Synthesis and Characterization of Transiently Stable Albumin-Coated Microbubbles via a Flow-Focusing Microfluidic Device

We describe a method for synthesizing albumin-shelled, large-diameter (>10 μm), transiently stable microbubbles using a flow-focusing microfluidic device (FFMD). The microfluidic device enables microbubbles to be produced immediately before insonation, thus relaxing the requirements for stability. Both reconstituted fractionated bovine serum albumin (BSA) and fresh bovine blood plasma were investigated as shell stabilizers. Microbubble coalescence was inhibited by the addition of either dextrose or glycerol and propylene glycol. Microbubbles were observed to have an acoustic half-life of approximately 6 s. Microbubbles generated directly within a vessel phantom containing flowing blood produced a 6.5-dB increase in acoustic signal within the lumen. Microbubbles generated in real time upstream of in vitro rat aortic smooth muscle cells under physiologic flow conditions successfully permeabilized 58% of the cells on insonation at a peak negative pressure of 200 kPa. These results indicate that transiently stable microbubbles produced via flow-focusing microfluidic devices are capable of image enhancement and drug delivery. In addition, successful microbubble production with blood plasma suggests the potential to use blood as a stabilizing shell.     

Albumin coated microbubble optimization: custom fabrication and comprehensive characterization

Gas microbubbles are used routinely to improve contrast in medical diagnostic imaging. The emerging fields of microbubble-enhanced quantitative imaging and microbubble-enhanced drug delivery have further enhanced the drive toward microbubble characterization and design techniques. The quest to improve efficiency, particularly in the field of drug delivery, presents a requirement to develop methods to manipulate microbubble properties to improve utility. This article presents an investigation in to the feasibility of influencing albumin shelled microbubble properties through the variation of albumin availability during fabrication. Microbubbles were fabricated from albumin suspensions of varying concentration before thorough physical and acoustic characterization. Microbubbles with shells fabricated from a 2% albumin suspension had a greater scattering to attenuation ratio (STAR) than 10% albumin preparations (4.4% and 2.2%, respectively) and approximately double the nonlinear STAR (from 0.7% to 1.5%). The 2% microbubbles also exhibited greater (up to 40%), more violent radial oscillations during high speed imaging than 5% and 10% preparations. The results show that microbubble characteristics can be simply manipulated in the lab and indicate that for a given application this may provide the opportunity to further enhance favorable characteristics.     

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the bloodstream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. Microbubbles generated by the flow-focusing microfluidic device were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 s, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n > 3). In addition, delivery was observed at all flow rates, with maximal delivery (>70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.     

Ultrasound microbubble contrast agents: Application to therapy for peripheral vascular disease

Ultrasound contrast agents are not only effective in ultrasonic imaging but are also important tools for drug or gene delivery. Ultrasound beams can disrupt microbubbles and cell membranes, offering the opportunity to locally deliver drugs or genes. Liposome-shelled microbubbles have many advantages and are widely used in many applications, while Lipofectamine™ (Invitrogen, Life Technologies, Carlsbad, CA, USA), as a material of microbubble membranes, has been used to enhance the effects of gene delivery. Ultrasound contrast agents that have therapeutic effects can be used for treating peripheral vascular diseases, particularly in thrombotic and angiogenic diseases. A combination of targeted contrast agent and drug-carrying contrast agent may be safer and more effective in treating thrombosis. Vascular endothelial growth factor-loaded microbubbles are expected to treat a variety of neovascular diseases such as severe limb ischemia and other diseases. Although there are several limitations in the application of therapeutic ultrasound microbubble contrast agents, it will offer a new hope for the treatment of peripheral vascular disease.     

Localized in Vivo Model Drug Delivery with Intravascular Ultrasound and Microbubbles

An intravascular ultrasound (IVUS) and microbubble drug delivery system was evaluated in both ex vivo and in vivo swine vessel models. Microbubbles with the fluorophore DiI embedded in the shell as a model drug were infused into ex vivo swine arteries at a physiologic flow rate (105 mL/min) while a 5-MHz IVUS transducer applied ultrasound. Ultrasound pulse sequences consisted of acoustic radiation force pulses to displace DiI-loaded microbubbles from the vessel lumen to the wall, followed by higher-intensity delivery pulses to release DiI into the vessel wall. Insonation with both the acoustic radiation force pulse and the delivery pulse increased DiI deposition 10-fold compared with deposition with the delivery pulse alone. Localized delivery of DiI was then demonstrated in an in vivo swine model. The theoretical transducer beam width predicted the measured angular extent of delivery to within 11%. These results indicate that low-frequency IVUS catheters are a viable method for achieving localized drug delivery with microbubbles.     

