A controlled in vitro study of optimal low intensity pulsed ultrasound fields for stimulation of proliferation in murine osteoblasts

Clinical, in vivo and in vitro studies have established that Low Intensity Pulsed Ultrasound (LIPUS) stimulates healing of fractured bone, but the mechanisms are not well understood. In vitro studies show cell proliferation, migration and many cellular markers are stimulated by LIPUS at frequencies of 1.0 - 1.5 MHz, even down to 45 kHz [1]. However, most trials did not control or measure the acoustic field, so the dose experienced by the cells across such studies cannot be compared. An in vitro ultrasound exposure method was developed to maintain control of the acoustic field. Murine osteoblasts (MC3T3-E1) were exposed to 20-minute LIPUS fields at the frequencies 1 MHz and 45 kHz and Mechanical Index from 0 (control) to 0.2. Cell proliferation was assessed by counting viable cells immediately before and twenty hours after LIPUS exposure in the centre of a custom-designed cell culture vessel. Initial results indicate that LIPUS fields increase cell proliferation at 1 MHz, 0.1 MI and more significantly at 45 kHz, 0.2 MI compared to controls, but has a detrimental or no effect otherwise. Future work will involve further repeats to acquire larger data sets, along with cell counts in other areas of the cell growth surface to increase data obtained from a single repeat. The study demonstrates the efficacy of the method for quantitative in vitro investigation of LPUS mechanisms and will be used in future work to find optimum LIPUS field characteristics, in terms of frequency, Mechanical Index, and pulse characteristics such as pulse repetition rate and pulse width

From animal models to patients : the role of placental microRNAs, miR-210, miR-126, and miR-148a/152 in preeclampsia

Placental microRNAs (miRNAs) regulate the placental transcriptome and play a pathological role in preeclampsia (PE), a hypertensive disorder of pregnancy. Three PE rodent model studies explored the role of placental miRNAs, miR-210, miR-126, and miR-148/152 respectively, by examining expression of the miRNAs, their inducers, and potential gene targets. This review evaluates the role of miR-210, miR-126, and miR-148/152 in PE by comparing findings from the three rodent model studies with in vitro studies, other animal models, and preeclamptic patients to provide comprehensive insight into genetic components and pathological processes in the placenta contributing to PE. The majority of studies demonstrate miR-210 is upregulated in PE in part driven by HIF-1a and NF-?Bp50, stimulated by hypoxia and/or immune-mediated processes. Elevated miR-210 may contribute to PE via inhibiting anti-inflammatory Th2-cytokines. Studies report an up- and downregulation of miR-126, arguably reflecting differences in expression between cell types and its multifunctional capacity.MiR-126 may play a pro-angiogenic role bymediating the PI3K-Akt pathway. Most studies report miR-148/152 family members are upregulated in PE. Evidence suggests they may inhibit DNA methylation of genes involved in metabolic and inflammatory pathways. Given the genetic heterogeneity of PE, it is unlikely that a single placental miRNA is a suitable therapeutic target for all patients. Investigating miRNAs in PE subtypes in patients and animal models may represent a more appropriate approach going forward. Developing methods for targeting placental miRNAs and specific placental cell types remains crucial for research seeking to target placental miRNAs as a novel treatment for PE

Rapid prototyped microvessel flow phantom for controlled investigation of ultrasound-mediated targeted drug delivery

The ability to undertake controlled and accurate investigation of the complex features that determine microvasculature-fluid systems is essential in our progress towards successful clinical exploitation of ultrasound mediated targeted drug delivery (UmTDD). We present an engineered platform to enable accurate understanding of the microvascular flow characteristics capable of influencing UmTDD. We develop novel 3D-printed flow phantoms, accurately replicating real microvascular structures, and a new approach to investigating UmTDD phenomena that places microbubble-microvessel interactions at the heart of the problem. Our aim is to establish a robust, lab-based platform for controlled and systematic investigation of microvessel architectures as key determinants in UmTDD efficiency

Ultrasound and microbubble gene delivery for targeting altered placental microRNAs in preeclampsia

