John R. Eisenbrey

Doxorubicin and paclitaxel loaded microbubbles for ultrasound triggered drug delivery

A polymer ultrasound contrast agent (UCA) developed in our lab has been shown to greatly reduce in size when exposed to ultrasound, resulting in nanoparticles less than 400 nm in diameter capable of escaping the leaky vasculature of a tumor to provide a sustained release of drug. Previous studies with the hydrophilic drug doxorubicin (DOX) demonstrated enhanced drug delivery to tumors when triggered with ultrasound. However the therapeutic potential has been limited due to the relatively low payload of DOX. This study compares the effects of loading the hydrophobic drug paclitaxel (PTX) on the agents acoustic properties, drug payload, tumoricidal activity, and the ability to deliver drugs through 400 nm pores. A maximum payload of 129.46 ± 1.80 μg PTX/mg UCA (encapsulation efficiency 71.92 ± 0.99%) was achieved, 20 times greater than the maximum payload of DOX (6.2 μg/mg), while maintaining the acoustic properties. In vitro, the tumoricidal activity of paclitaxel loaded UCA exposed to ultrasound was significantly greater than controls not exposed to ultrasound (p<0.0016). This study has shown that PTX loaded UCA triggered with focused ultrasound have the potential to provide a targeted and sustained delivery of drug to tumors.

Development and optimization of a doxorubicin loaded poly(lactic acid) contrast agent for ultrasound directed drug delivery

An echogenic, intravenous drug delivery platform is proposed in which an encapsulated chemotherapeutic can travel to a desired location and drug delivery can be triggered using external, focused ultrasound at the area of interest. Three methods of loading poly(lactic acid) (PLA) shelled ultrasound contrast agents (UCA) with doxorubicin are presented. Effects on encapsulation efficiency, in vitro enhancement, stability, particle size, morphology and release during UCA rupture are compared by loading method and drug concentration. An agent containing doxorubicin within the shell was selected as an ideal candidate for future hepatocellular carcinoma studies. The agent achieved a maximal drug load of 6.2 mg Dox/g PLA with an encapsulation efficiency of 20.5%, showed a smooth surface morphology and tight size distribution (poly dispersity index=0.309) with a peak size of 1865 nm. Acoustically, the agent provided 19 dB of enhancement in vitro at a dosage of 10 microg/ml, with a half life of over 15 min. In vivo, the agent provided ultrasound enhancement of 13.4+/-1.6 dB within the ascending aorta of New Zealand rabbits at a dose of 0.15 ml/kg. While the drug-incorporated agent is thought to be well suited for future drug delivery experiments, this study has shown that agent properties can be tailored for specific applications based on choice of drug loading method.

Delivery of Encapsulated Doxorubicin by Ultrasound-Mediated Size Reduction of Drug-Loaded Polymer Contrast Agents

Low delivery efficiency combined with systemic toxicity of traditional chemotherapy provides a need for improved chemotherapeutic delivery. Within our laboratory, we have developed polymer ultrasound contrast agents (1.2-1.8 ¿m in diameter) containing doxorubicin (Dox) within the shell (100-150 nm). In vivo this platform is expected to circulate through the vasculature until activated at the tumor site with external focused ultrasound (US). In vitro, the agent is responsive to US and when insonated at peak positive pressure amplitudes of 0.69 MPa and above, shows dramatic size reduction, eventually reaching a mean particle size of 350 nm, presumably due to fragmentation of, or gas release from the agent. The resulting Dox-polymer particles retain the drug and are small enough to pass through the leaky pores (350-400 nm) within the tumor vasculature, providing a sustained intratumoral release of chemotherapeutic as the polymer degrades. In vivo studies using a VX2 liver tumor model have shown that the combination of the agent and US results in nearly 50% less drug delivered to the nontargeted, healthy liver ( p = 0.009) and a 110% increase (p = 0.004) in Dox delivery to the viable peripheral tissue of the tumor, relative to the uninsonated controls. This study shows how US-mediated destruction of drug-loaded polymer contrast agent can be used to deliver encapsulated drug for potential sustained release. Penetration mechanisms of these resulting particles and their ability to provide a sustained release from the tumor interstia will be explored in the future.

