Microbubble Shell Research Articles

Engineering Responsive Ultrasound Contrast Agents Through Crosslinked Networks on Lipid-Shelled Microbubbles.

Ultrasound imaging with contrast agents, especially with lipid-shelled microbubbles, has become a vital tool in clinical diagnostics. Efforts to adapt these agents for molecular imaging have typically focused on targeted binding. More recently, crosslinking the lipid shell to alter its mechanical properties, followed by decrosslinking upon exposure to a stimulus, has been shown as a promising approach for imaging soluble molecular targets. Nevertheless, a systematic study of the influence of crosslinker concentration and structure on the mechanical properties of microbubbles has not been undertaken. An improved understanding of the role of these parameters is necessary to more effectively design contrast agents that detect proteases, an informative class of soluble disease markers. Here, the influence of crosslinker parameters on the acoustic properties of microbubbles, developing a model of crosslinker network formation on microbubble shells that explains the experimental observations, are studied. By incorporating cleavable elements that respond to UV light or proteolysis, kinetically resolved acoustic detection of these stimuli and the relevance of crosslinker design are demonstrated. The framework established in this study can be readily adapted to other protease-cleavable units and provides a basis for the future development of responsive ultrasound contrast agents for molecular imaging of proteolytic activity.

Theoretical Study of the Effects of Nanoparticles on the Acoustic Performance of Microbubbles.

There are growing investigations on incorporating solid nanoparticles (NPs) into the shell of microbubbles (MBs), because NPs may endow the MBs with other bio-functions, such as multimodality imaging and drug delivery. These novel MBs have been developed as hybrid MBs' contrast agents. Generally, the shell density of hybrid MBs was assumed to be the same as water in the studies of bubble dynamics. In fact, the NPs in the layer of MBs can change the density of the shell, which leads to the change of scattering characteristics of MBs under ultrasonic excitation. Thus, it is necessary to develop a new model to simulate dynamics of the hybrid MBs. Here, we have investigated scattering characteristics of the hybrid MB embedded with NPs based on a modified Rayleigh-Plesset model. The numerical and analytical solutions to this equation are obtained for oscillation response, harmonic components, and scattered cross section of hybrid MBs at small-amplitude oscillations. The results indicated that the shell density had a greater impact on the nonlinear harmonics than fundamental ones. Considering acoustic driving frequency and pulse lengths, the largest ultraharmonic amplitude is 14 times larger than the smallest value. Considering the effects of bubble equilibrium radius, the second scattering cross section of hybrid MBs increased first and then decreased with increasing bubble equilibrium radius. Therefore, the optimal values of shell density for hybrid MBs can be predicted to obtain higher scattered signals. This also offers more accurate assessment of scattering characteristics for hybrid MB contrast agents.

Strategies to Maximize Anthracycline Drug Loading in Albumin Microbubbles.

Human serum albumin (HSA) microbubbles (MBs) are attracting increasing attention as image-guided and stimuli-responsive drug delivery systems. To better understand and maximize drug encapsulation in HSA MBs, we investigated the impact of the loading strategy and the drugs' physicochemical properties on their entrapment in the MB shell. Regarding loading strategy, we explored preloading, i.e., incubating drugs with HSA prior to MB formation, as well as postloading, i.e., incubating drugs with preformed MB. Both strategies were utilized to encapsulate six anthracyclines with different physicochemical properties. We demonstrate that drug loading in the HSA MB shell profits from preloading as well as from employing drugs with high intrinsic HSA binding affinity. These findings exemplify the potential of exploiting the natural bioconjugation interactions between drugs and HSA to formulate optimally loaded MBs, and they promote the development of HSA MBs for ultrasound-triggered drug delivery.

The Impact of Surface Drug Distribution on the Acoustic Behavior of DOX-Loaded Microbubbles.