Ultrasound (US) and microbubble (MB) gene delivery has attracted growing interest as a clinically applicable gene therapy (GT). Though preclinical studies have investigated the system in various tissues, there is limited research in targeting the placenta. This is a potential therapeutic strategy for preeclampsia (PE), which has an underlying genetic basis and ineffective management strategies. Differentially expressed placental microRNAs (miRNAs) in PE may represent suitable targets for GT. Microbubbles (SonoVue) and plasmid (pGL3 or pGL4.13) were administered systemically to CD1 mice, followed by exposure of the heart to US (H14, 1.8 M.I., 1cm focal depth, 2 minutes), using Siemens Acuson Sequoia-512 system and 15L8 probe. Luciferase assays were performed to evaluate gene transfection. Significantly differentially expressed placental miRNAs in PE patients were identified as candidates based on detection by three or more screening studies. Expression of candidate miRNAs was measured by qRT-PCR in PE rat model placentas. In trial 1, low levels of luciferase activity were detected in the heart of treatment mouse 1, 2 and 3. In trial 2, luciferase activity was evident in the atria of treatment mouse 2. In trial 3, higher luciferase activity was detected in the ventricles of the treatment mouse and activity was also detected in the atria. The literature review identified eight candidate miRNAs. MiR-223 (1.46-fold increase) and miR-181a (0.81-fold decrease) were significantly differentially expressed in PE rat model placentas. MiR-223 and -181a may represent targets for US and MB gene delivery. Future studies will apply the US and MB gene delivery protocol for translation to targeting the placenta in our PE rodent model

Microbubble – Microvessel Interactions and their Influence on UmTDD: Simulation Study

No abstract available

Contrast-enhanced magnetomotive ultrasound imaging (CE-MMUS) for colorectal cancer staging : assessment of sensitivity and resolution to detect alterations in tissue stiffness

A key challenge in the treatment of colorectal cancer is identification of the sentinel draining lymph node. Magnetomotive ultrasound, MMUS, has identified lymph nodes in rat models: superparamagnetic iron oxide nanoparticles (SPIONs) accumulated in the lymph are forced to oscillate by an external magnetic field; the resulting axial displacement is recovered allowing structure delineation with potential to indicate alterations in tissue stiffness, but it is limited by small vibration amplitudes. We propose CE-MMUS using SPION loaded microbubbles (SPION-MBs) to enhance sensitivity, reduce toxicity, and offer additional diagnostic or perfusion information. Laser doppler vibrometry measurements was performed on SPION containing tissue mimicking material during magnetic excitation. These measurements show a vibration amplitude of 279 ± 113 μm in a material with Young's modulus of 24.3 ± 2.8 kPa, while the displacements were substantially larger, 426 ± 9 μm, in the softer material, with a Young's modulus of 9.6 ± 0.8 kPa. Magnetic field measurement data was used to calibrate finite element modelling of both MMUS and CE-MMUS. SPION-MBs were shown to be capable of inducing larger tissue displacements under a given magnetic field than SPIONs alone, leading to axial displacements of up to 2.3x larger. A doubling in tissue stiffness (as may occur in cancer) reduces the vibration amplitude. Thus, there is potential for CE-MMUS to achieve improved stiffness sensitivity. Our aim is to define the potential contribution of CE-MMUS in colorectal cancer diagnosis and surgical guidance

Ultrasound and Microbubbles Promote the Retention of Fluorescent Compounds in the Small Intestine

Focused ultrasound (US) is a novel means to increase the passage of medication through the wall of the small intestine. The purpose of this study was to determine whether US and microbubbles (MBs) can facilitate delivery of macromolecular therapeutic agents across the intestinal epithelium in vitro and in vivo. In vitro experiments involved delivery of compounds across a cell monolayer, namely Caco-2 cells cultured on ThinCert filters. The cells were cultured for a minimum of 3 weeks to mimic the polarised intestinal epithelium. A suspension of dextran with or without MBs, prepared in growth medium, was introduced into the apical chamber of the ThinCert with a syringe pump through a channel in the centre of a miniature focused US transducer (4 MHz, 1 MPa PNP). Each in vivo experiment involved a tethered endoscopic capsule with an US transducer and a delivery channel inserted into the small intestine of a terminally anaesthetised pig via a surgical stoma. The amount of fluorescent dextran delivered across the Caco-2 monolayer when employing US, MBs and dextran was higher than the amount delivered with dextran alone. With this approach, fluorescent marking of the wall of the small intestine was achieved in vivo by applying US and MBs. Our work indicates that US has potential for application in targeted treatment of gastrointestinal disease and oral drug delivery

