Lipid-shelled Microbubbles Research Articles

Effect of Microbubble Size, Composition, and Multiple Sonication Points on Sterile Inflammatory Response in Focused Ultrasound-Mediated Blood-Brain Barrier Opening.

Blood-brain barrier opening (BBBO) using focused ultrasound (FUS) and microbubbles (MBs) has emerged as a promising technique for delivering therapeutics to the brain. However, the influence of various FUS and MB parameters on BBBO and subsequent sterile inflammatory response (SIR) remains unclear. In this study, we investigated the effects of MB size and composition, as well as the number of FUS sonication points, on BBBO and SIR in an immunocompetent mouse model. Using MRI-guided MB + FUS, we targeted the striatum and assessed extravasation of an MRI contrast agent to assess BBBO and RNaseq to assess SIR. Our results revealed distinct effects of these parameters on BBBO and SIR. Specifically, at a matched microbubble volume dose (MVD), MB size did not affect the extent of BBBO, but smaller (1 μm diameter) MBs exhibited a lower classification of SIR than larger (3 or 5 μm diameter) MBs. Lipid-shelled microbubbles exhibited greater BBBO and a more pronounced SIR compared to albumin-shelled microbubbles, likely owing to the latter's poor in vivo stability. As expected, increasing the number of sonication points resulted in greater BBBO and SIR. Furthermore, correlation analysis revealed strong associations between passive cavitation detection measurements of harmonic and inertial MB echoes, BBBO, and the expression of SIR gene sets. Our findings highlight the critical role of MB and FUS parameters in modulating BBBO and subsequent SIR in the brain. These insights inform the development of targeted drug delivery strategies and the mitigation of adverse inflammatory reactions in neurological disorders.

PROTEUS: A Physically Realistic Contrast-Enhanced Ultrasound Simulator-Part I: Numerical Methods.

Ultrasound contrast agents have been used as vascular reporters for the past 40 years. The ability to enhance vascular features in ultrasound images with engineered lipid-shelled microbubbles has enabled break-throughs such as the detection of tissue perfusion or super-resolution imaging of the microvasculature. However, advances in the field of contrast-enhanced ultra-sound are hindered by experimental variables that are difficult to control in a laboratory setting, such as complex vascular geometries, the lack of ground truth, and tissue nonlinearities. In addition, the demand for large datasets to train deep learning-based computational ultrasound imaging methods calls for the development of a simulation tool that can reproduce the physics of ultrasound wave interactions with tissues and microbubbles. Here, we introduce a physically realistic contrast-enhanced ultrasound simulator (PROTEUS) consisting of four inter-connected modules that account for blood flow dynamics in segmented vascular geometries, intravascular microbubble trajectories, ultrasound wave propagation, and nonlinear microbubble scattering. The first part of this study describes numerical methods that enabled this development. We demonstrate that PROTEUS can generate contrast-enhanced radiofrequency data in various vascular architectures across the range of medical ultrasound frequencies. PROTEUS offers a customizable framework to explore novel ideas in the field of contrast-enhanced ultrasound imaging. It is released as an open-source tool for the scientific community.

Ultrasound trapping and navigation of microrobots in the mouse brain vasculature

The intricate and delicate anatomy of the brain poses significant challenges for the treatment of cerebrovascular and neurodegenerative diseases. Thus, precise local drug delivery in hard-to-reach brain regions remains an urgent medical need. Microrobots offer potential solutions; however, their functionality in the brain remains restricted by limited imaging capabilities and complications within blood vessels, such as high blood flows, osmotic pressures, and cellular responses. Here, we introduce ultrasound-activated microrobots for in vivo navigation in brain vasculature. Our microrobots consist of lipid-shelled microbubbles that autonomously aggregate and propel under ultrasound irradiation. We investigate their capacities in vitro within microfluidic-based vasculatures and in vivo within vessels of a living mouse brain. These microrobots self-assemble and execute upstream motion in brain vasculature, achieving velocities up to 1.5 µm/s and moving against blood flows of ~10 mm/s. This work represents a substantial advance towards the therapeutic application of microrobots within the complex brain vasculature.

