Michał Mleczko

Bursting Bubbles and Bilayers

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol) (PEG) - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those - not necessarily involving microubbles - to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly “theranostic” vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery.

Determination of microbubble cavitation threshold pressure as function of shell chemistry

AbstractThe sensitivity of inertial cavitation threshold to changes in shell viscosity and elasticity makes shell chemistry (here, polyethylene glycol (PEG) molecular weight and composition) a potential tuning parameter for microbubble based ultrasound contrast and drug delivery applications. It is anticipated that microbubble shell chemistry can be used to adjust the inertial cavitation threshold so as to either avoid or achieve cavitation at a given operating pressure. Here such ideas are tested by measuring the inertial cavitation threshold for populations of phospholipid shelled microbubbles suspended in aqueous media, and this method is used to quantify the influence of shell chemistry on the inertial cavitation threshold. The experimental cavitation data are fitted with a modification of the Herring equation, using shell viscosity and elasticity as the tuning parameters. It is concluded that the design and synthesis of microbubbles with a prescribed inertial cavitation threshold is feasible using PE...

Influence of shell composition on the resonance frequency of microbubble contrast agents.

The effect of variations in microbubble shell composition on microbubble resonance frequency is revealed through experiment. These variations are achieved by altering the mole fraction and molecular weight of functionalized polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell and measuring the microbubble resonance frequency. The resonance frequency is measured via a chirp pulse and identified as the frequency at which the pressure amplitude loss of the ultrasound wave is the greatest as a result of passing through a population of microbubbles. For the shell compositions used herein, we find that PEG molecular weight has little to no influence on resonance frequency at an overall PEG mole fraction (0.01) corresponding to a mushroom regime and influences the resonance frequency markedly at overall PEG mole fractions (0.050-0.100) corresponding to a brush regime. Specifically, the measured resonance frequency was found to be 8.4, 4.9, 3.3 and 1.4 MHz at PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.075. At an overall PEG mole fraction of just 0.01, on the other hand, resonance frequency exhibited no systematic variation, with values ranging from 5.7 to 4.9 MHz. Experimental results were analyzed using the Sarkar bubble dynamics model. With the dilatational viscosity held constant (10(-8) N·s/m) and the elastic modulus used as a fitting parameter, model fits to the pressure amplitude loss data resulted in elastic modulus values of 2.2, 2.4, 1.6 and 1.8 N/m for PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.010 and 4.2, 1.4, 0.5 and 0.0 N/m, respectively, at an overall PEG mole fraction of 0.075. These results are consistent with theory, which predicts that the elastic modulus is constant in the mushroom regime and decreases with PEG molecular weight to the inverse 3/5 power in the brush regime. Additionally, these results are consistent with inertial cavitation studies, which revealed that increasing PEG molecular weight has little to no effect on inethe rtial cavitation threshold in the mushroom regime, but that increasing PEG molecular weight decreases inertial cavitation markedly in the brush regime. We conclude that the design and synthesis of microbubbles with a prescribed resonance frequency is attainable by tuning PEG composition and molecular weight.

Coencapsulation of lipid microbubbles within polymer microcapsules for contrast applications

In this work, the authors examine the acoustic response generated by the coencapsulation of phospholipid shelled microbubbles within the aqueous core of polymer microcapsules and its feasibility as an ultrasound contrast agent. The addition of the polymer shell provides the added benefit of approximately doubling the inertial cavitation threshold of the microbubbles contained within. The feasibility of the utilisation of the coencapsulated contrast agent as a drug delivery vehicle is also discussed. It is concluded that the coencapsulated contrast agent provides contrast similar to that of unencapsulated microbubbles, both in acoustic response and image intensity of contrast to tissue.

Controlling cavitation for controlled release

We present a novel, long-lived ultrasound contrast vehicle with triggered drug release capability. The vehicle comprises shell-stabilized microbubbles encapsulated within the aqueous core of a vesicle-like microcapsule. Encapsulating microbubbles enhances their longevity as ultrasound contrast agents by shielding the microbubbles from dissolution in a bulk aqueous phase. Moreover, co-encapsulation of microbubbles with a drug offers a simple mechanism for controlled drug release; ultrasound-induced cavitation of the microbubbles within microcapsules causes leakage of the inner contents from the microcapsules into the surrounding medium. By controlling the extent of microbubble cavitation one can control when - and at what rate - the microcapsule releases drug. Here we describe construction of the vehicle, how one can tune the microbubble cavitation threshold via changes in microbubble shell elasticity, and how ultrasound-induced leakage varies with microcapsule shell type and composition. We show results for different microcapsule shell materials, including self-assembled bilayers of phospholipids (liposomes) and di-block copolymers (polymersomes) and a non-self-assembled, macromolecular polymer, using the fluorescent dye calcein as a drug mimic.