Subharmonic Contrast Microbubble Signals for Noninvasive Pressure Estimation under Static and Dynamic Flow Conditions

Our group has proposed the concept of subharmonic aided pressure estimation (SHAPE) utilizing microbubble-based ultrasound contrast agent signals for the noninvasive estimation of hydrostatic blood pressures. An experimental system for in vitro SHAPE was constructed based on two single-element transducers assembled confocally at a 60° angle to each other. Changes in the first, second and subharmonic amplitudes of five different ultrasound contrast agents were measured in vitro at static hydrostatic pressures from 0–186 mmHg, acoustic pressures from 0.35–0.60 MPa peak-to-peak and frequencies of 2.5–6.6 MHz. The most sensitive agent and optimal parameters for SHAPE were determined using linear regression analysis and implemented on a Logiq 9 scanner (GE Healthcare, Milwaukee, WI). This implementation of SHAPE was then tested under dynamic-flow conditions and compared to pressure-catheter measurements. Over the pressure range studied, the first and second harmonic amplitudes reduced approximately 2 dB for all contrast agents. Over the same pressure range, the subharmonic amplitudes decreased by 9–14 dB and excellent linear regressions were achieved with the hydrostatic pressure variations (r2 = 0.98, p < 0.001). Optimal sensitivity was achieved at a transmit frequency of 2.5 MHz and acoustic pressure of 0.35 MPa using Sonazoid (GE Healthcare, Oslo, Norway). A Logiq 9 scanner was modified to implement SHAPE on a convex transducer with a frequency range from 1.5–4.5 MHz and acoustic pressures from 0–3.34 MPa. Results matched the pressure catheter (r2 = 0.87). In conclusion, subharmonic contrast signals are a good indicator of hydrostatic pressure. Out of the five ultrasound contrast agents tested, Sonazoid was the most sensitive for subharmonic pressure estimation. Real-time SHAPE has been implemented on a commercial scanner and offers the possibility of allowing pressures in the heart and elsewhere to be obtained noninvasively.

Simultaneous grayscale and subharmonic ultrasound imaging on a modified commercial scanner

OBJECTIVE To demonstrate the feasibility of simultaneous dual fundamental grayscale and subharmonic imaging on a modified commercial scanner. MOTIVATION The ability to generate signals at half the insonation frequency is exclusive to ultrasound contrast agents (UCA). Thus, subharmonic imaging (SHI; transmitting at f(0) and receiving at f(0)/2) provides improved visualization of UCA within the vasculature via suppression of the surrounding tissue echoes. While this capability has proven useful in a variety of clinical applications, the SHI suppression of surrounding tissue landmarks (which are needed for sonographic navigation) also limits it use as a primary imaging modality. In this paper we present results using a commercial ultrasound scanner modified to allow imaging in both grayscale (f(0)=4.0 MHz) and SHI (f(0)=2.5 MHz, f(0)/2=1.25 MHz) modes in real time. METHODS A Logiq 9 ultrasound scanner (GE Healthcare, Milwaukee, WI) with a 4C curvilinear probe was modified to provide this capability. Four commercially available UCA (Definity, Lantheus Medical Imaging, North Billerica, MA; Optison, GE Healthcare, Princeton, NJ; SonoVue, Bracco Imaging, Milan, Italy; and Sonazoid, GE Healthcare, Oslo, Norway) were all investigated in vitro over an acoustic output range of 3.34 MPa. In vivo the subharmonic response of Sonazoid was investigated in the portal veins of four canines (open abdominal cavity) and four patients with suspected portal hypertension. RESULTS In vitro, the four UCA showed an average maximum subharmonic amplitude of 44.1±5.4 dB above the noise floor with a maximum subharmonic amplitude of 48.6±1.6 dB provided by Sonazoid. The average in vivo maximum signal above the noise floor from Sonazoid was 20.8±2.3 dB in canines and 33.9±5.2 dB in humans. Subharmonic amplitude as a function of acoustic output in both groups matched the S-curve behavior of the agent observed in vitro. The dual grayscale imaging provided easier sonographic navigation, while the degree of tissue suppression in SHI mode varied greatly on a case by case basis. CONCLUSIONS These results demonstrate the feasibility of dual grayscale and SHI on a modified commercial scanner. The ability to simultaneously visualize both imaging modes in real time should improve the applicability of SHI as a future primary clinical imaging modality.