Optical and acoustic characterisation of multimodal contrast agents for colorectal cancer lymph node detection

Introduction Localisation and characterisation of lymph nodes in colorectal cancer (CRC) is integral to staging, resection surgery and patient outcomes [1]. However, appropriate imaging technologies capable of providing detailed information to the oncologist to inform these processes remain severely limited. We are investigating magnetic ultrasound contrast-agents, delivered either as gas microbubbles or phase-change nanodroplets each with streptavidin-biotinylated magnetic nanoparticles, for combined contrast-enhanced and magneto-motive ultrasound imaging CE-MMUS and more sensitive disease detection. Methods Magnetic-microbubbles were prepared from Target-Ready Micromarker (Fujifilm, Visualsonics) and biotinylated magnetic nanoparticles [2] and condensed to produce phase-change magnetic-nanoparticles [3]. Contrast agents were sized, concentration and magnetic loading measured using dynamic light scattering and nanoparticle tracking analysis (Zetasizer & Nanosight NS300, Malvern Panalytical) to inform acoustic drive conditions required to activate phase-change, returning them to microbubbles and later confirmed using single-element excitation transducers (frequencies: 1, 3.5, 5 MHz) and a passive cavitation detection (Precision Acoustics). Phantoms mimicking acoustic and mechanical properties of lymph node tissue were fabricated using polyacrylamide [4] incorporating each contrast agent. Results Contrast enhanced ultrasound imaging was performed (18MHz, Vevo 3100, Fujifilm VisualSonics) in a wild type mouse to assess lymphatic drainage of magnetic microbubbles after bolus injection, with peak enhancement occurring at 3.7s. An externally applied magnetic field (solenoid, in-house fabrication; 4 & 20 Hz, 1.3T) was used to produce preliminary MMUS data demonstrating proof of concept of each formulation. Data were also used to inform a finite-element model to assess magneto-mechanical interactions of a magnetic microbubble with an elastic solid [5]. The estimated relationship between tissue displacement and microbubble / nanodroplet size was compared against formulation size distribution and predictions relating to tissue displacement were validated in tissue phantoms. Finally, tissue displacements generated and recovered via MMUS in our pre-clinical model for each formulations (magnetic microbubbles, magnetic nanodroplets) were investigated. Conclusions Multimodal magnetic contrast agents are easily fabricated from commercial formulations to support CE-MMUS. Phase-change agents are readily returned to microbubble-state once in a region of interest. We previously demonstrated perfusion dynamics can indicate lymph node metastatic involvement [6]. Smaller diameter agents, optimised for lymph node microvessel drainage may improve our technique sensitivity and preserve full utility in CE-MMUS

Development of Preclinical Ultrasound Imaging Techniques to Identify and Image Sentinel Lymph Nodes in a Cancerous Animal Model

Lymph nodes (LNs) are believed to be the first organs targeted by colorectal cancer cells detached from a primary solid tumor because of their role in draining interstitial fluids. Better detection and assessment of these organs have the potential to help clinicians in stratification and designing optimal design of oncological treatments for each patient. Whilst highly valuable for the detection of primary tumors, CT and MRI remain limited for the characterization of LNs. B-mode ultrasound (US) and contrast-enhanced ultrasound (CEUS) can improve the detection of LNs and could provide critical complementary information to MRI and CT scans; however, the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) guidelines advise that further evidence is required before US or CEUS can be recommended for clinical use. Moreover, knowledge of the lymphatic system and LNs is relatively limited, especially in preclinical models. In this pilot study, we have created a mouse model of metastatic cancer and utilized 3D high-frequency ultrasound to assess the volume, shape, and absence of hilum, along with CEUS to assess the flow dynamics of tumor-free and tumor-bearing LNs in vivo. The aforementioned parameters were used to create a scoring system to predict the likelihood of a disease-involved LN before establishing post-mortem diagnosis with histopathology. Preliminary results suggest that a sum score of parameters may provide a more accurate diagnosis than the LN size, the single parameter currently used to predict the involvement of an LN in disease

Ultrasound mediated delivery of quantum dots from a proof of concept capsule endoscope to the gastrointestinal wall

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focused ultrasound during in vivo experiments using porcine models. This study illustrates how such a device could be potentially used for gastrointestinal drug delivery and the challenges to be overcome before focused ultrasound and microbubbles could be used with this device for the oral delivery of biologic therapeutics