Effect of microbubble fragility on transit rate measurement by contrast echography

We sought to propose a simplified method to measure flow velocity based on ultrasonic microbubble destruction, and investigated the effect of microbubble shell fragility on such measurement. Acoustic density (AD) from the second harmonic short axis image of flow was obtained at variable velocities (2 to 73 mm/s) in an in vitro model during long (1000 ms) and short (33 ms) interval ultrasound (US) pulsing, allowing complete and partial microbubble replenishment between pulses, respectively. Microbubbles with shell elastic modulus of 0.4 MPa and 16 MPa were tested. By shortening pulsing interval, AD diminished gradually, rather than abruptly, to a plateau level for both microbubbles. The extent of AD decay was greater for the fragile than the strong microbubbles. A linear relationship existed between the magnitude of AD decay and flow velocity only in the higher and lower velocity range for the fragile and the strong microbubbles, respectively. Thus, difference in contrast intensities during long and short pulsing intervals, respectively, allowing complete and partial replenishment may provide for velocity measurement, in which choice of optimal microbubble fragility for the range of velocity to measure may increase the accuracy.     

Ultrasound Molecular Imaging of Cardiovascular Disease

Molecular imaging with ultrasound contrast agents relies on the detection of microbubbles within diseased tissue. Microbubbles produce an acoustic signal owing to their resonant properties in an ultrasound field. Microbubble targeting is accomplished by either manipulating the microbubble shell for attachment of microbubbles to activated leukocytes, or by conjugation of disease-specific ligands to the microbubble surface. Inflammation, angiogenesis, and thrombus formation are central pathophysiologic processes in many cardiovascular diseases and produce phenotypic changes in the vascular compartment that can be imaged with targeted ultrasound contrast agents. In the future, targeted contrast ultrasound could aid in the diagnosis of atherosclerosis, myocardial ischemia, transplant rejection, and thrombosis syndromes and could be used for assessing angiogenesis.     

Deformable gas-filled microbubbles targeted to P-selectin.

Ultrasound contrast microbubbles have been successfully targeted to a number of intravascular disease markers. We hypothesized that targeted delivery could be improved further, by making the microbubbles deformable, leading to increased microbubble-endothelium adhesion contact area and stabilized adhesion. Activated leukocytes utilize such strategy; they deform after binding to inflamed endothelium in the vasculature. Lipid-shell microbubbles were targeted to the endothelial inflammatory protein P-selectin with a monoclonal anti-P-selectin antibody attached to the microbubble shell. Deformable microbubbles were created by controlled pressurization with partial gas loss, which generated an average excess shell surface area of approximately 30% and the formation of outward-projected wrinkles and folds. Targeted microbubble adhesion and deformability were assessed in the parallel plate flow chamber under shear flow. Sustained adhesion of deformable microbubbles at wall shear stresses between 0.4 and 1.35 dyn/cm(2) was consistently better than adhesion of wrinkle-free microbubbles. Over this shear range, targeted wrinkled microbubbles were deformed by shear flow, unlike wrinkle-free microbubbles. In a murine cremaster inflammation model, a significant improvement of deformable microbubble targeting was observed by intravital microscopy. Overall, the mechanical aspects of adhesion, such as particle shape, deformability and surface microstructure, are important in engineering efficient site-targeted particle-based agents for medical imaging and therapy.     

Microbubble Stability is a Major Determinant of the Efficiency of Ultrasound and Microbubble Mediated in vivo Gene Transfer

In the search for an efficient nonviral gene therapy approach for the treatment of genetic disorders of cardiac and skeletal muscle such as Duchenne muscular dystrophy, ultrasound in combination with contrast enhancing microbubbles has emerged as a promising tool for safe and site-specific enhancement of gene delivery. Indeed, microbubble-enhanced gene transfer (MBGT) has been investigated for a wide variety of target sites using both reporter and therapeutic genes. Although a range of different microbubbles have been used for MBGT studies, comparison of their efficiencies is difficult because microbubble concentration and the ultrasound settings used for the application vary considerably. Only two studies to date have attempted a direct comparison of commercially available microbubbles, and both concluded that not all microbubbles show the same efficiencies with MBGT. Thus far, the reason for this is unclear. Here, the efficiency of three commercially available microbubbles—Optison, SonoVue and Sonazoid—was analyzed to understand the microbubble properties that are important for their function as an effective enhancer for gene transfer in vivo. In this study, plasmid DNA or antisense oligonucleotides were delivered by systemic injection with MBGT, focused on the heart. Gene delivery to the heart with equalized concentrations of the three microbubbles showed that Optison and Sonazoid are more efficient in MBGT compared with SonoVue, which showed the weakest gene transfer to the myocardium. Investigations into the properties of these microbubbles showed that size and shell composition did not directly influence MBGT, whereas the microbubbles with increased stability in an ultrasound field showed better MBGT results than those degrading faster. Moreover, the microbubble concentration used for MBGT was also found to be an important factor influencing the efficiency of MBGT. In conclusion, the stability of a microbubble was shown to be a major influential factor for its performance in MBGT, as is the concentration of the microbubbles used. These findings emphasize the importance of detailed investigations into the properties of microbubbles to allow the production of a microbubble specifically designed for optimum performance with MBGT. (E-mail: d.wells@imperial.ac.uk)     

