Lipid-shelled Microbubbles Research Articles

Temporal Stability of Lipid-Shelled Microbubbles During Acoustically-Mediated Blood-Brain Barrier Opening

Non-invasive blood-brain barrier (BBB) opening using focused ultrasound (FUS) is being tested as a means to locally deliver drugs into the brain. Such FUS therapies require injection of preformed microbubbles, currently used as contrast agents in ultrasound imaging. Although their behavior during exposure to imaging sequences has been well described, our understanding of microbubble stability within a therapeutic field is still not complete. Here, we study the temporal stability of lipid-shelled microbubbles during therapeutic FUS exposure in two timescales: the short time scale (i.e., μs of low-frequency ultrasound exposure) and the long time scale (i.e., days post-activation). We first simulated the microbubble response to low-frequency sonication, and found a strong correlation between viscosity and fragmentation pressure. Activated microbubbles had a concentration decay constant of 0.02 d−1 but maintained a quasi-stable size distribution for up to 3 weeks (< 10% variation). Microbubbles flowing through a 4-mm vessel within a tissue-mimicking phantom (5% gelatin) were exposed to therapeutic pulses (fc: 0.5 MHz, peak-negative pressure: 300 kPa, pulse length: 1 ms, pulse repetition frequency: 1 Hz, n=10). We recorded and analyzed their acoustic emissions, focusing on emitted energy and its temporal evolution, alongside the frequency content. Measurements were repeated with concentration-matched samples (107 microbubbles/ml) on day 0, 7, 14, and 21 after activation. Temporal stability decreased while inertial cavitation response increased with storage time both in vitro and in vivo, possibly due to changes in the shell lipid content. Using the same parameters and timepoints, we performed BBB opening in a mouse model (n=3). BBB opening volume measured through T1-weighted contrast-enhanced MRI was equal to 19.1 ± 7.1 mm3, 21.8 ± 14 mm3, 29.3 ± 2.5 mm3, and 38 ± 20.1 mm3 on day 0, 7, 14, and 21, respectively, showing no significant difference over time (p-value: 0.49). Contrast enhancement was 24.9 ± 1.7 %, 23.7 ± 11.7 %, 28.9 ± 5.3 %, and 35 ± 13.4 %, respectively (p-value: 0.63). In conclusion, the in-house made microbubbles studied here maintain their capacity to produce similar therapeutic effects over a period of 3 weeks after activation, as long as the natural concentration decay is accounted for. Future work should focus on stability of commercially available microbubbles and tailoring microbubble shell properties towards therapeutic applications.

Bactericidal Activity of Lipid-Shelled Nitric Oxide-Loaded Microbubbles.

The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro. MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure (in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure (in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFP-saturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications.

PD-L1-targeted microbubbles loaded with docetaxel produce a synergistic effect for the treatment of lung cancer under ultrasound irradiation.

Immunotherapy is gradually becoming as important as traditional therapy in the treatment of cancer, but adverse drug reactions limit patient benefits from PD1/PD-L1 checkpoint inhibitor drugs in the treatment of non-small cell lung cancer (NSCLC). As a chemotherapeutic drug for NSCLC, docetaxel (DTX) can synergize with PD1/PD-L1 checkpoint inhibitors but increase haematoxicity and neurotoxicity. Herein, anti-PD-L1 monoclonal antibody (mAb)-conjugated and docetaxel-loaded multifunctional lipid-shelled microbubbles (PDMs), which were designed with biologically safe phospholipids to produce synergistic antitumour effects, reduced the incidence of side effects and promoted therapeutic effects under ultrasound (US) irradiation. The PDMs were prepared by the acoustic-vibration method and then conjugated with an anti-PD-L1 mAb. The material features of the microbubbles and their cytotoxic effects, cellular apoptosis and cell cycle inhibition were studied. A subcutaneous tumour model was established to test the drug concentration-dependent and antitumour effects of the PDMs combined with US irradiation, and an orthotopic lung tumour model simultaneously confirmed the antitumour effect of this synergistic treatment. The PDMs achieved higher cellular uptake than free DTX, especially when combined with US irradiation. The PDMs combined with US irradiation also induced an increased rate of cellular apoptosis and an elevated G2-M arrest rate in cancer cells, which was positively correlated with PD-L1 expression. An in vivo study showed that synergistic treatment had relatively strong effects on tumour growth inhibition, increased survival time and decreased adverse effect rates. Our study possibly provides a well-controlled design for immunotherapy and chemotherapy and has promising potential for clinical application in NSCLC treatment.

