No abstract

Echogenic liposomes (ELIP) were developed as ultrasound-triggered targeted drug or gene delivery vehicles (Lanza et al., 1997;Huang et al., 2001). Recombinant tissue-type Plasminogen Activator (rt-PA), a thrombolytic, has been loaded into ELIP (Tiukinhoy-Laing et al., 2007). These vesicles have the potential to be used for ultrasound-enhanced thrombolysis in the treatment of acute ischemic stroke, myocardial infarction, deep vein thrombosis, or pulmonary embolus. A clinical diagnostic ultrasound scanner (Philips HDI 5000) equipped with a linear array transducer (L12-5) was employed for in vitro studies using rt-PA-loaded ELIP (T-ELIP). The goal of this study was to quantify ultrasound-triggered drug release from rt-PA-loaded echogenic liposomes. 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. T-ELIP were echogenic in the flow model (5 ml/min) for thirty minutes. The thrombolytic drug remained associated with the liposome when exposed to lowamplitude B-mode pulses over 60 min and was released when exposed to color Doppler pulses or Triton X-100. The rt-PA released from the liposomes had similar enzymatic activity as the free drug. These T-ELIP are robust and echogenic during continuous fundamental 6.9-MHz B-mode imaging at a low exposure output level (600 kPa). Furthermore, 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).

AbbreviationsC-ELIP, calcein-loaded echogenic liposome; ELIP, echogenic liposome; MDI, mean digital intensity; MI, mechanical index; P-ELIP, papaverine-loaded echogenic liposome; ROI, region of interest; rt-PA, recombinant tissue plasminogen activator he clinical need for organ-or tissue-specific drug delivery, also known as targeted drug delivery, arises when systemic delivery of a drug in sufficient doses to achieve a therapeutic effect at the target site results in deleterious systemic effects. Relevant clinical problems include delivery of chemotherapeutic drugs to tumors, delivery of thrombolytic drugs to the cerebral or coronary circulation during ischemic stroke T ArticleObjective. To achieve ultrasound-controlled drug delivery using echogenic liposomes (ELIPs), we assessed ultrasound-triggered release of hydrophilic and lipophilic agents in vitro using color Doppler ultrasound delivered with a clinical 6-MHz compact linear array transducer. Methods. Calcein, a hydrophilic agent, and papaverine, a lipophilic agent, were each separately loaded into ELIPs. Calceinloaded ELIP (C-ELIP) and papaverine-loaded ELIP (P-ELIP) solutions were circulated in a flow model and treated with 6-MHz color Doppler ultrasound or Triton X-100. Treatment with Triton X-100 was used to release the encapsulated calcein or papaverine content completely. The free calcein concentration in the solution was measured directly by spectrofluorimetry. The free papaverine in the solution was separated from liposome-bound papaverine by spin column filtration, and the resulting papaverine concentration was measured directly by absorbance spectrophotometry. Dynamic changes in echogenicity were assessed with low-output B-mode ultrasound (mechanical index, 0.04) as mean digital intensity. Results. Color Doppler ultrasound caused calcein release from C-ELIPs compared with flow alone (P < .05) but did not induce papaverine release from P-ELIPs compared with flow alone (P > .05). Triton X-100 completely released liposome-associated calcein and papaverine. Initial echogenicity was higher for C-ELIPs than P-ELIPs. Color Doppler ultrasound and Triton X-100 treatments reduced echogenicity for both CELIPs and P-ELIPs (P < .05). Conclusions. The differential efficiency of ultrasoundmediated pharmaceutical release from ELIPs for water-and lipid-soluble compounds suggests that water-soluble drugs are better candidates for the design and development of ELIP-based ultrasound-controlled drug delivery systems.

Objective-The purpose of this study was to identify the pressure threshold for the destruction of Optison (octafluoropropane contrast agent; Amersham Health, Princeton, NJ) using a laboratoryassembled 3.5-MHz pulsed ultrasound system and a clinical diagnostic ultrasound scanner.Methods-A 3.5-MHz focused transducer and a linear array with a center frequency of 6.9 MHz were positioned confocally and at 90° to each other in a tank of deionized water. Suspensions of Optison (5-8 × 10 4 microbubbles/mL) were insonated with 2-cycle pulses from the 3.5-MHz transducer (peak rarefactional pressure, or P r , from 0.0, or inactive, to 0.6 MPa) while being interrogated with fundamental B-mode imaging pulses (mechanical index, or MI, = 0.04). Scattering received by the 3.5-MHz transducer or the linear array was quantified as mean backscattered intensity or mean digital intensity, respectively, and fit with exponential decay functions (Ae −kt + N, where A + N was the amplitude at time 0; N, background echogenicity; and k, decay constant). By analyzing the decay constants statistically, a pressure threshold for Optison destruction due to acoustically driven diffusion was identified.Results-The decay constants determined from quantified 3.5-MHz radio frequency data and Bmode images were in good agreement. The peak rarefactional pressure threshold for Optison destruction due to acoustically driven diffusion at 3.5 MHz was 0.15 MPa (MI = 0.08). Furthermore, the rate of Optison destruction increased with increasing 3.5-MHz exposure pressure output.Conclusions-Optison destruction was quantified with a laboratory-assembled 3.5-MHz ultrasound system and a clinical diagnostic ultrasound scanner. The pressure threshold for acoustically driven diffusion was identified, and 3 distinct mechanisms of ultrasound contrast agent destruction were observed with acoustic techniques. Keywordsacoustically driven diffusion; Optison; rapid fragmentation; static diffusion; ultrasound contrast agent destruction Diagnostic ultrasound, combined with the injection of ultrasound contrast agents (UCAs) into the vasculature, provides the practitioner with an estimate of blood velocity and fractional blood volume in the myocardium, 1 the kidney, 2 and the brain. 3 Ultrasound imaging has shown promise for non-invasive quantification of tissue blood perfusion. The technique, known as perfusion imaging, takes advantage of the response of UCAs to low-and high-output acoustic pulses. The accuracy of this technique depends on complete and rapid loss of the contrast enhancement on high-power insonation and negligible microbubble destruction during the imaging phase. Rapid destruction of a UCA is possible through a process known as fragmentation, and pressure thresholds for fragmentation of several UCAs have been identified. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 4,5 A substantial loss of echogenicity is also possible at acoustic pressures lower than the fragmentation threshold. This type of destruction is caused by acoust...