Acoustic Characterization of Echogenic Liposomes

In our labs we have demonstrated that ELIP can encapsulate gases such as air, nitric oxide, argon and xenon. The entrapment of gas microbubbles allows for ELIP to be used as ultrasound contrast agents (UCAs). The amount of contrast enhancement provided by UCAs is dependent on the UCA size and shell properties as well as the ultrasound insonation parameters. We have quantified the frequency dependent attenutation and integrated backscatter from air-filled, non-drug-loaded ELIP. The scattering-to-attenuation ratio (STAR) was found to range from 8% to 22% between 6 MHz and 25 MHz. ELIP sizes were also measured using a Beckman Coulter Multisizer 3 and a dynamic light scattering system (Zetasizer). ELIP were found to be polydisperse ranging from 40 nm to 6 µm in diameter. The size of the encapsulated microbubbles were found to be 18% of the volume of the liposome. Due to the polydisperse size distribution of ELIP, they can be imaged using transvascular (3-10 MHz) as well as intravascular (20-40 MHz) systems. Further studies are being carried out to acoustically characterize different types of gas-loaded and drug-loaded ELIP.

Related References:
Kopechek J.A., et al. 2011, Journal of Acoustical Society of America, 130:3472-3481
Kopechek J. A., PhD Dissertation, 2011

Frequency-dependent attenuation of ELIP.
Attenuation data was obtained using two single-element broadband transducers at a peak output pressure of 33 kPa. (N=5)

Frequency-dependent backscatter of ELIP.
Backscatter was measured using three single-element broadband transducers at a peak output pressure of 33 kPa. (N=5)

Stability of Echogenic Liposomes as a Blood Pool Ultrasound Contrast Agent

Stability of the echogenicity of ELIP in physiologic conditions is crucial to their successful translation to clinical use. We determined the effects of the surrounding media’s dissolved air concentration, temperature transition and hydrodynamic pressure on the echogenicity of ELIP. ELIP samples were diluted in porcine plasma or whole blood and pumped through a pulsatile flow phantom. B-mode images were acquired using a diagnostic transducer (L12-5) of a Philips HDI 5000 scanner. The dissolved gas levels were controlled to be degassed, arterial equivalent or venous equivalent levels. The hydrodynamic pressure in the system was controlled to be 1 mmHg (no overpressure), 60/20 mmHg, 120/80 mmHg (normotensive) or 145/90 mmHg (stage 1 hypertensive). The plasma and system temperatures were both maintained at room temperature or body temperature (no temperature transition). To study the effect of temperature transition, the plasma with ELIP was warmed from room temperature to body temperature or cooled from body temperature. ELIP were found to be stable in plasma and whole blood at physiologic dissolved gas levels, body temperature and at normotensive and hypertensive pressures. However, they were rendered non-echogenic when diluted in degassed plasma, or subjected to temperature transitions from body temperature to room temperature. Thus ELIP require proper handling procedures to avoid loss of echogenicity.

Related References:
Radhakrishnan et al. 2012, Ultrasound in Medicine and Biology

Effect of hydrodynamic pressure on ELIP echogenicity.
ELIP diluted in plasma were flowed through a system at body temperature and subjected to various hydrodynamic pressures: 1 mmHg (No overpressure), 120/80 mmHg (Normotensive) and 145/90 mmHg (Stage 1 hypertensive). ELIP were echogenic at all three pressures.

Effect of temperature transition on ELIP echogenicity.
ELIP diluted in plasma were subjected to temperature transitions indicated by (&DeltaT). Mean digital intensities (MDI) were measured on Bmode images. A significant loss of echogenicity was observed in ELIP undergoing a temperature transition from body temperature to room temperature