A multifunctional theranostic contrast agent for ultrasound/near infrared fluorescence imaging-based tumor diagnosis and ultrasound-triggered combined photothermal and gene therapy

PurposeEncapsulated microbubbles (MBs) have been reported as new theranostic carriers for simultaneous imaging and ultrasound (US)-triggered therapy. Here, we designed a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. MethodsGold nanorods (AuNRs) resonant at 880 nm together with the NIR797 dye were first encapsulated in lipid-shelled MBs to construct fluorescent gold microbubbles (NIR797/AuMBs) via thin film hydration and mechanical shaking in the presence of sulfur hexafluoride (SF6) gas. Then, polyethylenimine (PEI)-DNA complexes were electrostatically conjugated onto the surface of the NIR797/AuMBs, forming theranostic encapsulated MBs (PEI-DNA/NIR797/AuMBs). The potential of the PEI-DNA/NIR797/AuMBs for use as a dual-modality contrast enhancement agent was evaluated in vitro and in vivo. The antitumor effect of US/NIR laser irradiation mediating double-fusion suicide gene and photothermal therapy was also investigated using Bel-7402 cells and xenografts. ResultsThe developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. And US + laser exposure group showed a much higher rate of cell inhibition, apoptosis and necrosis as well as a higher Bel-7402 xenograft inhibition rate than the single gene therapy or single exposure (US or laser) group. ConclusionsPEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy. Statement of SignificanceThis is an original paper focusing on developing a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. The developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. PEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

High-speed video microscopy and numerical modeling of bubble dynamics near a surface of urinary stone.

Ultra-high-speed video microscopy and numerical modeling were used to assess the dynamics of microbubbles at the surface of urinary stones. Lipid-shell microbubbles designed to accumulate on stone surfaces were driven by bursts of ultrasound in the sub-MHz range with pressure amplitudes on the order of 1 MPa. Microbubbles were observed to undergo repeated cycles of expansion and violent collapse. At maximum expansion, the microbubbles' cross-section resembled an ellipse truncated by the stone. Approximating the bubble shape as an oblate spheroid, this study modeled the collapse by solving the multicomponent Euler equations with a two-dimensional-axisymmetric code with adaptive mesh refinement for fine resolution of the gas-liquid interface. Modeled bubble collapse and high-speed video microscopy showed a distinctive circumferential pinching during the collapse. In the numerical model, this pinching was associated with bidirectional microjetting normal to the rigid surface and toroidal collapse of the bubble. Modeled pressure spikes had amplitudes two-to-three orders of magnitude greater than that of the driving wave. Micro-computed tomography was used to study surface erosion and formation of microcracks from the action of microbubbles. This study suggests that engineered microbubbles enable stone-treatment modalities with driving pressures significantly lower than those required without the microbubbles.

Time and Frequency Characteristics of Cavitation Activity Enhanced by Flowing Phase-Shift Nanodroplets and Lipid-Shelled Microbubbles During Focused Ultrasound Exposures

