Robust spatially resolved pressure measurements using MRI with novel buoyant advection-free preparations of stable microbubbles in polysaccharide gels

MRI of fluids containing lipid coated microbubbles has been shown to be an effective tool for measuring the local fluid pressure. However, the intrinsically buoyant nature of these microbubbles precludes lengthy measurements due to their vertical migration under gravity and pressure-induced coalescence. A novel preparation is presented which is shown to minimize both these effects for at least 25 min. By using a 2% polysaccharide gel base with a small concentration of glycerol and 1,2-distearoyl-sn-glycero-3-phosphocholine coated gas microbubbles, MR measurements are made for pressures between 0.95 and 1.44 bar. The signal drifts due to migration and amalgamation are shown to be minimized for such an experiment whilst yielding very high NMR sensitivities up to 38% signal change per bar

Subharmonic Behavior of Phospholipid-coated Ultrasound Contrast Agent Microbubbles

Coated microbubbles, unlike tissue are able to scatter sound subharmonically. Therefore, the subharmonic behavior of coated microbubbles can be used to enhance the contrast in ultrasound contrast imaging. Theoretically, a threshold amplitude of the driving pressure can be calculated above which subharmonic oscillations of microbubbles are initiated. Interestingly, earlier experimental studies on coated microbubbles demonstrated that the threshold for these bubbles is much lower than predicted by the traditional linear viscoelastic shell models. This paper presents an experimental study on the subharmonic behavior of differently sized individual phospholipid coated microbubbles. The radial subharmonic response of the microbubbles was recorded with the Brandaris ultra high-speed camera as a function of both the amplitude and the frequency of the driving pulse. Threshold pressures for subharmonic generation as low as 5 kPa were found near a driving frequency equal to twice the resonance frequency of the bubble. An explanation for this low threshold pressure is provided by the shell buckling model proposed by Marmottant et al. [J. Acoust. Soc. Am. 118, 3499–3505 (2005)]. It is shown that the change in the elasticity of the bubble shell as a function of bubble radius as proposed in this model, enhances the subharmonic behavior of the microbubbles

Ultrasound-responsive polymer-coated microbubbles that bind and protect DNA

Ultrasound in combination with microbubbles has recently been considered by gene delivery scientists to be an interesting approach to enhance gene transfer into cells. Its low toxicity and simplicity to apply in vivo without major complications make this technology (sonoporation) especially attractive. Sonoporation of DNA has been evaluated in vivo by the injection of free plasmid DNA (pDNA) together with microbubbles (as used in diagnostic imaging) in the bloodstream. However, the in vivo gene-transfer efficiency in these experiments remained rather low. Both the enzymatic degradation of the injected pDNA as well as the low pDNA concentration in the neighborhood of sonoporated cell membranes may explain this low efficiency. Therefore, we developed polymer-coated microbubbles that can bind and protect the pDNA. Coating albumin-shelled microbubbles with poly(allylamine hydrochloride) (PAH) makes the surface charge of the microbubbles positive without drastically affecting the size distribution of the microbubbles, thereby not affecting the ultrasound responsiveness and injectability. The cationic coating allowed both to bind up to 0.1 pg of DNA per microbubble as well as to protect the bound DNA against nucleases. Finally, the PAH coating significantly increased the lifetime of the microbubbles (half-life approximately 7 h), making them more convenient for in vivo applications because more microbubbles are expected to reach the target organ. Binding and nuclease protection of DNA by polymer-coated diagnostic microbubbles has, to our knowledge, never been demonstrated. We conclude that these LbL-coated microbubbles might be significant in the further development of ultrasound-mediated gene delivery

Acoustofluidic measurements on polymer-coated microbubbles: primary and secondary Bjerknes forces

The acoustically-driven dynamics of isolated particle-like objects in microfluidic environments is a well-characterised phenomenon, which has been the subject of many studies. Conversely, very few acoustofluidic researchers looked at coated microbubbles, despite their widespread use in diagnostic imaging and the need for a precise characterisation of their acoustically-driven behaviour, underpinning therapeutic applications. The main reason is that microbubbles behave differently, due to their larger compressibility, exhibiting much stronger interactions with the unperturbed acoustic field (primary Bjerknes forces) or with other bubbles (secondary Bjerknes forces). In this paper, we study the translational dynamics of commercially-available polymer-coated microbubbles in a standing-wave acoustofluidic device. At increasing acoustic driving pressures, we measure acoustic forces on isolated bubbles, quantify bubble-bubble interaction forces during doublet formation and study the occurrence of sub-wavelength structures during aggregation. We present a dynamic characterisation of microbubble compressibility with acoustic pressure, highlighting a threshold pressure below which bubbles can be treated as uncoated. Thanks to benchmarking measurements under a scanning electron microscope, we interpret this threshold as the onset of buckling, providing a quantitative measurement of this parameter at the single-bubble level. For acoustofluidic applications, our results highlight the limitations of treating microbubbles as a special case of solid particles. Our findings will impact applications where knowing the buckling pressure of coated microbubbles has a key role, like diagnostics and drug delivery

The use of microbubbles to target drug delivery

Ultrasound-mediated microbubbles destruction has been proposed as an innovative method for noninvasive delivering of drugs and genes to different tissues. Microbubbles are used to carry a drug or gene until a specific area of interest is reached, and then ultrasound is used to burst the microbubbles, causing site-specific delivery of the bioactive materials. Furthermore, the ability of albumin-coated microbubbles to adhere to vascular regions with glycocalix damage or endothelial dysfunction is another possible mechanism to deliver drugs even in the absence of ultrasound. This review focuses on the characteristics of microbubbles that give them therapeutic properties and some important aspects of ultrasound parameters that are known to influence microbubble-mediated drug delivery. In addition, current studies involving this novel therapeutical application of microbubbles will be discussed

Design and evaluation of drug loaded microbubbles for ultrasound guided cancer therapy

Cancer is a genetic disease, caused by mutations in the genome of normal cells. Chemical and physical damage to the cellular genome can induce these mutations resulting in the transformation of a healthy cell into a tumor cell. Over the past years, researchers have acquired a basic understanding of tumor onset. Several important proto-oncogenes and proteins involved in angiogenesis have been identified, leading to the development and clinical use of several new anticancer agents. We succeeded in preparing microbubbles which are able to carry genetic (pDNA, siRNA) and chemotherapeutic (doxorubicin) drugs. We proved that these microbubbles can selectively deliver their genetic or chemotherapeutic drugs to melanoma cells upon ultrasound exposure. We also showed that the activity of currently available drug carriers (pDNA or siRNA lipoplexes and doxorubicin-liposomes) was significantly improved by microbubble coupling and ultrasound exposure. This enables us to obtain a space and time controlled drug delivery guided by ultrasound. The fact that ultrasound and microbubbles have already been approved for ultrasound contrast imaging and are currently used in daily clinic, makes it very plausible that the microbubble concept reported in this thesis may find its way as an advanced drug delivery system

Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds

Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters—excitation intensity, frequency and duty cycle—to find 30 mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering