Lipid-coated Microbubbles Research Articles

Lipid microbubbles as a vehicle for targeted drug delivery using focused ultrasound-induced blood–brain barrier opening

Focused ultrasound in conjunction with lipid microbubbles has fully demonstrated its ability to induce non-invasive, transient, and reversible blood–brain barrier opening. This study was aimed at testing the feasibility of our lipid-coated microbubbles as a vector for targeted drug delivery in the treatment of central nervous system diseases. These microbubbles were labeled with the fluorophore 5-dodecanoylaminfluorescein. Focused ultrasound targeted mouse brains in vivo in the presence of these microbubbles for trans-blood–brain barrier delivery of 5-dodecanoylaminfluorescein. This new approach, compared to previously studies of our group, where fluorescently labeled dextrans and microbubbles were co-administered, represents an appreciable improvement in safety outcome and targeted drug delivery. This novel technique allows the delivery of 5-dodecanoylaminfluorescein at the region of interest unlike the alternative of systemic exposure. 5-dodecanoylaminfluorescein delivery was assessed by ex vivo fluorescence imaging and by in vivo transcranial passive cavitation detection. Stable and inertial cavitation doses were quantified. The cavitation dose thresholds for estimating, a priori, successful targeted drug delivery were, for the first time, identified with inertial cavitation were concluded to be necessary for successful delivery. The findings presented herein indicate the feasibility and safety of the proposed microbubble-based targeted drug delivery and that, if successful, can be predicted by cavitation detection in vivo.

Shape mode stability of lipid-coated ultrasound contrast agent microbubbles

Ultrasound contrast agents (UCAs) are shell encapsulated microbubbles developed originally for ultrasound imaging enhancement. More recently, UCAs are being exploited for therapeutic applications such as drug and gene delivery. Ultrasound transducer pulses can induce spherical (radial) UCA oscillations, translation, and nonspherical shape oscillations, the latter of which can lead to breakup. Breakup can facilitate drug or gene delivery, but should be minimized for imaging purposes to increase residence time and maximize diagnostic effect. Therefore, an understanding of the interplay between the acoustic driving and shape mode stability of UCAs is important for both diagnostic and therapeutic applications. The present work couples a radial model of a lipid-coated microbubble with a model for bubble translation and nonspherical shape oscillation to predict shape mode stability for ultrasound driving frequencies and pressure amplitudes of clinical interest. In addition, calculations of the stability of individual shape modes, residence time, maximum radius, and translation are provided with respect to acoustic driving parameters and compared to an unshelled bubble. The effects of shell elasticity, shell viscosity, and initial radius on stability are investigated. The results show greater stability at higher values of shell elasticity and viscosity and at smaller radius, and provide guidance for optimizing shell design and ultrasound driving parameters with respect to shape stability.

Effects of ultrasound in presence of microbubbles on cartilage tissue regeneration in three-dimensional printed scaffolds

Gas-filled microbubbles encapsulated with lipids and other surfactants are highly responsive to ultrasound, which has led to their effective role as ultrasound contrast agents (UCA). In this study, for the first time, we used lipid-coated microbubbles (MB) prepared in-house in order to better harness the beneficial effects of ultrasound stimulation on proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs) within a novel 3D printed poly (ethylene glycol) diacrylate (PEG-DA) hydrogel scaffolds. A significant increase in cell number (p <0.001) was observed with low intensity pulsed ultrasound (LIPUS) treatment in the presence of 0.5 % (v/v) MB after 1, 3, and 5 days of culture. MSC proliferation enhanced up to 40% after 5 days of culture in the presence of MB and LIPUS while this value was only 18% when excited with LIPUS alone. We investigated the effects of acoustic parameters such as excitation intensity, frequency, and pulse repetition period on MSC proliferation rate. Our 3-week chondrogenic differentiation results demonstrated that combining LIPUS with MB significantly enhanced both Glycosaminoglycan (GAG) and type II collagen production. Therefore, integrating LIPUS and MB appears to be a promising strategy for enhanced MSC growth and chondrogenic differentiation.

Unilateral Opening of Rat Blood-Brain Barrier Assisted by Diagnostic Ultrasound Targeted Microbubbles Destruction.

Objective. Blood-brain barrier (BBB) is a key obstacle that prevents the medication from blood to the brain. Microbubble-enhanced cavitation by focused ultrasound can open the BBB and proves to be valuable in the brain drug delivery. The study aimed to explore the feasibility, efficacy, and safety of unilateral opening of BBB using diagnostic ultrasound targeted microbubbles destruction in rats. Methods. A transtemporal bone irradiation of diagnostic ultrasound and intravenous injection of lipid-coated microbubbles were performed at unilateral hemisphere. Pathological changes were monitored. Evans Blue extravasation grades, extraction from brain tissue, and fluorescence optical density were quantified. Lanthanum nitrate was traced by transmission electron microscopy. Results. After diagnostic ultrasound mediated microbubbles destruction, Evans Blue extravasation and fluorescence integrated optical density were significantly higher in the irradiated hemisphere than the contralateral side (all p < 0.01). Erythrocytes extravasations were demonstrated in the ultrasound-exposed hemisphere (4 ± 1, grade 2) while being invisible in the control side. Lanthanum nitrate tracers leaked through interendothelial cleft and spread to the nerve fiber existed in the irradiation side. Conclusions. Transtemporal bone irradiation under DUS mediated microbubble destruction provides us with a more accessible, safer, and higher selective BBB opening approach in rats, which is advantageous in brain targeted drugs delivery.

Dynamic acousto-elastic testing applied to a highly dispersive medium and evidence of shell buckling of lipid-coated gas microbubbles.

Dynamic acousto-elastic testing is applied to a mixture of lipid-coated microbubbles in water. A dynamic change of ambient pressure is produced by a 16 kHz pressure wave having a peak pressure amplitude of 28 kPa. The induced changes of phase velocity and attenuation are captured by a sequence of short ultrasound pulses with a center frequency of 4 MHz. As a consequence of the dispersion brought about by the resonance of microbubbles at a frequency close to 2 MHz, time-domain approaches like the cross-correlation method are shown to be unsuited to determine the variation in ultrasound wavespeed. A frequency-domain analysis shows that the acousto-elastic effect (first order pressure derivative of ultrasound phase velocity) depends on the ultrasound frequency. The acousto-elastic effect tends to that measured in water for an ultrasound frequency above the resonance frequency of microbubbles, while it is two orders of magnitude larger for an ultrasound frequency close to or below the resonance frequency of microbubbles. Besides the large magnitude of the acousto-elastic effect observed for an ultrasound frequency below the resonance frequency of microbubbles, the first order pressure derivative of ultrasound phase velocity is negative. This supports the occurrence of shell buckling of lipid-coated microbubbles induced by the 16 kHz pressure wave.

Radial excursions of bound and non-bound targeted lipid-coated single microbubbles

One of the main challenges for ultrasound molecular imaging is acoustically distinguishing non-bound microbubbles from those that have bound to their molecular target. We previously showed that biotinylated DPPC-based microbubbles (16 C-atoms) had a larger binding area and a more domed shape when bound to a streptavidin-coated surface than DSPC-based microbubbles (18 C-atoms) [1]. In the present in vitro study, we used the Brandaris 128 ultrahigh-speed camera (~15 Mfps) to compare the acoustical responses of biotinylated DPPC and DSPC-based microbubbles in a non-bound configuration and bound to a streptavidin-coated membrane, aiming to acoustically discriminate them from each other. The microbubbles were driven at a pressure of 50 kPa and at frequencies between 1 and 4 MHz. The main difference between bound and non-bound microbubbles was the lower radial excursion at the fundamental frequency for bound microbubbles. Resonance frequencies and subharmonic responses were the same for bound and non-bound micr...