This study investigated and compared the time and frequency characteristics of cavitation activity between phase-shift nanodroplets (NDs) and lipid-shelled microbubbles (MBs) exposed to focused ultrasound (FUS) under physiologically relevant flow conditions. Root-mean-square (RMS) of broadband noise, spectrograms of the passive cavitation detection signals and inertial cavitation doses (ICDs) were calculated during FUS at varying mean flow velocities and two different peak-rarefactional pressures. At a lower pressure of 0.94 MPa, the mean values of the RMS amplitudes versus time for the NDs showed an upward trend but slowed down as the mean flow velocity increased. For flowing NDs, the rate of growth in RMS amplitudes within 2–5 MHz decreased more obviously than those within 5–8 MHz. At a higher pressure of 1.07 MPa, the increase in RMS amplitudes was accelerated as the mean flow velocity increased from 0 to 10 cm/s and slowed down as the mean flow velocity reached 15 cm/s. The general downward trends of RMS amplitudes for the MBs were retarded as the mean flow velocity increased at both acoustic pressures of 0.94 MPa and 1.07 MPa. At 0.94 MPa, the mean ICD value for the NDs decreased from 57 to 36 as the mean flow velocity increased from 0 to 20 cm/s. At 1.07 MPa, the mean ICD value initially increased from 45 to 57 as the mean flow velocity increased from 0 to 10 cm/s and subsequently decreased to 43 as the mean flow velocity reached 20 cm/s. For the MBs, the mean ICD value increased with increasing mean flow velocity at both acoustic pressures. These results could aid in future investigations of cavitation-enhanced FUS with the flowing phase-shift NDs and encapsulated, gas-filled MBs for various applications.

Multifunctional Theranostic Contrast Agent for US/NIRF-Based Tumor Diagnosis and US-Triggered Combined Photothermal and Gene Therapy

Purpose: Encapsulated microbubbles (MBs) have been reported asnew theranostic carriers for simultaneous imaging and ultrasound (US)-triggered therapy. Here, we designed a dual-modality US/NIRF contrastagent and extended its applications from image contrast enhancement tocombined diagnosis and therapy with US-directed and site-specifictargeting. Methods: Gold nanorods (AuNRs) resonant at 880 nm together with theNIR797 dye were first encapsulated in lipid-shelled MBs to constructfluorescent gold microbubbles (NIR797/AuMBs) via thin film hydration andmechanical shaking in the presence of sulfur hexafluoride (SF6) gas.Then, polyethylenimine (PEI)-DNA complexes were electrostaticallyconjugated onto the surface of the NIR797/AuMBs, forming theranosticencapsulated MBs (PEI-DNA/NIR797/AuMBs). The potential of the PEIDNA/NIR797/AuMBs for use as a dual-modality contrast enhancement agentwas evaluated in vitro and in vivo. The antitumor effect of US/NIR laserirradiation mediating double-fusion suicide gene and photothermal therapywas also investigated using Bel-7402 cells and xenografts. Results: The developed theranostic AuMB complexes could not only provideexcellent US and NIRF imaging to detect tumors but also serve as anefficient US-triggered carrier for gene delivery and photothermalablation of tumors in xenografted nude mice. And US + laser exposuregroup showed a much higher rate of cell inhibition, apoptosis andnecrosis as well as a higher Bel-7402 xenograft inhibition rate than thesingle gene therapy or single exposure (US or laser) group. Conclusions: PEI-DNA/NIR797/AuMBs would be of great value for providingmore comprehensive diagnostic information and to guide more accurate andeffective synergistic cancer therapy.

Characterization and Imaging of Lipid-Shelled Microbubbles for Ultrasound-Triggered Release of Xenon.

Xenon (Xe) is a bioactive gas capable of reducing and stabilizing neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for xenon loading and ultrasound-triggered release. Microbubbles loaded with either xenon (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) (9:1 v/v) were synthesized by high-shear mixing. The size distribution and the frequency-dependent attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband acoustic attenuation spectroscopy system, respectively. The Xe dose was evaluated using gas chromatography/mass spectrometry. The total Xe doses in Xe-MB and Xe-OFP-MB were 113.1 ± 13.5 and 145.6 ± 25.5μl per mg of lipid, respectively. Co-encapsulation of OFP increased the total xenon dose, attenuation coefficient, microbubble stability (in an undersaturated solution), and shelf life of the agent. Triggered release of gas payload was demonstrated with 6-MHz duplex Doppler and 220-kHz pulsed ultrasound. These results constitute the first step toward the use of lipid-shelled microbubbles for applications such as neuroprotection in stroke.

Molecular Effects of Glycerol on Lipid Monolayers at the Gas-Liquid Interface: Impact on Microbubble Physical and Mechanical Properties.