Technique for the Characterization of Phospholipid Microbubbles Coatings by Transmission Electron Microscopy

Gas microbubbles stabilized by a surfactant or polymer coating are of considerable clinical interest because of their imaging and drug delivery potential under ultrasound exposure. The utility of microbubbles for a given application is intrinsically linked to their structure and stability. These in turn are highly sensitive to coating composition and fabrication techniques. Various methods including fluorescence and atomic force microscopy have been applied to characterize microbubble properties, but direct observation of coating structure at the nanoscale still poses a considerable challenge. Here we describe a transmission electron microscopy (TEM) technique to observe the surface of microbubbles. Images from a series of phospholipid-coated microbubble systems, including those decorated with nanoparticles, are presented. They indicate that the technique enables visualization of the coating structure, in particular lipid discontinuities and nanoparticle distribution. This information can be used to better understand how microbubble surface structure relates to formulation and/or processing technique and ultimately to functionality.     

Endothelial Cells,First Target of Drug Delivery Using Microbubble-Assisted Ultrasound

Microbubble-assisted ultrasound has emerged as a promising method for local drug delivery. Microbubbles are intravenously injected and locally activated by ultrasound, thus increasing the permeability of vascular endothelium for facilitating extravasation and drug uptake into the treated tissue. Thereby, endothelial cells are the first target of the effects of ultrasound-driven microbubbles. In this review, the in vitro and in vivo bioeffects of this method on endothelial cells are described and discussed, including aspects on the permeabilization of biologic barriers (endothelial cell plasma membranes and endothelial barriers), the restoration of their integrity, the molecular and cellular mechanisms involved in both these processes, and the resulting intracellular and intercellular consequences. Finally, the influence of the acoustic settings, microbubble parameters, treatment schedules and flow parameters on these bioeffects are also reviewed.     

超声微泡造影剂对心肌组织的生物学效应及介导VEGF基因转染大鼠心肌的实验研究

目的 探讨超声微泡造影剂对心肌组织的生物学效应及其介导VEGF基因转染大鼠心肌的有效性。方法 18只健康雄性Wistar大鼠，取3只采用超声波在鼠胸壁破坏微泡造影剂，观察对心肌组织显微结构的影响。将另15只急性心肌梗死3天后的雄性Wistar大鼠分为3组，每组5只。第一组采用超声破坏微泡造影剂的方式，将pcDzVEGFm基因转染大鼠心肌至造影剂不再显影(约6min)；第二组尾静脉输入同等剂量携pcD。VEGF。基因的造影剂；第三组为对照。2周后，取缺血心肌组织行VEGF免疫组织化学染色，观察心肌组织血管内皮生长因子(VEGF)蛋白表达情况。结果超声波破坏微泡造影剂能使心肌组织充血，产生大量空泡，并有部分心肌细胞坏死。采用超声微泡造影剂介导的VEGF基因转染，能明显增强大鼠心肌组织VEGF蛋白的表达。结论 超声微泡造影剂能明显增强对组织的空化效应，其介导的VEGF基因治疗是一种无创、新型、高效的基因转移方法。     

Investigation of microbubble response to long pulses used in ultrasound-enhanced drug delivery

In current drug delivery approaches, microbubbles and drugs can be co-administered while ultrasound is applied. The mechanism of microbubble interaction with ultrasound, the drug and the cells is not fully understood. The aim of this study was to investigate microbubble response to long ultrasonic pulses used in drug delivery approaches. Two different in vitro set-ups were considered: with the microbubbles diluted in an enclosure and with the microbubbles flowing in a capillary tube. Acoustic streaming, which influences the observed bubble response, was observed in “typical” drug delivery conditions in the first set-up. With the capillary set-up, streaming effects were avoided and accurate bubble responses were recorded. The diffraction pattern of the source greatly influences the bubble response and in different locations of the field different bubble responses are observed. At low nondestructive pressures, microbubbles can oscillate for thousands of cycles repeatedly. At high acoustic pressures (at 1 MHz), most bubble activity disappeared within about 100 μs despite the length of the pulse, mainly due to violent bubble destruction and subsequent accelerated diffusion.     