An in vitro study of subharmonic emissions from monodisperse lipid-coated microbubbles

Subharmonic imaging is an ultrasound imaging method which utilizes the subharmonic response of a contrast agent to ultrasound excitation. It is possible to achieve high agent-to-tissue contrast because tissues do not emit subharmonic signals. In this project, we investigated the relationship between subharmonic emissions from monodisperse lipid-coated microbubbles and acoustic pressure. First, the resonance frequency for monodisperse microbubble suspension was determined from attenuation spectra measured using a through-transmission technique. Next, the microbubbles were excited at the resonance and at twice the resonance frequency. A transducer positioned orthogonal to the excitation transducer was used to detect acoustic emissions from the microbubbles at half the excitation frequency. It was found that the pressure required for subharmonic emissions was lower when monodisperse microbubbles were excited at twice the resonance frequency. For example, 6.5μm diameter microbubbles with a resonance frequency of 1.4 MHz had a pressure threshold for subharmonic emissions of approximately 30 kPa at 2.8 MHz excitation while those excited at 1.4 MHz could not be forced into subharmonic oscillations for the pressure range we used in this study (i.e., below 150 kPa). Implications of these results on the use of monodisperse lipid-coated microbubbles for subharmonic imaging will be discussed.

Non-linear Response and Viscoelastic Properties of Lipid-Coated Microbubbles: DSPC versus DPPC

For successful in vivo contrast-enhanced ultrasound imaging (CEUS) and ultrasound molecular imaging, detailed knowledge of stability and acoustical properties of the microbubbles is essential. Here, we compare these aspects of lipid-coated microbubbles that have either 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as their main lipid; the other components were identical. The microbubbles were investigated in vitro over the frequency range 1–4 MHz at pressures between 10 and 100 kPa, and their response to the applied ultrasound was recorded using ultrahigh-speed imaging (15 Mfps). Relative to DPPC-coated microbubbles, DSPC-coated microbubbles had (i) higher acoustical stability; (ii) higher shell elasticity as derived using the Marmottant model (DSPC: 0.26 ± 0.13 N/m, DPPC: 0.06 ± 0.06 N/m); (iii) pressure amplitudes twice as high at the second harmonic frequency; and (iv) a smaller amount of microbubbles that responded at the subharmonic frequency. Because of their higher acoustical stability and higher non-linear response, DSPC-coated microbubbles may be more suitable for contrast-enhanced ultrasound.

Internal polymer scaffolding in lipid-coated microbubbles for control of inertial cavitation in ultrasound theranostics.

A lipid-polymer composite structure was developed for tuning of inertial cavitation activity of microbubbles under ultrasound exposure. The incorporation of a thin layer of polymer networks inside the lipid monolayer resulted in marked reduction in the inertial cavitation dose. This strategy has the potential to increase the safety of ultrasound theranostic applications assisted by microbubble cavitation.

Acoustic characterization of monodisperse lipid-coated microbubbles: relationship between size and shell viscoelastic properties.

The acoustic attenuation spectrum of lipid-coated microbubble suspensions was measured in order to characterize the linear acoustic behavior of ultrasound contrast agents. For that purpose, microbubbles samples were generated with a very narrow size distribution by using microfluidics techniques. A performance as good as optical characterization techniques of single microbubbles was achieved using this method. Compared to polydispersions (i.e., contrast agents used clinically), monodisperse contrast agents have a narrower attenuation spectrum, which presents a maximum peak at a frequency value corresponding to the average single bubble resonance frequency. The low polydispersity index of the samples made the estimation of the lipid viscoelastic properties more accurate since, as previously reported, the shell linear parameters may change with the equilibrium bubble radius. The results showed the great advantage of dealing with monodisperse populations rather than polydisperse populations for the acoustic characterization of ultrasound contrast agents.

Condensation phase diagrams for lipid-coated perfluorobutane microbubbles.

The goal of this study was to explore the thermodynamic conditions necessary to condense aqueous suspensions of lipid-coated gas-filled microbubbles into metastable liquid-filled nanodrops as well as the physicochemical mechanisms involved with this process. Individual perfluorobutane microbubbles and their lipid shells were observed as they were pressurized at 34.5 kPa s(-1) in a microscopic viewing chamber maintained at temperatures ranging from 5 to 75 °C. The microbubbles contracted under pressure, ultimately leading to either full dissolution or microbubble-to-nanodrop condensation. Temperature-pressure phase diagrams conveying condensation and stability transitions were constructed for microbubbles coated with saturated diacylphosphatidylcholine lipids of varying acyl chain length (C16 to C24). The onset of full dissolution was shifted to higher temperatures with the use of longer acyl chain lipids or supersaturated media. Longer chain lipid shells resisted both dissolution of the gas core and mechanical compression through a pronounced wrinkle-to-fold collapse transition. Interestingly, the lipid shell also provided a mechanical resistance to condensation, shifting the vapor-to-liquid transition to higher pressures than for bulk perfluorobutane. This result indicated that the lipid shell can provide a negative apparent surface tension under compression. Overall, the results of this study will aid in the design and formulation of vaporizable fluorocarbon nanodrops for various applications, such as diagnostic ultrasound imaging, targeted drug delivery, and thermal ablation.