The production and stability of microbubbles (MBs) is enhanced by increasing the viscosity of both the formation and storage solution, respectively. Glycerol is a good candidate for biomedical applications of MBs, since it is biocompatible, although the exact molecular mechanisms of its action is not fully understood. Here, we investigate the influence glycerol has on lipid-shelled MB properties, using a range of techniques. Population lifetime and single bubble stability were studied using optical microscopy. Bubble stiffness measured by AFM compression is compared with lipid monolayer behavior in a Langmuir-Blodgett trough. We deduce that increasing glycerol concentrations enhances stability of MB populations through a 3-fold mechanism. First, binding of glycerol to lipid headgroups in the interfacial monolayer up to 10% glycerol increases MB stiffness but has limited impact on shell resistance to gas permeation and corresponding MB lifetime. Second, increased solution viscosity above 10% glycerol slows down the kinetics of gas transfer, markedly increasing MB stability. Third, above 10%, glycerol induces water structuring around the lipid monolayer, forming a glassy layer which also increases MB stiffness and resistance to gas loss. At 30% glycerol, the glassy layer is ablated, lowering the MB stiffness, but MB stability is further augmented. Although the molecular interactions of glycerol with the lipid monolayer modulate the MB lipid shell properties, MB lifetime continually increases from 0 to 30% glycerol, indicating that its viscosity is the dominant effect on MB solution stability. This three-fold action and biocompatibility makes glycerol ideal for therapeutic MB formation and storage and gives new insight into the action of glycerol on lipid monolayers at the gas-liquid interface.

Lipid-shelled microbubbles for ultrasound-triggered release of bioactive gases to treat stroke and cardiovascular disease

Ischemia-reperfusion-induced neurological injury is a primary cause of stroke disability. Xenon (Xe), a bioactive gas, has potential as an effective and nontoxic neuroprotectant for the treatment of ischemic stroke. Nitric oxide (NO) is a potent bioactive gas capable of inducing vasodilatory, anti-inflammatory, neuroprotectant and bactericidal effects. The goal of this work was to develop lipid-shelled microbubbles for site-specific release of Xe or NO upon pulsed ultrasound exposure. Gas-loaded microbubbles were synthesized by high-shear mixing of a lipid dispersion in a vial that contained, Xe or NO, and octafluoropropane (OFP) in combination. Attenuation spectroscopy measurements demonstrate the feasibility of 6-MHz pulsed Doppler ultrasound-triggered release of Xe or NO from microbubbles. The addition of OFP in the lipid-shelled microbubbles increased the number density, size, and stability of the microbubbles, particularly in undersaturated saline. Gas chromatography mass spectrometry was employed to measure Xe dose (127 ± 29 μl Xe/mg lipid). The payload of NO in the microbubbles (97 ± 12 μl NO/mg lipid) was assessed using an amperometric sensor. Intravenous administration of microbubbles carrying a neuroprotective or a vasodilatory gas in combination with ultrasound exposure has potential as a novel noninvasive strategy for local therapeutic delivery to modulate the effects or duration of cerebral ischemia.

Ultrasound-targeted microbubble destruction improved the antiangiogenic effect of Endostar in triple-negative breast carcinoma xenografts.