Ambient Pressure Sensitivity of the Subharmonic Response of Coated Microbubbles: Effects of Acoustic Excitation Parameters.

The sensitivity of the acoustic response of microbubbles, specifically a strong correlation between their subharmonic response and the ambient pressure, has motivated development of a non-invasive subharmonic-aided pressure estimation (SHAPE) method. However, this correlation has previously been found to vary depending on the microbubble type, the acoustic excitation and the hydrostatic pressure range. In this study, the ambient pressure sensitivity of microbubble response was investigated. The fundamental, subharmonic, second harmonic and ultraharmonic responses from an in-house lipid-coated microbubble were measured for excitations with peak negative pressures (PNPs) of 50-700 kPa and frequencies of 2, 3 and 4 MHz in the ambient overpressure range 0-25 kPa (0-187 mmHg) in an in vitro setup. The subharmonic response typically has three stages-occurrence, growth and saturation-with increasing excitation PNP. We find distinct decreasing and increasing variations of the subharmonic signal with overpressure that are closely related to the threshold of subharmonic generation in a lipid-shelled microbubble. Above the excitation threshold, that is, in the growth-saturation phase, subharmonic signals decreased linearly with slopes as high as -0.56 dB/kPa with ambient pressure increase; below the threshold excitation (at atmospheric pressure), increasing overpressure triggers subharmonic generation, indicating a lowering of subharmonic threshold, and therefore leads to an increase in subharmonic with overpressure, the maximum enhancement being ∼11 dB for 15 kPa overpressure at 2 MHz and 100 kPa PNP. This study indicates the possible development of novel and improved SHAPE methodologies.

QCM-D Investigations on Cholesterol-DNA Tethering of Liposomes to Microbubbles for Therapy.

Lipid-shelled microbubbles (MBs) offer potential as theranostic agents, capable of providing both contrast enhancement in ultrasound imaging as well as a route for triggered drug release and improved localized drug delivery. A common motif in the design of such therapeutic vehicles is the attachment of the drug carrier, often in the form of liposomes, to the microbubble. Traditionally, such attachments have been based around biotin-streptavidin and maleimide-PDP chemistries. Comparatively, the use of DNA-lipid tethers offers potential advantage. First, their specificity permits the construction of more complex architectures that might include bespoke combinations of different drug-loaded liposomes and/or targeting groups, such as affimers or antibodies. Second, the use of dual-lipid tether strategies should increase the strength of the individual tethers tethering the liposomes to the bubbles. The ability of cholesterol-DNA (cDNA) tethers for conjugation of liposomes to supported lipid bilayers has previously been demonstrated. For in vivo applications, bubbles and liposomes often contain a proportion of polyethylene glycol (PEG) to promote stealth-like properties and increase lifetimes. However, the associated steric effects may hinder tethering of the drug payload. We show that while the presence of PEG reduced the tethering affinity, cDNA can still be used for the attachment of liposomes to a supported lipid bilayer (SLB) as measured via QCM-D. Importantly, we show, for the first time, that QCM-D can be used to study the tethering of microbubbles to SLBs using cDNA, signified by a decrease in the magnitude of the frequency shift compared to liposomes alone due to the reduced density of the MBs. We then replicate this tethering interaction in the bulk and observe attachment of liposomes to the shell of a central MB and hence formation of a model therapeutic microbubble.

Material Properties, Dissolution and Time Evolution of PEGylated Lipid-Shelled Microbubbles: Effects of the Polyethylene Glycol Hydrophilic Chain Configurations.