Manipulating nanoscale features on the surface of dye-loaded microbubbles to increase their ultrasound-modulated fluorescence output.

The nanoscale surface features of lipid-coated microbubbles can dramatically affect how the lipids interact with one another as the microbubble diameter expands and contracts under the influence of ultrasound. During microbubble manufacturing, the different lipid shell species naturally partition forming concentrated lipid islands. In this study the dynamics of how these nanoscale islands accommodate the expansion of the microbubbles are monitored by measuring the fluorescence intensity changes that occur as self-quenching lipophilic dye molecules embedded in the lipid layer change their distance from one another. It was found that when the dye molecules were concentrated in islands, less than 5% of the microbubbles displayed measurable fluorescence intensity modulation indicating the islands were not able to expand sufficiently for the dye molecules to separate from one another. When the microbubbles were heated and cooled rapidly through the lipid transition temperature the islands were melted creating an even distribution of dye about the surface. This resulted in over 50% of the microbubbles displaying the fluorescence-modulated signal indicating that the dye molecules could now separate sufficiently to change their self-quenching efficiency. The separation of the surface lipids in these different formations has significant implications for microbubble development as ultrasound and optical contrast agents.

Lipid Shedding from Single Oscillating Microbubbles

Lipid-coated microbubbles are used clinically as contrast agents for ultrasound imaging and are being developed for a variety of therapeutic applications. The lipid encapsulation and shedding of the lipids by acoustic driving of the microbubble has a crucial role in microbubble stability and in ultrasound-triggered drug delivery; however, little is known about the dynamics of lipid shedding under ultrasound excitation. Here we describe a study that optically characterized the lipid shedding behavior of individual microbubbles on a time scale of nanoseconds to microseconds. A single ultrasound burst of 20 to 1000 cycles, with a frequency of 1 MHz and an acoustic pressure varying from 50 to 425 kPa, was applied. In the first step, high-speed fluorescence imaging was performed at 150,000 frames per second to capture the instantaneous dynamics of lipid shedding. Lipid detachment was observed within the first few cycles of ultrasound. Subsequently, the detached lipids were transported by the surrounding flow field, either parallel to the focal plane (in-plane shedding) or in a trajectory perpendicular to the focal plane (out-of-plane shedding). In the second step, the onset of lipid shedding was studied as a function of the acoustic driving parameters, for example, pressure, number of cycles, bubble size and oscillation amplitude. The latter was recorded with an ultrafast framing camera running at 10 million frames per second. A threshold for lipid shedding under ultrasound excitation was found for a relative bubble oscillation amplitude >30%. Lipid shedding was found to be reproducible, indicating that the shedding event can be controlled.

Response to ultrasound of two types of lipid-coated microbubbles observed with a high-speed optical camera

Microbubbles (MBs) can be coated with different lipids, but exact influences on acoustical responses remain unclear. The distribution of lipids in the coating of homemade MBs is heterogeneous for DSPC and homogeneous for DPPC-based MBs, as observed with 4Pi confocal microscopy. In this study, we investigated whether DSPC and DPPC MBs show a different vibrational response to ultrasound. MBs composed of main lipid DSPC or DPPC (2 C-atoms less) with a C4F10 gas core, were made by sonication. Microbubble spectroscopy was performed by exciting single MBs with 10-cycle sine wave bursts having a frequency from 1 to 4 MHz and a peak negative pressure of 10, 20, and 50 kPa. The vibrational response to ultrasound was recorded with the Brandaris 128 high-speed camera at 15 Mfps. Larger acoustically induced deflation was observed for DPPC MBs. For a given resting diameter, the resonance frequency was higher for DSPC, resulting in higher shell elasticity of 0.26 N/m as compared to 0.06 N/m for DPPC MBs. Shell viscosity was similar (~10-8 kg/s) for both MB types. Non-linear behavior was characterized by the response at the subharmonic and second harmonic frequencies. More DPPC (71%) than DSPC MBs (27%) showed subharmonic response, while the behavior at the second harmonic frequency was comparable. The different acoustic responses of DSPC and DPPC MBs are likely due to the choice of the main lipid and the corresponding spatial distribution in the MB coating.

