Shengping Qin

Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering

Microbubble contrast agents and the associated imaging systems have developed over the past 25 years, originating with manually-agitated fluids introduced for intra-coronary injection. Over this period, stabilizing shells and low diffusivity gas materials have been incorporated in microbubbles, extending stability in vitro and in vivo. Simultaneously, the interaction of these small gas bubbles with ultrasonic waves has been extensively studied, resulting in models for oscillation and increasingly sophisticated imaging strategies. Early studies recognized that echoes from microbubbles contained frequencies that are multiples of the microbubble resonance frequency. Although individual microbubble contrast agents cannot be resolved-given that their diameter is on the order of microns-nonlinear echoes from these agents are used to map regions of perfused tissue and to estimate the local microvascular flow rate. Such strategies overcome a fundamental limitation of previous ultrasound blood flow strategies; the previous Doppler-based strategies are insensitive to capillary flow. Further, the insonation of resonant bubbles results in interesting physical phenomena that have been widely studied for use in drug and gene delivery. Ultrasound pressure can enhance gas diffusion, rapidly fragment the agent into a set of smaller bubbles or displace the microbubble to a blood vessel wall. Insonation of a microbubble can also produce liquid jets and local shear stress that alter biological membranes and facilitate transport. In this review, we focus on the physical aspects of these agents, exploring microbubble imaging modes, models for microbubble oscillation and the interaction of the microbubble with the endothelium.

Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall

Many thousands of contrast ultrasound studies have been conducted in clinics around the world. In addition, the microbubbles employed in these examinations are being widely investigated to deliver drugs and genes. Here, for the first time, the oscillation of these microbubbles in small vessels is directly observed and shown to be substantially different than that predicted by previous models and imaged within large fluid volumes. Using pulsed ultrasound with a center frequency of 1 MHz and peak rarefactional pressure of 0.8 or 2.0 MPa, microbubble expansion was significantly reduced when microbubbles were constrained within small vessels in the rat cecum (p<0.05). A model for microbubble oscillation within compliant vessels is presented that accurately predicts oscillation and vessel displacement within small vessels. As a result of the decreased oscillation in small vessels, a large resting microbubble diameter resulting from agent fusion or a high mechanical index was required to bring the agent shell into contact with the endothelium. Also, contact with the endothelium was observed during asymmetrical collapse, not during expansion. These results will be used to improve the design of drug delivery techniques using microbubbles.

Acoustic response of compliable microvessels containing ultrasound contrast agents

The existing models of the dynamics of ultrasound contrast agents (UCAs) have largely been focused on an UCA surrounded by an infinite liquid. Preliminary investigations of a microbubbles oscillation in a rigid tube have been performed using linear perturbation, under the assumption that the tube diameter is significantly larger than the UCA diameter. In the potential application of drug and gene delivery, it may be desirable to fragment the agent shell within small blood vessels and in some cases to rupture the vessel wall, releasing drugs and genes at the site. The effect of a compliant small blood vessel on the UCAs oscillation and the microvessels acoustic response are unknown. The aim of this work is to propose a lumped-parameter model to study the interaction of a microbubble oscillation and compliable microvessels. Numerical results demonstrate that in the presence of UCAs, the transmural pressure through the blood vessel substantially increases and thus the vascular permeability is predicted to be enhanced. For a microbubble within an 8 to 40 microm vessel with a peak negative pressure of 0.1 MPa and a centre frequency of 1 MHz, small changes in the microbubble oscillation frequency and maximum diameter are observed. When the ultrasound pressure increases, strong nonlinear oscillation occurs, with an increased circumferential stress on the vessel. For a compliable vessel with a diameter equal to or greater than 8 microm, 0.2 MPa PNP at 1 MHz is predicted to be sufficient for microbubble fragmentation regardless of the vessel diameter; however, for a rigid vessel 0.5 MPa PNP at 1 MHz may not be sufficient to fragment the bubbles. For a centre frequency of 1 MHz, a peak negative pressure of 0.5 MPa is predicted to be sufficient to exceed the stress threshold for vascular rupture in a small (diameter less than 15 microm) compliant vessel. As the vessel or surrounding tissue becomes more rigid, the UCA oscillation and vessel dilation decrease; however the circumferential stress is predicted to increase. Decreasing the vessel size or the centre frequency increases the circumferential stress. For the two frequencies considered in this work, the circumferential stress does not scale as the inverse of the square root of the acoustic frequency va as in the mechanical index, but rather has a stronger frequency dependence, 1/va.

