Therapeutic Bubbles

Ultrasound contrast agents consist of gas microbubbles (1 – 10 μm) that are coated by a protein, lipid or polymer. In addition to their diagnostic value, microbubbles have great potential as local drug delivery systems. Generally, two methods can be distinguished. The first is co-administration, i.e. the simultaneously administration of drugs and microbubbles where the vibrating microbubbles induce a transient increase in permeability of cell membranes and/or tissues. Elucidating this mechanism was one of the aims of this thesis. We found a direct correlation between vibrating microbubble-induced cell deformation and cell membrane permeability. Microbubbles induced transient pore formation, but also increased endocytosis. Endocytosis contribution was found to be dependent on the molecular size of the drug. Both intracellular calcium and reactive oxygen species were found to play a role in the mechanism of microbubble-induced increase in endothelial layer permeability. Targeted microbubbles could also increase cell membrane permeability, indicating that molecular imaging and drug delivery can be combined. The second class of therapeutic bubbles is the incorporation of a drug in the microbubble. Polymer microbubbles were constructed containing gas and therapeutics. Using ultrasound, the therapeutics were released from the microbubbles and taken up by cells. In addition, these microbubbles were found to induce an increase in endothelial cell membrane permeability as also observed with our co-administration studies. The research described in this thesis aids in understanding how to utilise bubbles for therapy in the most optimal way. However, many technical and pharmaceutical issues still need to be resolved before microbubble-mediated treatments in humans become available. For now, therapeutic bubbles are excitingly vibrant and bursting of great potential

Ultrasound-mediated targeted microbubble sonoporation of endothelial cells

Molecular imaging using ultrasound makes use of targeted microbubbles. In this study we investigated whether these microbubbles could also be used to induce drug uptake in endothelial cells. CD31-targeted microbubbles insonified at 1 MHz induced sonoporation when they were larger than 3.0 μm or when their relative vibration amplitude was larger than 0.5. No relationship was found between the position of the microbubble on the cell and the induction of sonoporation

Corrigendum to "In vivo characterization of ultrasound contrast agents: microbubble spectroscopy in a chicken embryo" (Ultrasound Med Biol 2012;38:1608-1617)

The authors regret that there was a mistake in reporting the mol% of the microbubble coating composition used. For all experiments, the unit in mg/mL was utilized, and the conversion mistake occurred only when converting to mol% to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol (Shen et al. 2008; Kilic and Bolukcu 2018), not 328.53 g/mol. On page 1610, the sentence should read “The coating was composed of DSPC (84.8 mol%; P6517, Sigma-Aldrich, Zwijndrecht, The Netherlands); PEG-40 stearate (8.2 mol%; P3440, Sigma-Aldrich); DSPE-PEG(2000) (5.9 mol%; 880125P, Avanti Polar Lipids, Alabaster, AL, USA); and DSPE-PEG(2000)-biotin (1.1 mol%; 880129C, Avanti Polar Lipids).” This correction does not change the conclusions published in this work. The authors apologize for any inconvenience caused

Ultrasonic Characterization of Ibidi μ-Slide I Luer Channel Slides for Studies With Ultrasound Contrast Agents

Understanding and controlling the ultrasound contrast agent&amp;#x2019;s response to an applied ultrasound pressure field is crucial when investigating ultrasound imaging sequences and therapeutic applications. The magnitude and frequency of the applied ultrasonic pressure waves affect the oscillatory response of the ultrasound contrast agent. Therefore, it is important to have an ultrasound compatible and optically transparent chamber in which the acoustic response of the ultrasound contrast agent can be studied. The aim of our study was to determine the &lt;italic&gt;in-situ&lt;/italic&gt; ultrasound pressure amplitude in the ibidi &amp;#x03BC;-slide I Luer channel, an optically transparent chamber suitable for cell culture including culture under flow, for all microchannel heights (200, 400, 600, 800 &amp;#x03BC;m). First, the &lt;italic&gt;in-situ&lt;/italic&gt; pressure field in the 800 &amp;#x03BC;m-high channel, was experimentally characterized using Brandaris 128 ultra-high-speed camera recordings of microbubbles and a subsequent iterative processing method, upon insonification at 2 MHz, 45&amp;#x00B0; incident angle, and 50 kPa peak negative pressure. Control studies in another cell culture chamber, the CLINIcell, were compared to the obtained results. The pressure amplitude was &amp;#x2013;3.7 dB with respect to the pressure field without the ibidi &amp;#x03BC;-slide. Second, using finite element analysis, we determined the &lt;italic&gt;in-situ&lt;/italic&gt; pressure amplitude in the ibidi with the 800 &amp;#x03BC;m channel (33.1 kPa) which was comparable to the experimental value (34 kPa). The simulations were extended to the other ibidi channel heights (200, 400, 600 &amp;#x03BC;m) with either 35&amp;#x00B0; or 45&amp;#x00B0; incident angle, and at 1 MHz and 2 MHz. The predicted &lt;italic&gt;in-situ&lt;/italic&gt; ultrasound pressure fields were between -8.7 dB to -1.1 dB of the incident pressure field depending on the listed configurations of ibidi slides with different channel heights, applied ultrasound frequencies, and incident angles. In conclusion, the determined ultrasound &lt;italic&gt;in-situ&lt;/italic&gt; pressures demonstrate the acoustic compatibility of the ibidi &amp;#x03BC;-slide I Luer for different channel heights, thereby showing its potential for studying the acoustic behavior of ultrasound contrast agents for imaging and therapy.</p