The stable nonlinear acoustic response of free-floating lipid-coated microbubbles

The stability of the microbubbles maintained by the lipid coating is crucial for diagnostic contrast-enhanced ultrasound imaging. We present a study of the stability of the dynamic response of single free-floating microbubbles (DSPC-based homemade microbubbles) with an acoustical camera. A 30 MHz probing wave (800 μs duration) measures the dynamic response of single microbubbles to 42 short sine bursts (1 MHz, 10 μs duration, 3 μs interval between each excitation) at three different peak pressures (25, 100, and 200 kPa). For each microbubble exposed to the 42 consecutive pulses, the following parameters were calculated: the radial strain at the driving frequency (ɛf), at the second/third harmonic frequencies (ɛ2f,ɛ3f), the ratio of radial excursion in expansion over that in compression (EoC) and the dc offset in the time-domain response. Nearly all 1500 individual microbubbles measured showed stable vibrational response. As expected ɛ2f and ɛ3f increase with ɛf, but they reach a plateau when ɛf exceeds about 30%. For ɛf smaller than 15%, we observed compression-dominant behaviors (dc offset &amp;lt; 0 and EoC &amp;lt; 1), while microbubbles show expansion-dominant responses (dc offset &amp;gt; 0 and EoC &amp;gt; 1) for ɛf larger than 15%.

Low-Amplitude Non-linear Volume Vibrations of Single Microbubbles Measured with an “Acoustical Camera”

Contrast-enhanced ultrasound imaging is based on the detection of non-linear vibrational responses of a contrast agent after its intravenous administration. Improving contrast-enhanced images requires an accurate understanding of the vibrational response to ultrasound of the lipid-coated gas microbubbles that constitute most ultrasound contrast agents. Variations in the volume of microbubbles provide the most efficient radiation of ultrasound and, therefore, are the most important bubble vibrations for medical diagnostic ultrasound imaging. We developed an “acoustical camera” that measures the dynamic volume change of individual microbubbles when excited by a pressure wave. In the work described here, the technique was applied to the characterization of low-amplitude non-linear behaviors of BR14 microbubbles (Bracco Research, Geneva, Switzerland). The amplitude dependence of the resonance frequency and the damping, the prevalence of efficient subharmonic and ultraharmonic vibrations and the amplitude dependence of the response at the fundamental frequency and at the second harmonic frequency were investigated. Because of the large number of measurements, we provide a statistical characterization of the low-amplitude non-linear properties of the contrast agent.

Intracerebral administration of ultrasound-induced dissolution of lipid-coated GDNF microbubbles provides neuroprotection in a rat model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic neurons in the substantia nigra. Neurotrophic factors, such as glial cell derived neurotrophic factor (GDNF), have been shown to provide a neuroprotective effect in PD rats. We have previously reported that ultrasound-induced lipid-coated GDNF microspheres, which release GDNF in a sustained manner after low frequency ultrasound stimulation, can reduce hypoxic-ischemic injury in neonatal rats. In the present study, we investigated whether lipid-coated GDNF microspheres can provide a neuroprotective effect in a rat model of PD. After a rat model of PD was produced by 6-hydroxydompamine (6-OHDA) injections, lipid-coated GDNF microspheres (1.5mg/kg) were injected into the striatum of PD rats. We found that GDNF levels were increased in the striatum of PD rats after lipid-coated GDNF microspheres administration following low frequency ultrasound stimulation (20kHz, 5min per day, daily for 4 weeks). Moreover, GDNF microspheres reduced apomorphine-induced rotations, and increased striatal dopamine and nigral tyrosine hydroxylase (TH) levels in PD rats. Additionally, GDNF microspheres reduced caspase-3, tumor necrosis factor-alpha, matrix metalloproteinase 9 (MMP-9) and OX-6 levels induced by 6-OHDA injections in PD rats. These data indicated that lipid-coated GDNF microspheres can provide a neuroprotective effect in PD rats.