Targeted Gene Transfection from Microbubbles into Vascular Smooth Muscle Cells Using Focused,Ultrasound-Mediated Delivery

We investigated a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0 to 950 kPa) and pulse durations (0 to 100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA or ultrasound alone. Microbubbles in combination with ultrasound (500-kPa, 1-MHz, 50-cycle bursts at a pulse repetition frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 ± 27%) and without (19 ± 8%). Maximal gene transfection (∼1% of cells) occurred using 50-cycle, 1-MHz pulses at 300 kPa, which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound. (E-mail: jh7fj@virginia.edu)     

Nanointerrogation of ultrasonic contrast agent microbubbles using atomic force microscopy

Predicting the acoustic response of an encapsulated microbubble to ultrasound requires an accurate assessment of the mechanical properties of the microbubble shell. Atomic force microscopy (AFM) provides an unprecedented spatial and force resolution of the order of Angstroms and subnanonewtons, respectively. It is introduced here as a means to interrogate microbubbles manufactured for ultrasonic imaging. The advantage of AFM over scanning electron microscopy (SEM) is that the microbubbles need not be subjected to a low temperature or low-pressure environment. The microbubbles were interrogated in a liquid environment, which could potentially be a simulated physiological environment. AFM was used in tapping mode imaging to reveal topographical detail of biSphere microbubbles. Because microbubbles are large objects compared with the overall size of usual AFM tips, a convolution between the AFM tip and the microbubble was typical of the acquired topographies. However, a part of the top half of the bubble was imaged with nanometer resolution, and roughness measurements are reported. Force-distance curves were captured using contact mode AFM. The range of stiffness or effective spring constant of biSphere was found to be between 1 and 6 N m(-1). In conclusion, the AFM is proposed here for the first time as a tool to image the surface of bubbles at the nanometer range in liquid and to perform reproducible measurements on the mechanical properties of individual microbubbles.     

Lipid Microbubbles as Ultrasound-Stimulated Oxygen Carriers for Controllable Oxygen Release for Tumor Reoxygenation

Microbubbles are proposed as a potentially novel method for oxygen delivery in vivo in initial studies. The lack of commercial microbubbles for oxygen delivery in preclinical research prompted us to fabricate an oxygen-loaded lipid microbubble. We aimed to extend the innovative strategy to modulate the tumor hypoxic microenvironment, using microbubbles intravenously as an oxygen carrier for the controllable tumor-specific delivery of oxygen by ultrasound (US). In our experiment, an oxygen-loaded lipid-coated microbubble (OLM) with mixed gas (O₂/C₃ F₈, 5:1 v/v) was fabricated and exhibited a higher rate of oxygen release to a desaturated solution through burst by US than that in the absence of US. Although in in vivo studies, OLMs could be imaged and triggered by US to elevate the pO₂ level in the breast VX2 tumor dramatically within a matter of minutes. The added presence of US-activated OLMs elicited a nearly six-fold increase in pO₂ levels within 1?min compared with that of the pre-injection. Owing to the high oxygen payload, great acoustic stability and acoustic properties, OLMs may be proposed as an ideal radio-sensitizer. We conclude that oxygen release mediated by ultrasound-targeted microbubble destruction is feasible and shows potential in image-guided, site-specific cancer radiotherapy.     

超声微泡造影剂介导EGFP质粒转染大鼠视网膜的实验研究

目的探讨超声破坏微泡介导EGFP质粒转染大鼠视网膜的效率及可行性，为实现外源基因高效、定向的转移目的奠定基础。方法将30只Long-evans大鼠分为6组，第1组仅以0．5w／cm。的超声波辐照大鼠眼球，第2组于尾静脉输入适当剂量的微泡造影剂，并立即以相同能量的超声波辐照大鼠眼球，第3组于尾静脉输入质粒，第4组于尾静脉输入质粒，并以超声辐照大鼠眼球，第5组于尾静脉输入质粒与微泡，第6组尾静脉输入质粒、微泡，并用超声辐照眼球。转染2周后，在激光共聚焦显微镜下观察EGFP表达情况。结果超声微泡介导的EGFP质粒对大鼠视网膜的转染效率，明显高于其他实验组。一定能量和时间的超声波辐照，及适当浓度的微泡，对大鼠视网膜脉络膜无明显损伤。结论利用低频率和一定能量的超声击碎携带EGFP质粒的超声微泡造影剂，能够有效地提高EGFP质粒在大鼠视网膜的转染效率。     