Polyethylene glycol (PEG) is often added to the lipid coating of a contrast microbubble to prevent coalescence and improve circulation. At high surface density, PEG chains are known to undergo a transition from a mushroom configuration to an extended brush configuration. We investigated the effects of PEG chain configuration on attenuation and dissolution of microbubbles by varying the molar ratio of the PEGylated lipid in the shell with three (0%, 2% and 5%) in the mushroom configuration and two (10% and 20%) in the brush configuration. We measured attenuation through the bubble suspensions and used it to obtain the characteristic rheological properties of their shells according to two interfacial rheological models. The interfacial elasticity was found to be significantly lower in the brush regime (∼0.6 N/m) than in the mushroom regime (∼1.3 N/m), but similar in value within each regime. The dissolution behavior of microbubbles under acoustic excitation inside an air-saturated medium was studied by measuring the time-dependent attenuation. Total attenuation recorded a transient increase because of growth resulting from air influx and an eventual decrease caused by dissolution. Microbubble shell composition with varying PEG concentrations had significant effects on dissolution dynamics.

Pressure Measurement in a Bladder Phantom Using Contrast-Enhanced Ultrasonography-A Path to a Catheter-Free Voiding Cystometrogram.

The long-term goal of this study is to investigate the efficacy of a novel, ultrasound-based technique called subharmonic-aided pressure estimation (SHAPE) to measure bladder pressure as a part of a cystometrogram (CMG) in a urodynamic test (ie, pressure-flow study). SHAPE is based on the principle that subharmonic emissions from ultrasound contrast microbubbles (MBs) decrease linearly with an increase in ambient pressure. We hypothesize that, using the SHAPE technique, we can measure voiding bladder pressure catheter-free. This is of importance because the CMG catheter, due to its space-occupying property and non-physiological effects, can undermine the reliability of the test during voiding and cause misdiagnosis. In this study, we tested this hypothesis and optimized the protocol in a controlled benchtop environment. A bladder phantom was designed and built, capable of simulating clinically relevant bladder pressures. Laboratory-made lipid-shelled MBs (similar in composition to the commercial agent, DEFINITY) was diluted in 0.9% normal saline and infused into the bladder phantom using the CMG infusion system. A typical simulated CMG consists of 1 filling and 4 post-filling events. During CMG events, the bladder phantom is pressurized multiple times at different clinically relevant levels (small, medium, and large) to simulate bladder pressures. Simultaneous with pressurization, MB subharmonic signal was acquired. For each event, the change in MB subharmonic amplitude was correlated linearly with the change in bladder phantom pressure, and the SHAPE conversion factor (slope of the linear fit) was determined. In doing so, a specific signal processing technique (based on a small temporal window) was used to account for time-decay of MB subharmonic signal during a simulated CMG. A strong inverse linear relationship was found to exist between SHAPE and bladder phantom pressures for each of the CMG filling and post-filling events ( r2> 0.9, root mean square error < 0.3 dB, standard error <0.01 dB, and P < 0.001). SHAPE showed a transient behavior in measuring bladder phantom pressure. The SHAPE conversion factor (in dB/cm H 2 O) varied between filling and post-filling events, as well as by post-filling time. The magnitude of the SHAPE conversion factor tended to increase immediately after filling and then decreases with time. Microbubble subharmonic emission is an excellent indicator of bladder phantom pressure variation. The strong correlation between SHAPE signal and bladder phantom pressure is indicative of the applicability of this method in measuring bladder pressure during a CMG. Our results suggest that different SHAPE conversion factors may be needed for different events during a CMG (ie, at different time points of a CMG). These findings will help us better protocolize this method for introduction into human subjects and allow us to take the next step toward developing a catheter-free voiding CMG using SHAPE.

Aptamer-Functionalized Microbubbles Targeted to P-selectin for Ultrasound Molecular Imaging of Murine Bowel Inflammation.