Acoustic investigation of pressure-dependent resonance and shell elasticity of lipid-coated monodisperse microbubbles

In this study, frequency-dependent attenuation was measured acoustically for monodisperse lipid-coated microbubble suspensions as a function of excitation pressure and radius. The resonance frequency was identified from the attenuation spectra and had an inverse relationship with mean microbubble diameter and excitation pressure. A reduction in the estimated shell elasticity constant from 0.50 N/m to 0.29 N/m was observed as the excitation pressure was increased from 25 kPa to 100 kPa, respectively, which suggests a nonlinear relationship exists between lipid shell stiffness and applied strain. These findings support the viewpoint that lipid shells coating microbubbles exist as heterogeneous mixtures that undergo dynamic and rapid variations in mechanical properties under applied strains.

Dual-frequency intravascular ultrasound transducer design for contrast agent imaging

Intravascular ultrasound (IVUS) is a unique diagnostic tool for assessing the composition and degree of stenosis of atheromata in cardiovascular disease. As of yet, contrast enhanced IVUS has not been clinically implemented despite its potential to provide functional information, such as degree of plaque vascularization, in conjunction with standard anatomical mapping. In this study, we demonstrate that nonlinear microbubble signals can be detected with minimal signal processing using an ultra-broadband IVUS transducer composed of a low frequency transmit and a high frequency receive combined element. Raw signals were acquired from a single element transducer (1 × 3 mm, fc = 6.5/35 MHz) after aligning to an acoustically transparent 200 µm inner diameter cellulose tube. Poly-disperse, (1–10 µm diameter) lipid-coated microbubbles were injected at high concentration (4.8 × 108 MBs/mL) and peak negative pressures were varied from −0.3 to −1.8 MPa while transmission cycles were increased from 1to 5. In vitro results indicate signal to noise ratios of 11 dB are attainable at 2nd harmonics and similar SNRs (10–8 dB) at higher harmonics. The results of this study indicate that contrast enhanced IVUS imaging is possible using a single transmit pulsing scheme enabled by using a dual- frequency broadband transducer design.

Temperature-Dependent Biphasic Shrinkage of Lipid-Coated Bubbles in Ultrasound

Lipid-coated microbubbles and emulsions are of interest as possible ultrasound-mediated drug delivery vehicles and for their interesting behaviors and fundamental properties. We and others have noted that bubbles coated with the long chain saturated phospholipid distearoylphosphatidylcholine (DSPC) rapidly shrink to a quasistable size when repeatedly insonated with short ultrasound pulses; such stability may adversely affect the bubble's subsequent ability to deliver its pharmacological cargo. Bubbles coated with the unsaturated lipid dioleoylphosphatidylcholine (DOPC) did not show stability but did undergo an abrupt change from rapid initial shrinkage to a slow persistent shrinkage, leading ultimately to dissolution or dispersion. As DOPC and DSPC differ not only in chain saturation but also phase behavior, we performed additional studies using dimyristoyl PC (DMPC) as a coat lipid and controlled the solution temperature to study bubble behavior on exposure to repeated ultrasound pulses for the same coat, in both fluid and gel phases. We find, first, that essentially all bubbles show an initially rapid shrinkage, in which gas loss exceeds the limit imposed by gas diffusion into the surrounding medium; this rapid shrinkage may be evidence of nanoscopic bubble fragmentation. Second, upon reaching a fraction of their initial size, bubbles begin a slower shrinkage with a shrinkage rate that depends on the resting phase state of the coat lipid: fluid DMPC monolayers give a more rapid shrinkage than gel phase. DOPC-coated bubbles showed no temperature-dependent responses in the same temperature range. The results are especially interesting in that bubble compression during the pulse is likely to adiabatically heat the bubble and fluidize the coat, regardless of its initial phase state; thus, some structural feature of the resting coat, such as defect lines in the gel phase, may be important in the subsequent response to the ~3 μs ultrasound pulse.