Ultrasound-targeted microbubble destruction (UTMD) has been reported to be a meritorious technique for drug targeting delivery. In this study, we aimed to evaluate the synergistic antiangiogenic effect of UTMD combined with Endostar on triple-negative breast carcinoma tumors. The lipid-shelled microbubbles (MBs) conjugated with Endostar were constructed using a biotin-avidin bridging chemistry method, and the morphological characteristics and drug-conjugating content were determined. MBs were administered intravenously to nude mice bearing MDA-MB-231 breast carcinoma xenografts and ultrasound exposure followed. The tumor microcirculation was observed by contrast-enhanced ultrasonography (CEUS) and the Endostar biodistribution was detected by enzyme-linked immunosorbent assay. Twenty-four breast carcinoma-bearing nude mice were divided into four groups. After treatment, every 3days for 15days the in vivo antitumor effects were assessed by calculating the tumor growth inhibition rate (TGIR). The tumor microcirculation was observed by CEUS, the tumor microvessel density (MVD) was calculated by immunohistochemistry under a microscope, and the vascular endothelial growth factor (VEGF) gene expression was detected by real-time quantitative polymerase chain reaction. The prepared Endostar-conjugated MBs were round and well-dispersed with a mean size of 2.8 ± 0.7µm and a drug conjugating content of 800.72 ± 70.53µg/108MBs. UTMD blocked the tumor microcirculation, and improved Endostar release in the targeted tumor tissue with a drug content of 1.12 ± 0.43µg/gram protein, which was about three times higher than that in Endostar group or Endostar conjugated MBs group. Endostar-conjugated MBs combined with UTMD treatment achieved the optimal antitumor effects in vivo with a TGIR of 46.29%, and apparent antiangiogenic effects with minimal tumor blood perfusion, MVD and VEGF gene expression level. UTMD can improve Endostar delivery in the targeting tumor tissue and mediate synergistic antiangiogenetic and antitumor effects, which may be a potential therapeutic strategy for refractory breast cancer.

Enhanced microbubble contrast agent oscillation following 250\u2009kHz insonation

Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. Following 250 kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh–Plesset equation for a clean bubble or the Marmottant model of a lipid-shelled microbubble. The expansion ratio reached 30-fold with 250 kHz at a peak negative pressure of 400 kPa, as compared to a measured expansion ratio of 1.6 fold for 1 MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100 kPa) for the 250 kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75–150 kPa. For pressures above 150 kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250 kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

InVivoMolecular Ultrasound Assessment of Glioblastoma Neovasculature with Endoglin-Targeted Microbubbles

Objectives Glioblastoma, as one of the most malignant cancer in the world, usually shows substantially increased angiogenesis. Endoglin (CD105), which is an alternative proangiogenic growth factor, has been remarkably upregulated on the proliferating glioblastoma neovasculature. However, little is known on the noninvasive assessment of the expression levels of CD105 during glioblastoma progression. Herein, we investigated the potential of the molecular ultrasound imaging for the noninvasive assessment of the expression levels of the biomarker CD105 during the glioblastoma progression. Materials and Methods The CD105-targeted perfluorocarbon-containing lipid-shelled microbubbles (MBs) were prepared. A parallel flow chamber was employed, in which the CD105-targeted and non-targeted MBs were tested across the CD105 ± expression cell lines. In vivo molecular US imaging was conducted based on a subcutaneous xenograft tumor model (n=9). Finally, the statistical analysis was conducted to quantitatively correlate the attachment numbers of MBs in the parallel flow chamber test with the CD105 expression levels of the cells in the flow cytometry test and the in vivo molecular ultrasound signals with the ex vivo expression levels of CD105 in the immunohistochemical test. Results and Discussion The attachment numbers of the CD105-targeted MBs significantly correlated with the CD105 expression levels of the cells in the parallel flow chamber test. There was a good correlation between the in vivo molecular ultrasound signals with the CD105-targeted MBs and the ex vivo expression levels of CD105 in the immunohistochemical test. The results indicate that the molecular US imaging is much potential to assess the progression of the glioblastoma neovasculature noninvasively.