Sonopermeation Enhances Uptake and Therapeutic Effect of Free and Encapsulated Cabazitaxel

Delivery of drugs and nanomedicines to tumors is often heterogeneous and insufficient and, thus, of limited efficacy. Microbubbles in combination with ultrasound have been found to improve delivery to tumors, enhancing accumulation and penetration. We used a subcutaneous prostate cancer xenograft model in mice to investigate the effect of free and nanoparticle-encapsulated cabazitaxel in combination with ultrasound and microbubbles with a lipid shell or a shell of nanoparticles. Sonopermeation reduced tumor growth and prolonged survival (26%–100%), whether the free drug was co-injected with lipid-shelled microbubbles or the nanoformulation was co-injected with lipid-shelled or nanoparticle-shelled microbubbles. Coherently with the improved therapeutic response, we found enhanced uptake of nanoparticles directly after ultrasound treatment that lasted several weeks (2.3 × –15.8 × increase). Neither cavitation dose nor total accumulation of nanoparticles could explain the variation within treatment groups, emphasizing the need for a better understanding of the tumor biology and mechanisms involved in ultrasound-mediated treatment.     

Ultrasound Imaging of Microbubble Activity during Sonoporation Pulse Sequences

Ultrasound-mediated drug delivery using the mechanical action of oscillating and/or collapsing microbubbles has been studied on many different experimental platforms, both in vitro and in vivo; however, the mechanisms remain to be elucidated. Many groups use sterile, enclosed chambers, such as Opticells and Clinicells, to optimize acoustic parameters in vitro needed for effective drug delivery in vivo, as well as for mechanistic investigation of sonoporation or the use of sound to permeate cell membranes. In these containers, cell monolayers are seeded on one side, and the remainder of the volume is filled with a solution containing microbubbles and a model drug. Ultrasound is then applied to study the effect of different parameters on model drug uptake in cell monolayers. Despite the simplicity of this system, the field has been unable to appropriately address what parameters and microbubble concentrations are most effective at enhancing drug uptake and minimizing cellular toxicity. In this work, a common in vitro sonoporation experimental setup was characterized through quantitative analysis of microbubble-dependent acoustic attenuation in combination with high-frame-rate and high-resolution imaging of bubble activity during sonoporation pulse sequences. The goal was to visualize the effect that ultrasound parameters have on microbubble activity. It was observed that under literature-derived sonoporation conditions (0.1–1 MPa, 20–1000 cycles and 10,000 to 10,000,000 microbubbles/mL), there is strong and non-linear acoustic attenuation, as well as bubble destruction, gas diffusion and bubble motion resulting in spatiotemporal pressure and concentration gradients. Ultimately, it was found that the acoustic conditions in common in vitro sonoporation setups are much more complex and confounding than often assumed.     

Effect of surface architecture on in vivo ultrasound contrast persistence of targeted size-selected microbubbles

Ultrasound molecular imaging is a powerful diagnostic modality using microbubbles coated with targeting ligands specific for endothelial biomarkers. The circulation persistence of ligand-bearing contrast agents is a key determinant in their contrast enhancement and targeting capability. Prior studies have shown that targeted microbubbles with ligands attached to the shell using the conventional exposed-ligand architecture (ELA) could trigger undesired ligand-induced complement activation and decreased circulation time. Microbubbles with the buried-ligand architecture (BLA), however, were found to inhibit complement activation and prolong circulation time. In the present study, we extended the stealth BLA microbubble design to size-selected (4 to 5-μm diameter) microbubbles targeted with cyclic RGD peptide using the postlabeling technique. Microbubble circulation persistence was measured in the healthy mouse kidney using a Visualsonics Vevo 770 scanner operating at 40 MHz in fundamental mode. The circulation persistence for targeted BLA microbubbles was significantly longer compared with their ELA counterparts and similar to no-ligand controls. Use of the BLA instead of the ELA increased the circulation half-life approximately two-fold. Analysis of the time-intensity and time-fluctuation curves with a two-compartment pharmacokinetic model showed a minimal degree of nonspecific vascular adhesion for any group. These results demonstrate the importance of surface architecture in the design of targeted microbubbles for ultrasound molecular imaging.