Our objectives were to develop a targeted microbubble with an anti-P-selectin aptamer and assess its ability to detect bowel inflammation in two murine models of acute colitis. Lipid-shelled microbubbles were prepared using mechanical agitation. A rapid copper-free click chemistry approach (azide-DBCO) was used to conjugate the fluorescent anti-P-selectin aptamer (Fluor-P-Ap) to the microbubble surface. Bowel inflammation was chemically induced using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in both Balb/C and interleukin-10-deficient (IL-10 KO) mice. Mouse bowels were imaged using non-linear contrast mode following an i.v. bolus of 1 × 108 microbubbles. Each mouse received a bolus of aptamer-functionalized and non-targeted microbubbles. Mouse phenotypes and the presence of P-selectin were validated using histology and immunostaining, respectively. Microbubble labelling of Fluor-P-Ap was complete after 20min at 37 ̊C. We estimate approximately 300,000 Fluor-P-Ap per microbubble and confirmed fluorescence using confocal microscopy. There was a significant increase in ultrasound molecular imaging signal from both Balb/C (p = 0.003) and IL-10 KO (p = 0.02) mice with inflamed bowels using aptamer-functionalized microbubbles in comparison to non-targeted microbubbles. There was no signal in healthy mice (p = 0.4051) using either microbubble. We constructed an aptamer-functionalized microbubble specific for P-selectin using a clinically relevant azide-DBCO click reaction, which could detect bowel inflammation in vivo. Aptamers have potential as a next generation targeting agent for developing cost-efficient and clinically translatable targeted microbubbles.

Rapid Copper-free Click Conjugation to Lipid-Shelled Microbubbles for Ultrasound Molecular Imaging of Murine Bowel Inflammation.

Microbubbles are ultrasound contrast agents that can adhere to disease-related vascular biomarkers when functionalized with binding ligands such as antibodies or peptides. The biotin-streptavidin approach has predominantly been used as the microbubble labeling approach in preclinical imaging. However, due to the immunogenicity of avidin in humans, it is not suitable for clinical translation. What would aid clinical translation is a simple and effective microbubble functionalization approach that could be directly translated from animals to humans. We developed a targeted microbubble to P-selectin, a vascular inflammatory marker, labeled using a strain-promoted [3 + 2] azide-alkyne (azide-DBCO) reaction, comparing its ability to detect bowel inflammation to that of P-selectin targeted microbubbles labeled with a traditional biotin-streptavidin approach. Bowel inflammation was chemically induced using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in Balb/C mice. Each mouse received both non-targeted and P-selectin targeted microbubbles (either biotin-streptavidin or azide-DBCO). Using the biotin-streptavidin reaction, there was a significant increase in the ultrasound molecular imaging signal in inflamed mice using P-selectin targeted (2.30 ± 0.91 a.u.) compared to isotype control microbubbles (1.14 ± 0.7 a.u.) (p = 0.009). Using the azide-DBCO reaction, there was a similar increase in the ultrasound molecular imaging signal in inflamed mice (2.54 ± 0.56 a.u) compared to the isotype control (0.44 ± 0.25 a.u) (p = 0.009). There were no significant differences between the two labeling approaches between non-targeted and P-selectin targeted microbubbles. Mouse inflammatory phenotypes and expression of P-selectin were validated using histology and immunostaining. We constructed P-selectin targeted microbubbles using an azide-DBCO click reaction, which could detect bowel inflammation in vivo. This reaction generated a similar ultrasound molecular imaging signal to biotin-strepavidin-labeled microbubbles. These data show the potential of click chemistry conjugation (azide-DBCO) as a quick, cost-efficient, and clinically translatable approach for developing targeted microbubbles.

Sensitivity of the subharmonic responses from contrast microbubbles to ambient pressure

Microbubbles are well-known ultrasound contrast agents which can produce nonlinear oscillation leading to harmonic and subharmonic responses. There has been an interest in utilizing this phenomenon for noninvasive pressure estimation and its use in diagnosis of diseases such as portal hypertension. The subharmonic response of a microbubble display three different regimes with increasing excitation: occurrence, growth, and saturation. In this study, subharmonic responses of lipid-shelled microbubbles were investigated with varying ambient pressures at different excitation parameters. We observed that subharmonic sensitivity to ambient pressure varied differently in different excitation ranges. We also noted that applying overpressure could induce subharmonic generation where there was no subharmonic without overpressure. At occurrence stage, the sensitivity of subharmonic to ambient pressure was significantly higher. This property can be utilized for improved pressure estimation with high sensitivity even at low acoustic excitation.