Characterization of lipid-encapsulated microbubbles for delivery of nitric oxide

Nitric oxide (NO) is a potent bioactive gas capable of inducing vasodilatory, anti-inflammatory, and bactericidal effects. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to develop a technique to sequester NO within lipid-shelled microbubbles. Microbubbles loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) were synthesized by high-shear mixing of 1 mL lipids with either 1 mL of NO, or a mixture of 0.9 mL NO and 0.1 mL OFP. The size distribution and attenuation coefficient of NO-MB and NO-OFP-MB were measured using a Coulter counter and a broadband acoustic attenuation spectroscopy system, respectively. The payload of NO in the microbubbles was assessed using an amperometric micro-electrode sensor. Co-encapsulation of NO with OFP increased the number density, attenuation coefficient, and temporal stability of lipid-shelled microbubbles. However, the amount of NO loaded in NO-MB and NO-OFP-MB was similar (0.91 ± 0.03 μM and 0.93 ± 0.1 μM, respectively). These results suggest that NO can be encapsulated within lipid-shelled microbubbles. However, co-encapsulation of OFP does not enhance the NO payload or the temporal stability, despite an increase in attenuation and number density of lipid-shelled microbubbles. Nitric oxide (NO) is a potent bioactive gas capable of inducing vasodilatory, anti-inflammatory, and bactericidal effects. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to develop a technique to sequester NO within lipid-shelled microbubbles. Microbubbles loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) were synthesized by high-shear mixing of 1 mL lipids with either 1 mL of NO, or a mixture of 0.9 mL NO and 0.1 mL OFP. The size distribution and attenuation coefficient of NO-MB and NO-OFP-MB were measured using a Coulter counter and a broadband acoustic attenuation spectroscopy system, respectively. The payload of NO in the microbubbles was assessed using an amperometric micro-electrode sensor. Co-encapsulation of NO with OFP increased the number density, attenuation coefficient, and temporal stability of lipid-shelled microbubbles. However, the amount of NO loaded in NO-MB and NO-OFP-MB wa...

Lipid-shelled microbubbles for ultrasound-triggered release of xenon

Xenon is a cellular protectant shown to stabilize or reduce neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for ultrasound-triggered xenon release. Microbubbles loaded with either xenon alone (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) were synthesized by high-shear mixing lipids with either 100% xenon, or 90% (v/v) xenon and 10% (v/v) octafluoropropane. The size distribution and the attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband attenuation spectroscopy system, respectively. Gas chromatography/mass spectrometry (GC/MS) was performed to assess the dose and stability of encapsulated xenon. The feasibility of xenon release using 5-MHz pulsed Doppler ultrasound and 220-kHz pulsed ultrasound was tested by ultrasound attenuation spectroscopy. Co-encapsulation of OFP increased the number density, attenuation coefficient, and temporal stability of microbubbles. The GC/MS measurements revealed that 143 ± 20 μL/mg and 131 ± 33 μL/mg of xenon was loaded in Xe-MB and Xe-OFP-MB, respectively. Xe-MB and Xe-OFP-MB retained 54 ± 11% and 66 ± 1% of the xenon payload within 15 min of activation, respectively. Attenuation measurements confirmed ultrasound-triggered xenon release. These results suggest that lipid-shelled microbubbles with OFP could serve as ultrasound-triggered xenon delivery agents to attenuate cellular breakdown.

Experimental observations and numerical modeling of lipid-shell microbubbles with stone targeting moieties for minimally-invasive treatment of urinary stones

Products incorporating stone-targeting microbubbles have recently entered human clinical trials as a new minimally-invasive approach to treat urinary stones. Lipid-shell, gas-core microbubbles can be introduced into the urinary tract through a catheter. Calcium-binding moieties incorporated into the lipid shell can facilitate binding to stones. The microbubbles can be excited by an extracorporeal source of low-intensity ultrasound. Alternatively, the microbubbles can be excited by an intraluminal source, such as a fiber-optic laser. With either excitation technique, stone-targeting microbubbles can significantly increase rates of erosion, pitting, and fragmentation of stones, as has recently been reported for in-vitro experiments with synthetic stones [Wiener et al., J. Urology, v.199, no.4S, e322 (2018)]. We report here on new experiments using high-speed photography to characterize microbubbles expansion of cracks within a stone and resultant breaking-off of stone fragments. Numerical modeling shows that the direction of microjets produced by collapsing stone-bound microbubbles depends strongly on bubble shape and stand-off distance. For a wide range of stand-off distances and bubble shapes, microbubble collapse is associated with pressure increases of some two orders of magnitude compared to the excitation source pressures. This in-vitro study provides key insights into the use of stone-targeting microbubbles in treatment of urinary stones.

Novel Perfluorinated Triblock Amphiphilic Copolymers for Lipid-Shelled Microbubble Stabilization.

