores.

Sonophoresis Using Ultrasound Contrast Agents: Dependence on Concentration.

Sonophoresis can increase skin permeability to various drugs in transdermal drug delivery. Cavitation is recognized as the predominant mechanism of sonophoresis. Recently, a new logical approach to enhance the efficiency of transdermal drug delivery was tried. It is to utilize the engineered microbubble and its resonant frequency for increase of cavitation activity. Actively-induced cavitation with low-intensity ultrasound (less than ~1 MPa) causes disordering of the lipid bilayers and the formation of aqueous channels by stable cavitation which indicates a continuous oscillation of bubbles. Furthermore, the mutual interactions of microbubble determined by concentration of added bubble are also thought to be an important factor for activity of stable cavitation, even in different characteristics of drug. In the present study, we addressed the dependence of ultrasound contrast agent concentration using two types of drug on the efficiency of transdermal drug delivery. Two types of experiment were designed to quantitatively evaluate the efficiency of transdermal drug delivery according to ultrasound contrast agent concentration. First, an experiment of optical clearing using a tissue optical clearing agent was designed to assess the efficiency of sonophoresis with ultrasound contrast agents. Second, a Franz diffusion cell with ferulic acid was used to quantitatively determine the amount of drug delivered to the skin sample by sonophoresis with ultrasound contrast agents. The maximum enhancement ratio of sonophoresis with a concentration of 1:1,000 was approximately 3.1 times greater than that in the ultrasound group without ultrasound contrast agent and approximately 7.5 times greater than that in the control group. These results support our hypothesis that sonophoresis becomes more effective in transdermal drug delivery due to the presence of engineered bubbles, and that the efficiency of transdermal drug delivery using sonophoresis with microbubbles depends on the concentration of microbubbles in case stable cavitation is predominant.

Aberration correction by nonlinear beam mixing: generation of a pseudo point sound source.

Nonlinear beam mixing with microbubbles was explored to create a pseudo point source for aberration correction of therapeutic ultrasound. A damping coefficient for a bubble driven by a dual frequency sound field was derived by revisiting Prosperetti's linearized damping model. As a result, the overall damping term for dual frequency was obtained by linear summation of two damping terms for each frequency. The numerical simulation based on the bubble model suggests that the most efficient size range to generate a 1 MHz frequency from 4 MHz and 5 MHz sound sources is 2.6 to 3.0 microm. Furthermore, this size range constitutes the primary distribution of a specific ultrasound contrast agent. When a chamber of 0.1% of the diluted agent is sonified by 4 MHz and 5 MHz sound beams with 80 degrees incident angle between them, an approximately 100 Pa, 1 MHz difference frequency signal can be measured approximately 10 cm away. In addition, the received 1 MHz difference frequency signal shows omni-directional characteristics, even though the overlap zone of the two sound beams is on the order of the difference frequency wavelength. Therefore, the induced sound source can be considered as a pseudo point source and is expected to be useful for aberration correction for therapeutic ultrasound.

Effects of contrast agent infusion rates on thresholds for tissue damage produced by single exposures of high-intensity ultrasound.

Stabilized microbubble ultrasound contrast agents (UCA) have potential to aid tissue ablation during ultrasound surgery by enhancing both cavitational and thermal damage mechanisms. Previously, we showed UCA infused at a rate of 1 microL/kg/min prior to ultrasound exposure could reduce the total energy required to produce tissue damage by up to two orders of magnitude. In this paper, we evaluate thresholds for macroscopic tissue damage with UCA infusion rates (IR) of 0.1, 0.3, 1, 3, and 10 microL/kg/min to determine IR potentially effective for ultrasound therapy. Canine kidneys were surgically externalized and insonified with single exposures of focused ultrasound. Incident exposures were 1.44 MHz tone bursts, either 250 ms in duration with intensity between 500 W/cm2 and 3200 W/cm2, or 100 micros to 1 s in duration with intensity equal to 3200 W/cm2. Probabilities of tissue damage occurrence were determined for each set of exposure conditions (intensity, duration, and IR). A threshold intensity and threshold duration, defined as the quantities for which tissue damage occurred with probability equal to 0.5, were estimated for each IR. Results show that, as IR increased from 0.1 to 10 microL/kg/min, the threshold intensity decreased by up to a factor of 3, and threshold duration decreased by up to a factor of 200. Microbubble introduction at IR up to 10 microL/kg/min thus may be effective in aiding ultrasound therapy.