Ultrasound increases nanoparticle delivery by reducing intratumoral pressure and increasing transport in epithelial and epithelial-mesenchymal transition tumors.

Acquisition of the epithelial-mesenchymal transition (EMT) tumor phenotype is associated with impaired chemotherapeutic delivery and a poor prognosis. In this study, we investigated the application of therapeutic ultrasound methods available in the clinic to increase nanotherapeutic particle accumulation in epithelial and EMT tumors by labeling particles with a positron emission tomography tracer. Epithelial tumors were highly vascularized with tight cell-cell junctions, compared with EMT tumors where cells displayed an irregular, elongated shape with loosened cell-cell adhesions and a reduction in E-cadherin and cytokeratins 8/18 and 19. Without ultrasound, the accumulation of liposomal nanoparticles administered to tumors in vivo was approximately 1.5 times greater in epithelial tumors than EMT tumors. When ultrasound was applied, both nanoaccumulation and apparent tumor permeability were increased in both settings. Notably, ultrasound effects differed with thermal and mechanical indices, such that increasing the thermal ultrasound dose increased nanoaccumulation in EMT tumors. Taken together, our results illustrate how ultrasound can be used to enhance nanoparticle accumulation in tumors by reducing their intratumoral pressure and increasing their vascular permeability.

Enhanced in vivo bioluminescence imaging using liposomal luciferin delivery system

To provide a continuous and prolonged delivery of the substrate D-luciferin for bioluminescence imaging in vivo, luciferin was encapsulated into liposomes using either the pH gradient or acetate gradient method. Under optimum loading conditions, 0.17 mg luciferin was loaded per mg of lipid with 90-95% encapsulation efficiency, where active loading was 6 to 18-fold higher than that obtained with passive loading. Liposomal luciferin in a long-circulating formulation had good shelf stability, with 10% release over 3-month storage at 4 degrees C. Pharmacokinetic profiles of free and liposomal luciferin were then evaluated in transgenic mice expressing luciferase. In contrast to rapid in vivo clearance of free luciferin (t(1/2)=3.54 min), luciferin encapsulated into long-circulating liposomes showed a prolonged release over 24h. The first-order release rate constant of luciferin from long-circulating liposomes, as estimated from the best fit of the analytical model to the experimental data, was 0.01 h(-1). Insonation of luciferin-loaded temperature-sensitive liposomes directly injected into one tumor of Met1-luc tumor-bearing mice resulted in immediate emission of light. Systemic injection of luciferin-loaded long-circulating liposomes into Met1-luc tumor-bearing mice, followed by unilateral ultrasound-induced hyperthermia, produced a gradual increase in radiance over time, reaching a peak at 4-7 h post-ultrasound.

Microbubble tunneling in gel phantoms

Insonified microbubbles were observed in vessels within a gel with a Youngs modulus similar to that of tissue, demonstrating shape instabilities, liquid jets, and the formation of small tunnels. In this study, tunnel formulation occurred in the direction of the propagating ultrasound wave, where radiation pressure directed the contact of the bubble and gel, facilitating the activity of the liquid jets. Combinations of ultrasonic parameters and microbubble concentrations that are relevant for diagnostic imaging and drug delivery and that lead to tunnel formation were applied and the resulting tunnel formation was quantified.

An imaging-driven model for liposomal stability and circulation.

Simultaneous labeling of the drug compartment and shell of delivery vehicles with optical and positron emission tomography (PET) probes is developed and employed to inform a hybrid physiologically based pharmacokinetic model. Based on time-dependent estimates of the concentration of these tracers within the blood pool, reticuloendothelial system (RES) and tumor interstitium, we compare the stability and circulation of long-circulating and temperature-sensitive liposomes. We find that rates of transport to the RES for long-circulating and temperature-sensitive particles are 0.046 and 0.19 h(-1), respectively. Without the application of exogenous heat, the rates of release from the long-circulating and temperature-sensitive particles circulating within the blood pool are 0.003 and 0.2 h(-1), respectively. Prolonged lifetime in circulation and slow drug release from liposomes result in a significantly greater drug area under the curve for the long-circulating particles. Future studies will couple these intrinsic parameters with exogenous heat-based release. Finally, we develop a transport constant for the transport of liposomes from the blood pool to the tumor interstitium, which is on the order of 0.01 h(-1) for the Met-1 tumor system.

