Jonathan A. Kopechek

Ultrasound-Triggered Release of Recombinant Tissue-Type Plasminogen Activator from Echogenic Liposomes

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; Philips Medical Systems, Bothell, WA, USA) 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 30 min. 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. 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). (E-mail: denise.smith@uc.edu).

Passive imaging with pulsed ultrasound insonations

Previously, passive cavitation imaging has been described in the context of continuous-wave high-intensity focused ultrasound thermal ablation. However, the technique has potential use as a feedback mechanism for pulsed-wave therapies, such as ultrasound-mediated drug delivery. In this paper, results of experiments and simulations are reported to demonstrate the feasibility of passive cavitation imaging using pulsed ultrasound insonations and how the images depend on pulsed ultrasound parameters. The passive cavitation images were formed from channel data that was beamformed in the frequency domain. Experiments were performed in an invitro flow phantom with an experimental echo contrast agent, echogenic liposomes, as cavitation nuclei. It was found that the pulse duration and envelope have minimal impact on the image resolution achieved. The passive cavitation image amplitude scales linearly with the cavitation emission energy. Cavitation images for both stable and inertial cavitation can be obtained from the same received data set.

Ultrasound-Mediated Release of Hydrophilic and Lipophilic Agents From Echogenic Liposomes

Objective. 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. Calcein‐loaded 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 C‐ELIPs and P‐ELIPs (P < .05). Conclusions. The differential efficiency of ultrasound‐mediated 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.

Acoustic characterization of echogenic liposomes: Frequency-dependent attenuation and backscatter

Ultrasound contrast agents (UCAs) are used clinically to aid detection and diagnosis of abnormal blood flow or perfusion. Characterization of UCAs can aid in the optimization of ultrasound parameters for enhanced image contrast. In this study echogenic liposomes (ELIPs) were characterized acoustically by measuring the frequency-dependent attenuation and backscatter coefficients at frequencies between 3 and 30 MHz using a broadband pulse-echo technique. The experimental methods were initially validated by comparing the attenuation and backscatter coefficients measured from 50-μm and 100-μm polystyrene microspheres with theoretical values. The size distribution of the ELIPs was measured and found to be polydisperse, ranging in size from 40 nm to 6 μm in diameter, with the highest number observed at 65 nm. The ELIP attenuation coefficients ranged from 3.7  ±  1.0 to 8.0  ±  3.3 dB/cm between 3 and 25 MHz. The backscatter coefficients were 0.011  ±  0.006 (cm str)(-1) between 6 and 9 MHz and 0.023 ± 0.006 (cm str)(-1) between 13 and 30 MHz. The measured scattering-to-attenuation ratio ranged from 8% to 22% between 6 and 25 MHz. Thus ELIPs can provide enhanced contrast over a broad range of frequencies and the scattering properties are suitable for various ultrasound imaging applications including diagnostic and intravascular ultrasound.

Delivery of stem cells to porcine arterial wall with echogenic liposomes conjugated to antibodies against CD34 and intercellular adhesion molecule-1

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.

Triggered drug release from superhydrophobic meshes using high-intensity focused ultrasound.

Application of high-intensity focused ultrasound to drug-loaded superhydrophobic meshes affords triggered drug release by displacing an entrapped air layer. The air layer within the superhydrophobic meshes is characterized using direct visualization and B-mode imaging. Drug-loaded superhydrophobic meshes are cytotoxic in an in vitro assay after ultrasound treatment.

The Impact of Bubbles on Measurement of Drug Release from Echogenic Liposomes

Echogenic liposomes (ELIP) encapsulate gas bubbles and drugs within lipid vesicles, but the mechanisms of ultrasound-mediated drug release from ELIP are not well understood. The effect of cavitation activity on drug release from ELIP was investigated in flowing solutions using two fluorescent molecules: a lipophilic drug (rosiglitazone) and a hydrophilic drug substitute (calcein). ELIP samples were exposed to pulsed Doppler ultrasound from a clinical diagnostic ultrasound scanner at pressures above and below the inertial and stable cavitation thresholds. Control samples were exposed to a surfactant, Triton X-100 (positive control), or to flow alone (negative control). Fluorescence techniques were used to detect release. Encapsulated microbubbles reduced the measured fluorescence intensity and this effect should be considered when assessing drug release from ELIP. The origin of this effect is not specific to ELIP. Release of rosiglitazone or calcein compared to the negative control was only observed with detergent treatment, but not with ultrasound exposure, despite the presence of stable and inertial cavitation activity. Release of rosiglitazone or calcein from ELIP exposed to diagnostic ultrasound was not observed, even in the presence of cavitation activity. Ultrasound-mediated drug delivery strategies with ELIP will thus rely on passage of the drug-loaded liposomes to target tissues.