Parametric Imaging Using Subharmonic Signals From Ultrasound Contrast Agents in Patients With Breast Lesions

Parametric maps showing perfusion of contrast media can be useful tools for characterizing lesions in breast tissue. In this study we show the feasibility of parametric subharmonic imaging (SHI), which allows imaging of a vascular marker (the ultrasound contrast agent) while providing near complete tissue suppression. Digital SHI clips of 16 breast lesions from 14 women were acquired. Patients were scanned using a modified LOGIQ 9 scanner (GE Healthcare, Waukesha, WI) transmitting/receiving at 4.4/2.2 MHz. Using motion‐compensated cumulative maximum intensity (CMI) sequences, parametric maps were generated for each lesion showing the time to peak (TTP), estimated perfusion (EP), and area under the time‐intensity curve (AUC). Findings were grouped and compared according to biopsy results as benign lesions (n = 12, including 5 fibroadenomas and 3 cysts) and carcinomas (n = 4). For each lesion CMI, TTP, EP, and AUC parametric images were generated. No significant variations were detected with CMI (P = .80), TTP (P = .35), or AUC (P = .65). A statistically significant variation was detected for the average pixel EP (P = .002). Especially, differences were seen between carcinoma and benign lesions (mean ± SD, 0.10 ± 0.03 versus 0.05 ± 0.02 intensity units [IU]/s; P = .0014) and between carcinoma and fibroadenoma (0.10 ± 0.03 versus 0.04 ± 0.01 IU/s; P = .0044), whereas differences between carcinomas and cysts were found to be nonsignificant. In conclusion, a parametric imaging method for characterization of breast lesions using the high contrast to tissue signal provided by SHI has been developed. While the preliminary sample size was limited, results show potential for breast lesion characterization based on perfusion flow parameters.

Ultrasound triggered cell death in vitro with doxorubicin loaded poly lactic-acid contrast agents

Traditional chemotherapy generally results in systemic toxicity, which also limits drug levels at the area of need. Two ultrasound contrast agents (UCA), with diameters between 1-2 microm in diameter and shell thicknesses of 100-200 nm, composed of poly lactic-acid (PLA), one loaded by surface adsorption and the other loaded by drug incorporation in the shell, were compared in vitro for potential use in cancer therapy. These poly lactic-acid (PLA) UCA platforms contain a gas core that in an ultrasound (US) field can cause the UCA to oscillate or rupture. Following a systemic injection of drug loaded UCA with external application of US focused at the area of interest, this platform could potentially increase drug toxicity at the area of need, while protecting healthy tissue through microencapsulation of the drug. In vitro toxicity in MDA-MB-231 breast cancer cells of the surface-adsorbed and shell-incorporated doxorubicin (Dox) loaded UCA were examined at 5 MHz insonation using a pulse repetition frequency of 100 Hz at varying pressure amplitudes. Both platforms resulted in equivalent cell death compared to free Dox and US when insonated at peak positive pressure amplitudes of 1.26 MPa and above. While no significant changes in cell death were seen for surface adsorbed Dox-UCA with or without insonation, cell death using the platform with Dox incorporated within the shell increased from 16.12% to 25.78% (p=0.0272), approaching double the potency of the platform when insonated at peak positive pressure amplitudes of 1.26 MPa and above. This mechanism is believed to be the result of UCA rupture at higher insonation pressure amplitudes, resulting in more exposed drug and shell surface area as well as increased cellular uptake of Dox containing polymer shell fragments. This study has shown that a polymer UCA with drug housed within the shell may be used for US-triggered cell death. US activation can be used to make a carrier significantly more potent once in the area of need.