Laboratory-Scale Production of Sterile Targeted Microbubbles.

Perfluorocarbon gas-filled microbubbles are clinically used as ultrasound contrast agents. We have been developing targeted microbubbles based BACS (buoyancy activated cell sorting) or BUBLES (buoyancy enabled separation) for ex vivo cell isolation from bloods for circulating tumor cell (CTC) detection and hematopoietic cell isolation. Recently, we further applied targeted microbubbles for multimarker cell sorting, and as artificial antigen presenting cells (aAPC) for T cell activation and expansion by taking advantage of a number of interesting properties of lipid-shelled microbubbles. This chapter will describe the process of manufacturing sterile targeted microbubbles for research applications.

Detection and Characterization of Sentinel Lymph Node by Ultrasound Molecular Imaging with B7-H3-Targeted Microbubbles in Orthotopic Breast Cancer Model in Mice.

Accurate identification and assessment of sentinel lymph node (SLN) using noninvasive imaging methods can play a vital role in tumor staging, surgical planning, and prognostic evaluation. In this study, we assessed the efficacy of B7-H3-targeted molecular-ultrasound imaging for the early SLN detection, and characterization in a mouse model of orthotopic breast cancer. We established a mouse breast cancer model with lymph node metastasis by injecting MAD-MB 231 cells which were engineered to express firefly luciferase reporter gene into the fat pad of the right 4th mammary gland in female BALB/c nude mice. The sole lymph node (LN) close to the tumor was regarded as the SLN for imaging investigation, which included metastatic and non-metastatic SLNs. The LN in the right 4th mammary gland from normal mice was used as normal control (normal mice LN). The commercially available preclinical streptavidin-coated, perfluorocarbon-containing lipid-shelled microbubbles (VisualSonics, Toronto, Canada) were used to generate B7-H3-targeted microbubbles (MBB7-H3) and control microbubbles (MBControl). Then, ultrasound molecular imaging (USMI) was performed using a high-resolution transducer (MS250; center frequency, 21MHz; Vevo 2100; VisualSonics, Toronto, Canada) after intravenous injection of microbubbles. The SLN was clearly detected and located under conventional (B-mode) and contrast-enhanced ultrasonography with microbubble injection. The metastatic SLNs showed a markedly higher signal from B7-H3-targeted microbubbles (MBB7-H3) compared to the non-metastatic SLNs and normal LNs. The metastatic SLN was further confirmed by ex vivo bioluminescence imaging and eventually verified by histological analysis. Our findings suggest the potential value of USMI using B7-H3 targeted microbubbles in breast cancer and establish an effective imaging method for the non-invasive detection and characterization of SLN.

Manufacturing Anti-CD19 CAR-Tscm Cells for Immunotherapy Using Innovative Microbubble-Based Technologies for Precision Cell Processing