A method for the determination of the inertial cavitation threshold of ultrasound contrast agents

Ultrasound contrast agents consist of microbubbles with diameters in the micron range. In recent developments, targeted drug delivery is effectuated using microbubbles. Microbubbles may be destroyed by inertial cavitation if the insonification amplitude is chosen high enough. The minimum threshold for inertial cavitation of a microbubble population is thus an important characteristic of an ultrasound contrast agent. This article proposes a method for the determination of ultrasound contrast agent destruction by inertial cavitation. Detection is implemented by transmission of a destruction and a probing pulse with a fixed time delay. Using an expectation maximization algorithm, modulated Gaussian pulses are fitted to the received waveform. By consideration of the time offset between each pulse, the responses to the individual pulses can be seperated and destroyed bubbles can be counted. A destruction curve for the contrast agent Definity was recorded at an insonification frequency of 2.25 MHz. At a peak negative pressure of 1.11 MPa, 18.81% of the recorded population was destroyed.

Microcapsules: Reverse Sonoporation and Long-lasting, Safe Contrast

We present a novel vehicle designed to serve the dual roles of enhanced ultrasound contrast and ultrasound-triggered drug delivery. The vehicle is comprised of a microcapsule that is filled with water in whose aqueous core a population of freely floating, phospholipid-coated microbubbles is suspended. At ultrasound intensities below the inertial cavitation threshold of the microbubbles, the microbubbles provide enhanced ultrasound contrast. The measured contrast is comparable in strength with SonoVue®. Encapsulation of microbubbles within microcapsules putatively eliminates – or at least significantly slows – dissolution of gas in the bulk aqueous medium, thereby avoiding disappearance of microbubbles that would otherwise occur due to pressure-induced gas diffusion across the surfactant monolayer coating the microbubble-water interface. Results suggest that our vehicle might provide longer lasting contrast in a clinical setting. We demonstrate that encapsulation of the microbubbles within microcapsules causes at least a doubling of the ultrasound intensity necessary to induce inertial cavitation. Moreover, no cell death was observed when cells were insonified in the presence of microbubble-containing microcapsules, whereas appreciable cell death occurs with unencapsulated microbubbles. These results point toward a potential safety benefit during ultrasound contrast imaging by using encapsulated microbubbles. Studies are underway to investigate the feasibility of ultrasound-triggered release of drug from the microcapsules, owing to inertial- or stable-cavitation, or both. Whereas leakage from polymeric microcapsule shells, such as poly(lactic acid), seemingly requires shell rupture and is exceedingly difficult to achieve, leakage across a lipid bilayer microcapsule shells appears feasible. Leakage across a bilayer shell has the additional benefit that the leakage mechanism can be tuned via phase behavior (liquid-ordered versus liquid-disordered) and cavitation mechanism (stable versus inertial).

Evaluation of an analytical solution to the Burgers equation based on Volterra series

A simple, well-interpretable, and explicit analytical solution to the Burgers equation based on Volterra series is derived. Its region of convergence is investigated and a method for the computationally efficient numerical evaluation of the associated Volterra polynomials is presented. For a given boundary condition, numerical results are compared to a widely-used numerical standard solution. After a propagation distance of 10 cm in steps of 5 mm the Volterra polynomials of degree 2 and 3 achieve relative errors in terms of the L2-norm of 4.22% and 1.35%, respectively.

Mutual Attraction of Oscillating Microbubbles

The driving of contrast microbubbles towards a boundary by means of primary radiation forces has been of interest for ultrasound-assisted drug delivery. Secondary radiation forces, resulting from oscillating microbubbles under ultrasound insonification, may cause the mutual attraction and subsequent coalescence of contrast microbubbles. This phenomenon has been less studied. Microbubbles with a negligible shell can be forced to translate towards each other at relatively low mechanical indices (MI). Thick-shelled microbubbles would require a higher MI to be moved. However, at high MI, microbubble disruption is expected. We investigated if thick-shelled contrast agent microbubbles can be forced to cluster at high-MI. The thick-shelled contrast agent M1639, inserted through a cellulose capillary, was subjected to 3 MHz, high-MI pulsed ultrasound from a commercial ultrasound machine, and synchronously captured through a high numerical aperture microscope. The agent showed the ultrasound-induced formation of bubble clusters, and the translation thereof towards the capillary boundary. Hence, forced translation and clustering of thick-shelled contrast microbubbles is feasible. The phase difference between the excursion of the oscillating bubble and the incident sound field was computed for free and encapsulated bubbles. There is a transition in phase difference for encapsulated bubbles, owing to the friction of the shell. Therefore, approach velocities of encapsulated bubbles may not be comparable to those of free gas bubbles.

Size distribution of microbubbles as a function of shell composition.

The effect of modifying the shell composition of a population of microbubbles on their size demonstrated through experiment. Specifically, these variations include altering both the mole fraction and molecular weight of functionalized polymer, polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell (1-15 mol% PEG, and 1000-5000 g/mole, respectively). The size distribution is measured with an unbiased image segmentation program written in MATLAB which identifies and sizes bubbles from micrographs. For a population of microbubbles with a shell composition of 5 mol% PEG2000, the mean diameter is 1.42 μm with a variance of 0.244 μm. For the remainder of the shell compositions studied herein, we find that the size distributions do not show a statistically significant correlation to either PEG molecular weight or mole fraction. All the measured distributions are nearly Gaussian in shape and have a monomodal peak.