Amphiphilic triblock (Atri) copolymers made of perfluorinated alkyl chain linked to hydrocarbon chain and methoxy-poly(ethylene glycol) of three different molecular weights were synthesized. In vitro evaluation demonstrated that these new compounds were noncytotoxic. Characterization and interaction of each triblock copolymer with a branched polyamine myristoyl lipid (2-{3[bis-(3-amino-propyl)-amino]-propylamino}- N-ditetradecyl carbamoyl methyl-acetamide, DMAPAP) were studied by the Langmuir film method and thermal analysis. The triblock copolymer/cationic lipids (1:10, w/w) were mixed with perfluorobutane gas to form microbubbles (MBs). The latter were characterized by optical microscopy to get the microbubble size and concentration by densimetry to determine the amount of encapsulated gas and by ultrasound to assess oscillation properties. Atri with the lowest and intermediate weights were shown to interact with the cationic lipid DMAPAP and stabilize the Langmuir film. In that case, monodisperse microbubbles ranging from 2.3 ± 0.1 to 2.8 ± 0.1 μm were obtained. The proportion of encapsulated gas within the MB shell increased up to 3 times after the incorporation of the copolymer with the lowest and intermediate weights. Moreover, the acoustic response of the microbubbles was maintained in the presence of the copolymers.

Spatially Uniform Tumor Treatment and Drug Penetration by Regulating Ultrasound with Microbubbles.

Tumor microenvironment has different morphologies of vessels in the core and rim regions, which influences the efficacy of tumor therapy. Our study proposed to improve the spatial uniformity of the antivascular effect and drug penetration within the tumor core and rim in combination therapies by regulating ultrasound-stimulated microbubble destruction (USMD). Focused ultrasound at 2 MHz and lipid-shell microbubbles (1.12 ± 0.08 μm, mean ± standard deviation) were used to perform USMD. The efficiency of the antivascular effect was evaluated by intravital imaging to determine the optimal USMD parameters. Tumor perfusion and histological alterations in the tumor core and rim were used to analyze the spatial uniformity of the antivascular effect and liposomal-doxorubicin (5 mg/kg) penetration in the combination therapy. Tumor vessels of specific sizes were disrupted by regulating USMD: vessels with sizes of 11 ± 3, 14 ± 5, 19 ± 7, and 23 ± 10 μm were disrupted by stimulation at acoustic pressures of 3, 5, 7, and 9 MPa, respectively (each p < 0.05). The effective treatment time of USMD (at 2 × 107 microbubbles/mouse, 7 MPa, and three cycles) was 60-120 min, which resulted in the disruption of 21-44% of vessels smaller than 50 μm. The reductions in perfusion and vascular density after combination therapy did not differ significantly between the tumor core and rim. This study found that regulating USMD can result in homogeneous antivascular effects and drug penetration within tumors and thereby improve the efficacy of combination therapies.

Experimental observations and numerical modeling of lipid-shell microbubbles with calcium-adhering moieties for minimally-invasive treatment of urinary stones.

A novel treatment modality incorporating calcium-adhering microbubbles has recently entered human clinical trials as a new minimally-invasive approach to treat urinary stones. In this treatment method, lipid-shell gas-core microbubbles can be introduced into the urinary tract through a catheter. Lipid moities with calcium-adherance properties incorporated into the lipid shell facilitate binding to stones. The microbubbles can be excited by an extracorporeal source of quasi-collimated ultrasound. Alternatively, the microbubbles can be excited by an intraluminal source, such as a fiber-optic laser. With either excitation technique, calcium-adhering microbubbles can significantly increase rates of erosion, pitting, and fragmentation of stones. We report here on new experiments using high-speed photography to characterize microbubble expansion and collapse. The bubble geometry observed in the experiments was used as one of the initial shapes for the numerical modeling. The modeling showed that the bubble dynamics strongly depends on bubble shape and stand-off distance. For the experimentally observed shape of microbubbles, the numerical modeling showed that the collapse of the microbubbles was associated with pressure increases of some two-to-three orders of magnitude compared to the excitation source pressures. This in-vitro study provides key insights into the use of microbubbles with calcium-adhering moieties in treatment of urinary stones.