INTRODUCTIONChimeric antigen receptor (CAR) T cell therapy provides a potential cure for patients who don't respond to standard treatments. One of the major challenges for current mainstream CAR-T therapy is lack of in vivo persistence of transferred cells. Intriguingly, more differentiated effector T cells that acquire enhanced in vitro anti-tumor properties were found to be paradoxically less effective for in vivo tumor treatment. It has become clear that the persistence of adoptive transferred T cells is crucial for in vivo activities. A significant linear correlation was found between the differentiation status of infused T cells and the potency of tumor regression, in the order of Tscm > Tcm >Tem. While promising, CAR-Tscm cell therapy demands technical innovation for precision cell processing and production. Here, we show CAR-Tscm cells generated by microbubble-based technologies for precision T cell sorting and activation and viral transduction are more robust than those generated by the conventional method in in vitro assays and in vivo animal study using the NSG mouse model.METHODSNaïve T cells were isolated by sequential sorting with 3 targeted lipid shell microbubbles (conjugated with anti-CD8, anti-CD45RA, and anti-CD62L). Briefly, CD8+ T cells were floated by anti-CD8 conjugated microbubbles and separated from nontargeted cells in PBMC. The floated CD8+ cells returned to solution after the microbubbles had undergone dissolution by increasing ambient pressure. These cells were subsequently floated by anti-CD45RA and anti-CD62L microbubbles sequentially, as described earlier. To generate CD19-CAR-modified Tscm-enriched cells, isolated naïve T cells were activated by anti-CD3/CD28 conjugated microbubbles and transduced by CD19-CAR retrovirus via Retronectin conjugated microbubbles. Transduced naïve T cells were cultured in media with IL-7, IL-21, and TWS119. Control CD19-CAR T cells were produced from same donors' PBMCs using standard culture conditions (soluble IL-2 and anti-CD3/CD28 coated culture flasks). Raji cells were co-cultured with effector cells (Tscm-enriched and standard CD19 CAR T cells) at a gradient ET ratio for in vitro cytotoxicity assay. Tscm-enriched and standard CD19-CAR T cells were injected into Raji-luc-bearing NSG mice. Tumor burden was measured by the XenogenIVIS system.RESULTSNaïve CD8+ T lymphocytes were enriched by sequential isolation of CD8+, CD62L+, and CD45RA+ cells from healthy donor PBMCs using respective antibody conjugated microbubbles. The CD8, CD62L, and CD45RA triple positive population was enriched from initial 10.08%± 2.4% to 71.84%±1.9% (n=4). We discovered that Retronectin conjugated microbubbles are advantageous to replace spinoculation technique to simplify viral transduction procedures. The average transduction efficiency was 27.71%± 8.7% using unconcentrated retroviral vectors. The Tscm-like cells were significantly enriched after two-week culture, compared to the standard method (37.78± 14.5% vs. 4.38± 0.7%). Tscm-enriched CD19-CAR T cells exhibited a stronger in vitro cytotoxicity towards Raji cells in comparison with standard CD19-CAR T cells (Figure 1). Consistently, in the NSG mice engrafted with Raji-Luc cancer cells, the CD19-CAR-modified Tscm-enriched cells showed longer-lasting antitumor responses than the CD19-CAR-T cells generated by the standard manufacturing process (Figure 2).CONCLUSIONWe have developed a bead-free, multi-positive selection system for CAR-Tscm production, and demonstrated that these Tscm cells effectively cause tumor regression and prolong diseased animal lifespan. A closed, automated, microbubble-based CAR-T cell manufacturing system, combining T cell isolation, activation, and transduction will be developed to improve performance and reduce costs. [Display omitted] DisclosuresFu: Diagnologix LLC: Current Employment. Shi: Diagnologix LLC: Current Employment. Liu: Diagnologix LLC: Current Employment.

Regadenoson Mycardial Contrast Echocardiography Perfusion Imaging for Detection of Allograft Coronary Vasculopathy

<h3>Purpose</h3> Due to the often asymptomatic nature of cardiac allograft vasculopathy (CAV) serial surveillance is recommended for all cardiac transplant (OHT) recipients. Ideally, CAV surveillance should be non-invasive, not involve ionizing radiation, and discriminate diffuse CAV from focal obstructions. We hypothesized that vasodilator stress myocardial contrast echocardiography (MCE) perfusion imaging can non-invasively detect microvascular impact of diffuse or focal CAV. <h3>Methods</h3> The study was a retrospective, single center analysis of OHT recipients undergoing MCE between 1/20 and 10/20 (n=11). MCE at rest and after regadenoson was performed using a continuous infusion of lipid-shelled microbubbles. Images were analyzed by evaluating time-intensity after a destructive pulse sequence in all vascular territories. MCE data were correlated with wall motion assessment by conventional stress echocardiography and invasive coronary angiography (ICA). Grading of MCE as normal, mild, severe; and focal vs. diffuse was performed by one experienced reviewer in a blinded fashion. <h3>Results</h3> Mean age was 63 ± 3.3 years post OHT, at an average of 10.8 ± 3.3 years post OHT. Severe perfusion abnormalities during MCE were able to distinguish high grade CAV (2+) from low grade CAV (0-1) utilizing ICA as the gold standard. While stress echocardiographic wall motion assessment failed to indicate early CAV grade 1, abnormal MCE perfusion was found in 3 of 7 cases with angiographic CAV grade 1. <h3>Conclusion</h3> MCE is a practical, non-invasive perfusion imaging technique that may be useful for detecting significant CAV in OHT recipients. Perfusion deficits correlate with significant coronary artery stenosis identified by standard stress echocardiography and ICA, and may identify diffuse small vessel disease more effectively than current noninvasive techniques. Larger, prospective studies are needed to support this hypothesis and ultimately provide a rationale for routine use of MCE for CAV surveillance.