Three-Dimensional Subharmonic Ultrasound Imaging In Vitro and In Vivo

RATIONALE AND OBJECTIVES Although contrast-enhanced ultrasound imaging techniques such as harmonic imaging (HI) have evolved to reduce tissue signals using the nonlinear properties of the contrast agent, levels of background suppression have been mixed. Subharmonic imaging (SHI) offers near complete tissue suppression by centering the receive bandwidth at half the transmitting frequency. The aims of this study were to demonstrate the feasibility of three-dimensional (3D) SHI and to compare it to 3D HI. MATERIALS AND METHODS Three-dimensional HI and SHI were implemented on a Logiq 9 ultrasound scanner with a 4D10L probe. Four-cycle SHI was implemented to transmit at 5.8 MHz and receive at 2.9 MHz, while two-cycle HI was implemented to transmit at 5 MHz and receive at 10 MHz. The ultrasound contrast agent Definity was imaged within a flow phantom and the lower pole of two canine kidneys in both HI and SHI modes. Contrast-to-tissue ratios and rendered images were compared offline. RESULTS SHI resulted in significant improvement in contrast-to-tissue ratios relative to HI both in vitro (12.11 ± 0.52 vs 2.67 ± 0.77, P< .001) and in vivo (5.74 ± 1.92 vs 2.40 ± 0.48, P = .04). Rendered 3D subharmonic images provided better tissue suppression and a greater overall view of vessels in a flow phantom and canine renal vasculature. CONCLUSIONS The successful implementation of SHI in 3D allows imaging of vascular networks over a heterogeneous sample volume and should improve future diagnostic accuracy. Additionally, 3D SHI provides improved contrast-to-tissue ratios relative to 3D HI.

Noninvasive LV Pressure Estimation Using Subharmonic Emissions From Microbubbles

To develop a new noninvasive approach to quantify left ventricular (LV) pressures using subharmonic emissions from microbubbles, an ultrasound scanner was used in pulse inversion grayscale mode; unprocessed radiofrequency data were obtained with pulsed wave Doppler from the aorta and/or LV during Sonazoid infusion. Subharmonic data (in dB) were extracted and processed. Calibration factor (mm Hg/dB) from the aortic pressure was used to estimate LV pressures. Errors ranged from 0.19 to 2.50 mm Hg when estimating pressures using the aortic calibration factor, and were higher (0.64 to 8.98 mm Hg) using a mean aortic calibration factor. Subharmonic emissions from ultrasound contrast agents have the potential to noninvasively monitor LV pressures.

Chronic Liver Disease: Noninvasive Subharmonic Aided Pressure Estimation of Hepatic Venous Pressure Gradient

PURPOSE To compare subharmonic aided pressure estimation (SHAPE) with pressure catheter-based measurements in human patients with chronic liver disease undergoing transjugular liver biopsy. MATERIALS AND METHODS This HIPAA-compliant study had U.S. Food and Drug Administration and institutional review board approval, and written informed consent was obtained from all participants. Forty-five patients completed this study between December 2010 and December 2011. A clinical ultrasonography (US) scanner was modified to obtain SHAPE data. After transjugular liver biopsy with pressure measurements as part of the standard of care, 45 patients received an infusion of a microbubble US contrast agent and saline. During infusion, SHAPE data were collected from a portal and hepatic vein and were compared with invasive measurements. Correlations between data sets were determined by using the Pearson correlation coefficient, and statistical significance between groups was determined by using the Student t test. RESULTS The 45 study patients included 27 men and 18 women (age range, 19-71 years; average age, 55.8 years). The SHAPE gradient between the portal and hepatic veins was in good overall agreement with the hepatic venous pressure gradient (HVPG) (R = 0.82). Patients at increased risk for variceal hemorrhage (HVPG ≥ 12 mm Hg) had a significantly higher mean subharmonic gradient than patients with lower HVPGs (1.93 dB ± 0.61 [standard deviation] vs -1.47 dB ± 0.29, P < .001), with a sensitivity of 100% and a specificity of 81%, indicating that SHAPE may be a useful tool for the diagnosis of clinically important portal hypertension. CONCLUSION Preliminary results show SHAPE to be an accurate noninvasive technique for estimating portal hypertension.