Ultrasound-mediated liposome delivery in an Ex Vivo Murine Artery

Echogenic liposomes (ELIP) have been used to entrap micro- and nanobubbles, enabling enhanced echogenicity and cavitation nucleation. The use of ultrasound to fragment drug-loaded ELIP near the target tissue, rather than relying on more gradual passive release, has the potential to produce a large temporal peak in drug or therapeutic effect. This is particularly important at the endothelium where the constant flow of blood may carry away the released drug rapidly, making it unavailable for uptake across the endothelium. Fluorescently-labeled and anti-ICAM-1-targeted ELIP were flowed through ex-vivo atheromatous murine arteries and treated with 1-MHz ultrasound at pressures above the stable cavitation threshold. Delivery of ELIP into and beyond the endothelium was quantified by examining the fluorescence in the intravascular and extravascular fluids separately. This platform provides a model system for evaluation of delivery methodologies to the arterial wall.


Related References:
Hitchcock K. E. et al. 2010, Journal of Controlled Release, 144:288-295
Hitchcock K. E., PhD Dissertation, 2010



Ex vivo murine aortas treated with Rhodamine-labeled ELIP delivered via a proximal injection of Rh-ELIP into an intravascular flow of 0.5% BSA at 5.6 mL/min.
A -C show an artery treated with Rh-ELIP alone, while D - F show an artery treated with a combination of Rh-ELIP and CW ultrasound (0.49 MPa peak to peak amplitude). Panels A and D show arteries stained with factor VIII to highlight the endothelium. Panels B and E show the arterial walls as viewed with a blue filter, superimposed over the same segment viewed with a red filter. Panels C and F show the red-filtered image alone, where the increased fluorescence in Panel F is due to the presence of Rhodamine. The full scale bar is 100 Ám.




Ultrasound-triggered release of tissue plasminogen activator from echogenic liposomes

Recombinant tissue-type Plasminogen Activator (rt-PA), a thrombolytic, was loaded into ELIP. A linear array transducer of a clinical diagnostic ultrasound scanner was used to study ultrasound triggered rt-PA enzymatic activity from rt-PA-loaded ELIP (T-ELIP). T-ELIP samples were exposed to 6.9-MHz B-mode pulses at a low pressure amplitude (600 kPa) to track the echogenicity over time under four experimental conditions: 1) flow alone to monitor gas diffusion from the T-ELIP, 2) pulsed 6.0-MHz color Doppler exposure above the acoustically driven threshold (0.8 MPa) to force gas out of the liposome gently, 3) pulsed 6.0-MHz color Doppler above the rapid fragmentation threshold (2.6 MPa), or 4) Triton X-100 to rupture the T-ELIP chemically as a positive control. Release of rt-PA for each ultrasound exposure protocol was assayed spectrophotometrically. The thrombolytic drug remained associated with the liposome when exposed to low-amplitude B-mode pulses over 60 min and was released when exposed to color Doppler pulses or Triton X-100. A therapeutic concentration of rt-PA can be released by fragmenting the T-ELIP with pulsed 6.0-MHz color Doppler ultrasound above the rapid fragmentation threshold (1.59 MPa).


Related References:
Smith D.A.B., et al. 2010, Ultrasound in Medicine and Biology, 36:145-157
Smith D.A.B., PhD Dissertation, 2008



Loss of echogenicity over time for T-ELIP suspensions
Continuously scanned fundamental B-mode pulses at a center frequency of 6.9-MHz (MI = 0.04, Pr = 600 kPa) in four separate exposures conditions; 1) flow alone, 2) color 6.0-MHz Doppler pulses above acoustically driven threshold (MI = 0.22, Pr = 0.8 MPa), 3) color 6.0-MHz Doppler pulses above rapid fragmentation threshold (MI = 0.7, Pr = 2.6 MPa), and 4) Triton X-100 (0.07% v/v). The T-ELIP were reconstituted at time zero. Note that the data shown at time points (2, 37 and 57 min) correspond to the echogenicity of T-ELIP suspensions (N = 5) with flow alone before being exposed to the color Doppler pulses for the next three minutes.

Concentration of rt-PA over time for T-ELIP suspensions
Continuously scanned fundamental B-mode pulses at a center frequency of 6.9-MHz (MI = 0.04, Pr = 600 kPa) in four separate exposures conditions; 1) flow alone, 2) color 6.0-MHz Doppler pulses above acoustically driven threshold (MI = 0.22, Pr = 0.8 MPa), 3) color 6.0-MHz Doppler pulses above rapid fragmentation threshold (MI = 0.7, Pr = 2.6 MPa), and 4) Triton X-100 (0.07% v/v).