Augmentation of Tissue Perfusion with Contrast Ultrasound: Influence of Three-Dimensional Beam Geometry and Conducted Vasodilation

Cavitation of microbubble contrast agents with ultrasound produces shear-mediated vasodilation and an increase in tissue perfusion. We investigated the influence of the size of the cavitation volume by comparing flow augmentation produced by two-dimensional (2D) versus three-dimensional (3D) therapeutic ultrasound. We also hypothesized that cavitation could augment flow beyond the ultrasound field through release of vasodilators that are carried downstream. In 11 rhesus macaques, cavitation of intravenously administered lipid-shelled microbubbles was performed in the proximal forearm flexor muscles unilaterally for 10min. Ultrasound cavitation (1.3MHz, 1.5MPa peak negative pressure) was performed with 2D or 3D transmission with beam elevations of 5 and 25mm, respectively, and pulsing intervals (PIs) sufficient to allow complete postdestruction refill (5 and 12sec for 2D and 3D, respectively). Contrast ultrasound perfusion imaging was performed before and after cavitation, using multiplane assessment within and beyond the cavitation field in 1.5-cm increments. Cavitation in the hindlimb of mice using 2D ultrasound at a PI of 1 or 5sec was performed to examine microvascular flow changes from cavitation in only arteries versus the microcirculation. In primates, the degree of muscle flow augmentation in the center of the cavitation field was similar for 2D and 3D conditions (five- to sixfold increase for both, P<.01 vs baseline). The spatial extent of flow augmentation was only modestly greater for 3D cavitation because of an increase in perfusion with 2D transmission that was detected outside of the cavitation field. In mice, cavitation in the microvascular compartment (PI 5sec) produced the greatest degree of flow augmentation, yet cavitation in the arterial compartment (PI 1sec) still produced a three- to fourfold increase in flow (P<.001 vs control). The mechanism for flow augmentation beyond the cavitation zone was investigated by invitro studies that demonstrated cavitation-related release of vasodilators, including adenosine triphosphate and nitric oxide, from erythrocytes and endothelial cells. Compared with 2D transmission, 3D cavitation of microbubbles generates a similar degree of muscle flow augmentation, possibly because of a trade-off between volume of cavitation and PI, and only modestly increases the spatial extent of flow augmentation because of the ability of cavitation to produce conducted effects beyond the ultrasound field.

Sonoporation generates downstream cellular impact after membrane resealing

Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1 MHz frequency, 0.50 MPa peak negative pressure; 10% duty cycle; 30 s exposure period; 29.1 J/cm2 acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24 h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24 h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12 h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3 h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.

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.

Developing a Microbubble-Based Contrast Agent for Synchrotron In-Line Phase Contrast Imaging.