Ultrasonic Characterization of Ibidi μ-Slide I Luer Channel Slides for Studies With Ultrasound Contrast Agents

Understanding and controlling the ultrasound contrast agent&amp;#x2019;s response to an applied ultrasound pressure field is crucial when investigating ultrasound imaging sequences and therapeutic applications. The magnitude and frequency of the applied ultrasonic pressure waves affect the oscillatory response of the ultrasound contrast agent. Therefore, it is important to have an ultrasound compatible and optically transparent chamber in which the acoustic response of the ultrasound contrast agent can be studied. The aim of our study was to determine the &lt;italic&gt;in-situ&lt;/italic&gt; ultrasound pressure amplitude in the ibidi &amp;#x03BC;-slide I Luer channel, an optically transparent chamber suitable for cell culture including culture under flow, for all microchannel heights (200, 400, 600, 800 &amp;#x03BC;m). First, the &lt;italic&gt;in-situ&lt;/italic&gt; pressure field in the 800 &amp;#x03BC;m-high channel, was experimentally characterized using Brandaris 128 ultra-high-speed camera recordings of microbubbles and a subsequent iterative processing method, upon insonification at 2 MHz, 45&amp;#x00B0; incident angle, and 50 kPa peak negative pressure. Control studies in another cell culture chamber, the CLINIcell, were compared to the obtained results. The pressure amplitude was &amp;#x2013;3.7 dB with respect to the pressure field without the ibidi &amp;#x03BC;-slide. Second, using finite element analysis, we determined the &lt;italic&gt;in-situ&lt;/italic&gt; pressure amplitude in the ibidi with the 800 &amp;#x03BC;m channel (33.1 kPa) which was comparable to the experimental value (34 kPa). The simulations were extended to the other ibidi channel heights (200, 400, 600 &amp;#x03BC;m) with either 35&amp;#x00B0; or 45&amp;#x00B0; incident angle, and at 1 MHz and 2 MHz. The predicted &lt;italic&gt;in-situ&lt;/italic&gt; ultrasound pressure fields were between -8.7 dB to -1.1 dB of the incident pressure field depending on the listed configurations of ibidi slides with different channel heights, applied ultrasound frequencies, and incident angles. In conclusion, the determined ultrasound &lt;italic&gt;in-situ&lt;/italic&gt; pressures demonstrate the acoustic compatibility of the ibidi &amp;#x03BC;-slide I Luer for different channel heights, thereby showing its potential for studying the acoustic behavior of ultrasound contrast agents for imaging and therapy.</p

Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.</p

An in vitro proof-of-principle study of sonobactericide

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro flow study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent with or without other therapeutics. We developed a model of IE biofilm using human whole-blood clots infected with patient-derived S. aureus (infected clots). Histology and live-cell imaging revealed a biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% loss, which was greater than the other treatment arms. Effluent size measurements suggested low likelihood of emboli formation. These results support the continued investigation of sonobactericide as a therapeutic strategy for IE

Very different performance of the power Doppler modalities of several ultrasound machines ascertained by a microvessel flow phantom

Introduction: In many patients with rheumatoid arthritis (RA) subclinical disease activity can be detected with ultrasound (US), especially using power Doppler US (PDUS). However, PDUS may be highly dependent on the type of machine. This could create problems both in clinical trials and in daily clinical practice. To clarify how the PDUS signal differs between machines we created a microvessel flow phantom.Methods: The flow phantom contained three microvessels (150, 1000, 2000 microns). A syringe pump was used to generate flows. Five US machines were used. Settings were optimised to assess the lowest detectable flow for each US machine.Results: The minimal detectable flow velocities showed very large differences between the machines. Only two of the machines may be able to detect the very low flows in the capillaries of inflamed joints. There was no clear relation with price. One of the lower-end machines actually performed best in all three vessel sizes.Conclusions: We created a flow phantom to test the sensitivity of US machines to very low flows in small vessels. The sensitivity of the power Doppler modalities of 5 different machines was very different. The differences found between the machines are probably caused by fundamental differences in processing of the PD signal or internal settings inaccessible to users. Machines considered for PDUS assessment of RA patients should be tested using a flow phantom similar to ours. Within studies, only a single machine type should be used