Multimodal imaging enables early detection and characterization of changes in tumor permeability of brain metastases

Our goal was to develop strategies to quantify the accumulation of model therapeutics in small brain metastases using multimodal imaging, in order to enhance the potential for successful treatment. Human melanoma cells were injected into the left cardiac ventricle of immunodeficient mice. Bioluminescent, MR and PET imaging were applied to evaluate the limits of detection and potential for contrast agent extravasation in small brain metastases. A pharmacokinetic model was applied to estimate vascular permeability. Bioluminescent imaging after injecting d-luciferin (molecular weight (MW) 320 D) suggested that tumor cell extravasation had already occurred at week 1, which was confirmed by histology. 7T T1w MRI at week 4 was able to detect non-leaky 100 μm sized lesions and leaky tumors with diameters down to 200 μm after contrast injection at week 5. PET imaging showed that (18)F-FLT (MW 244 Da) accumulated in the brain at week 4. Gadolinium-based MRI tracers (MW 559 Da and 2.066 kDa) extravasated after 5 weeks (tumor diameter 600 μm), and the lower MW agent cleared more rapidly from the tumor (mean apparent permeabilities 2.27 × 10(-5)cm/s versus 1.12 × 10(-5)cm/s). PET imaging further demonstrated tumor permeability to (64)Cu-BSA (MW 65.55 kDa) at week 6 (tumor diameter 700 μm). In conclusion, high field T1w MRI without contrast may improve the detection limit of small brain metastases, allowing for earlier diagnosis of patients, although the smallest lesions detected with T1w MRI were permeable only to d-luciferin and the amphipathic small molecule (18)F-FLT. Different-sized MR and PET contrast agents demonstrated the gradual increase in leakiness of the blood tumor barrier during metastatic progression, which could guide clinicians in choosing tailored treatment strategies.

Positron emission tomography imaging of the stability of Cu-64 labeled dipalmitoyl and distearoyl lipids in liposomes

Changes in lipid acyl chain length can result in desorption of lipid from the liposomal anchorage and interaction with blood components. PET studies of the stability of such lipids have not been performed previously although such studies can map the pharmacokinetics of unstable lipids non-invasively in vivo. The purpose of this study was to characterize the in vivo clearance of (64)Cu-labeled distearoyl- and dipalmitoyl lipid included within long circulating liposomes. Distearoyl and dipalmitoyl maleimide lipids (1mol%) in liposomes were labeled with a (64)Cu-incorporated bifunctional chelator (TETA-PDP) after the activation of pyridine disulfide to thiol by TCEP. Long circulating liposomes containing HSPC:DSPE-PEG2k-OMe:cholesterol: x (55:5:39:1), where x was (64)Cu-DSPE or (64)Cu-DPPE, or HSPC:DSPE-PEG2k-OMe:cholesterol:(64)Cu-DSPE:DPPC (54:5:39:1:1) were evaluated in serum (in vitro) and via intravenous injection to FVB mice. The time-activity curves for the blood, liver, and kidney were measured from PET images and the biodistribution was performed at 48h. In vitro assays showed that (64)Cu-DPPE transferred from liposomes to serum with a 7.9h half-life but (64)Cu-DSPE remained associated with the liposomes. The half clearance of radioactivity from the blood pool was 18 and 5h for (64)Cu-DSPE- and (64)Cu-DPPE liposome-injected mice, respectively. The clearance of radioactivity from the liver and kidney was significantly greater following the injection of (64)Cu-DPPE-labeled liposomes than (64)Cu-DSPE-labeled liposomes at 6, 18 and 28h. Forty eight hours after injection, the whole body radioactivity was 57 and 17% ID/cc for (64)Cu-DSPE and (64)Cu-DPPE, respectively. These findings suggest that the acyl chain length of the radiolabel should be considered for liposomal PET studies and that PET is an effective tool for evaluating the stability of nanoformulations in vivo.

Longitudinal Investigation of Permeability and Distribution of Macromolecules in Mouse Malignant Transformation using PET

Purpose: We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease. Experimental Design: Albumin and liposomes were labeled with 64Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability. Results: From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased ∼2.5-fold to 6.6 × 10−8 and 1.3 × 10−8 cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of ∼6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 ± 8.1 and 7.1 ± 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin. Conclusions: PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability. Clin Cancer Res; 17(3); 550–9. ©2010 AACR.