X-ray phase contrast imaging generates contrast from refraction of X-rays, enhancing soft tissue contrast compared to conventional absorption-based imaging. Our goal is to develop a contrast agent for X-ray in-line phase contrast imaging (PCI) based on ultrasound microbubbles (MBs), by assessing size, shell material, and concentration. Polydisperse perfluorobutane-core lipid-shelled MBs were synthesized and size separated into five groups between 1 and 10μm. We generated two size populations of polyvinyl-alcohol (PVA)-MBs, 2-3μm and 3-4μm, whose shells were either coated or integrated with iron oxide nanoparticles (SPIONs). Microbubbles were then embedded in agar at three concentrations: 5×107, 5×106 and 5×105 MBs/ml. In-line phase contrast imaging was performed at the Canadian Light Source with filtered white beam micro-computed tomography. Phase contrast intensity was measured by both counting detectable MBs, and comparing mean pixel values (MPV) in minimum and maximum intensity projections of the overall samples. Individual lipid-MBs 6-10μm, lipid-MBs 4-6μm and PVA-MBs coated with SPIONs were detectable at each concentration. At the highest concentration, lipid-MBs 6-10μm and 4-6μm showed an overall increase in positive contrast, whereas at a moderate concentration, only lipid-MBs 6-10μm displayed an increase. Negative contrast was also observed from two largest lipid-MBs at high concentration. These data indicate that lipid-MBs larger than 4μm are candidates for PCI, and 5×106 MBs/ml may be the lowest concentration suitable for generating visible phase contrast in vivo. Identifying a suitable MB for PCI may facilitate future clinical translation.

Ultrasound targeting of microbubble-bound anti PD-L1 mAb to enhance anti-tumor effect of cisplatin in cervical cancer xenografts treatment

AimsAnti-PD-L1 monoclonal antibody (mAb)-conjugated ultrasound (US) lipid-shelled microbubbles (PD-L1-MBs) were successfully synthesized to investigate whether that PD-L1-MBs could enhance anti-tumor effect in combination therapy with cisplatin (CDDP) under ultrasound mediation. Main methodsBased on affinity between biotin and streptavidin, we prepared microbubbles conjugated with anti-PD-L1 mAb by membrane hydration and mechanical oscillation. A subcutaneous tumor model was established to test the anti-tumor effect and immunological activity of this combination therapy. Bax and Bcl-2 expression were detected by RT-qPCR and Immunohistochemistry. Cells undergoing apoptosis in tissue section were determined by TUNEL. Proliferation of splenocytes was analyzed by Flow cytometry. A cytotoxic T lymphocyte assay was performed by CTL. Expression of PD-L1 and CD8 in tissue section was examined by immunologfluorescence. Expression of IFN-γ, TNF-α, CD86 and CD80 was also detected by RT-qPCR. Key findingsWe observed that the growth of the subcutaneous tumor was significantly slower in combined group than that in the group treated with either drug or microbubbles. Moreover, higher antitumor activity was observed in the combined group than that in cisplatin alone, which could be reflected by the number of apoptotic cells in tumor tissues and over expression of bax in the combined group. This combination treatment also exhibited a better immunological activity, increasing the infiltration of CD8+ T cells and the expression of several revelant cytokines. SignificanceThe ultrasound lipid-shelled PD-L1-MBs may enhance anti-tumor effects of cisplatin by blocking the PD-L1 site and improving immune function.

Ultrasound-responsive lipid microbubbles for drug delivery: A review of preparation techniques to optimise formulation size, stability and drug loading.

Lipid-shelled microbubbles have received extensive interest to enhance ultrasound-responsive drug delivery outcomes due to their high biocompatibility. While therapeutic effectiveness of microbubbles is well established, there remain limitations in sample homogeneity, stability profile and drug loading properties which restrict these formulations from seeing widespread use in the clinical setting. In this review, we evaluate and discuss the most encouraging leads in lipid microbubble design and optimisation. We examine current applications in drug delivery for the systems and subsequently detail shell compositions and preparation strategies that improve monodispersity while retaining ultrasound responsiveness. We review how excipients and storage techniques help maximise stability and introduce different characterisation and drug loading techniques and evaluate their impact on formulation performance. The review concludes with current quality control measures in place to ensure lipid microbubbles can be reproducibly used in drug delivery.