Accumulation of Phase-Shift Nanoemulsions to Enhance MR-Guided Ultrasound-Mediated Tumor Ablation In Vivo

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is being explored as a non-invasive technology to treat solid tumors. However, the clinical use of HIFU for tumor ablation applications is currently limited by the long treatment times required. Phase-shift nanoemulsions (PSNE), consisting of liquid perfluorocarbon droplets that can be vaporized into microbubbles, are being developed to accelerate HIFU-mediated heating. The purpose of this study was to examine accumulation of PSNE in intramuscular rabbit tumors in vivo. MR images were acquired before and after intravenous injection of gadolinium-containing PSNE. MR signal enhancement was observed in rabbit tumors up to six hours after injection, indicating that PSNE accumulated in the tumors. In addition, PSNE vaporization was detected in the tumor with B-mode ultrasound imaging, and MR thermometry measurements indicated that PSNE accelerated the rate of HIFU-mediated heating. These results suggest that PSNE could dramatically improve the efficiency and clinical feasibility of MRgHIFU.

Synthesis, Acoustic Stability, and Pharmacologic Activities of Papaverine-Loaded Echogenic Liposomes for Ultrasound Controlled Drug Delivery

Background: development of encapsulated therapeutics that could be released upon ultrasound exposure has strong implications for enhancing drug effects at the target site. We have developed echogenic liposomes (ELIP) suitable for ultrasound imaging of blood flow and ultrasound-mediated intravascular drug release. Papaverine was chosen as the test drug because its clinical application requires high concentration in the target vascular bed but low concentration in the systemic circulation. Methods: the procedure for preparation of standard ELIP was modified by including Papaverine hydrochloride in the lipid hydration solution, followed by three freeze-thaw cycles to increase encapsulation of the drug. Sizing and encapsulation pharmacokinetics were performed using a Coulter counter and a phosphodiesterase activity assay. Stability of Papaverine-loaded ELIP (PELIP) was monitored with a clinical diagnostic ultrasound scanner equipped with a linear array transducer at a center frequency of 4.5 MHz by assessing the mean digital intensity within a region of interest over time. The stability of PELIP was compared to those of standard ELIP and Optison™. Results: relative to standard ELIP, PELIP were larger (median diameter = 1.88 ± 0.10 μm for PELIP vs 1.08 ± 0.15 μm for ELIP) and had lower Mean Gray Scale Values (MGSV) (92 ± 24.8 for PELIP compared to 142.3 ± 10.7 for ELIP at lipid concentrations of 50 μg/ml). The maximum loading efficiency and mean encapsulated concentration were 24% ± 7% and 2.1 ± 0.7 mg/ml, respectively. Papaverine retained its phosphodiesterase inhibitory activity when associated with PELIP. Furthermore, a fraction of this activity remained latent until released by dissolution of liposomal membranes with detergent. The stability of both PELIP and standard ELIP were similar, but both are greater than that of Optison™. Conclusions: our results suggest that PELIP have desirable physical, biochemical, biological, and acoustic characteristics for potential in vivo administration and ultrasound-controlled drug delivery.

Ultrasound-mediated delivery of echogenic immunoliposomes to porcine vascular smooth muscle cells in vivo

Vascular smooth muscle cells (VSMCs) are important targets in the treatment of atherosclerosis. However, the arterial media, where the majority of VSMCs reside, have proven to be a difficult target for drug/gene delivery. We have demonstrated that ultrasound enhances drug/gene delivery to VSMCs in vitro by using echogenic immunoliposomes (ELIPs) as the vector. This study aimed to evaluate whether ultrasound can similarly enhance the delivery of an agent to VSMCs, particularly within the arterial media, in vivo, using ELIP. Anti–smooth-muscle cell actin-conjugated calcein-loaded ELIP were injected into the peripheral arteries of Yucatan miniswine (n = 8 arterial pairs). The right-sided porcine arteries were treated with 1-MHz continuous-wave ultrasound at a peak-to-peak pressure amplitude of 0.23 ± 0.05 MPa for 2 minutes. The contralateral arteries served as controls. Arteries were harvested after 30 minutes and imaged with fluorescence microscopy. Image data were converted to grayscale and analyzed by using computer-assisted videodensitometry. There was significant improvement in calcein uptake in all three arterial layers in the arteries exposed to ultrasound (> 300%). This enhanced uptake was site specific and appeared limited to the ultrasound-treated arterial segment. We have demonstrated enhanced delivery of a small molecule to VSMCs in all arterial wall layers, particularly the arterial media, using ultrasound and targeted ELIP. The combined effect of ultrasound exposure and ELIP as a contrast agent and a drug/gene-bearing vector has the potential for site-specific therapy directed at VSMC function.