Previous studies have reported substantial improvement of microbubble (MB)-mediated drug delivery with ultrasound when drugs are loaded onto the MB shell compared with a physical mixture. However, drug loading may affect shell properties that determine the acoustic responsiveness of MBs, producing unpredictable outcomes. The aim of this study is to reveal how the surface loaded drug (doxorubicin, DOX) affects the acoustic properties of MBs. A suitable formulation of MBs for DOX loading was first identified by regulating the proportion of two lipid materials (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol sodium salt (DSPG)) with distinct electrostatic properties. We found that the DOX loading capacity of MBs was determined by the proportion of DSPG, since there was an electrostatic interaction with DOX. The DOX payload reduced the lipid fluidity of MBs, although this effect was dependent on the spatial uniformity of DOX on the MB shell surface. Loading DOX onto MBs enhanced acoustic stability 1.5-fold, decreased the resonance frequency from 12–14 MHz to 5–7 MHz, and reduced stable cavitation dose by 1.5-fold, but did not affect the stable cavitation threshold (300 kPa). Our study demonstrated that the DOX reduces lipid fluidity and decreases the elasticity of the MB shell, thereby influencing the acoustic properties of MBs.

Investigating CXCR4 expression of tumor cells and the vascular compartment: A multimodal approach.

The C-X-C chemokine receptor 4 (CXCR4) is G protein-coupled receptor that upon binding to its cognate ligand, can lead to tumor progression. Several CXCR4-targeted therapies are currently under investigation, and with it comes the need for imaging agents capable of accurate depiction of CXCR4 for therapeutic stratification and monitoring. PET agents enjoy the most success, but more cost-effective and radiation-free approaches such as ultrasound (US) imaging could represent an attractive alternative. In this work, we developed a targeted microbubble (MB) for imaging of vascular CXCR4 expression in cancer. A CXCR4-targeted MB was developed through incorporation of the T140 peptide into the MB shell. Binding properties of the T140-MB and control, non-targeted MB (NT-MB) were evaluated in MDA-MB-231 cells where CXCR4 expression was knocked-down (via shRNA) through optical imaging, and in the lymphoma tumor models U2932 and SuDHL8 (high and low CXCR4 expression, respectively) by US imaging. PET imaging of [18F]MCFB, a tumor-penetrating CXCR4-targeted small molecule, was used to provide whole-tumor CXCR4 readouts. CXCR4 expression and microvessel density were performed by immunohistochemistry analysis and western blot. T140-MB were formed with similar properties to NT-MB and accumulated sensitively and specifically in cells according to their CXCR4 expression. In NOD SCID mice, T140-MB persisted longer in tumors than NT-MB, indicative of target interaction, but showed no difference between U2932 and SuDHL8. In contrast, PET imaging with [18F]MCFB showed a marked difference in tumor uptake at 40–60 min post-injection between the two tumor models (p<0.05). Ex vivo analysis revealed that the large differences in CXCR4 expression between the two models are not reflected in the vascular compartment, where the MB are restricted; in fact, microvessel density and CXCR4 expression in the vasculature was comparable between U2932 and SuDHL8 tumors. In conclusion, we successfully developed a T140-MB that can be used for imaging CXCR4 expression in the tumor vasculature.

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.

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0–12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.

Fluorocarbon-assisted nanoparticle-decorated microbubble design

In addition to the use in cardiovascular diagnostic procedures, microbubbles (MBs) have potential in diagnosis of tumours, vascular and blood flow abnormalities, as well as for targeted, ultrasound-triggered drug delivery and as intravascular mechanical devices. All commercial soft, phospholipid-shelled MBs contain a fluorocarbon (FC) gas. Both experimental data and theoretical models confirm that the cohesion pressure in phospholipid monolayers is decreased by FCs, supporting a strong shell film fluidizing effect. The co-surfactant activity of FC gases towards MB shell components or therapeutic cargos, including polymers, proteins, fluorinated drugs, biomarkers and tracers, will be discussed. A new approach allowed immobilization of fluorinated tracers in phospholipid-shelled MBs. Perfluorohexane allowed preparation of stable spherical hydrophobin HFBII-coated MBs, while only inadequate mixtures of aggregates and elongated MBs are obtained under air. Nanodiamonds, iron oxide and cerium oxide nanoparticles can be driven to the air/water interface when exposed to a FC vapor. This new phenomenon enables production of stable nanoparticle-shelled MBs without need for a surfactant. Such microbubbles have potential as multimodal contrast agents (MRI, optical, photoacoustics, PET/ultrasound), as image-guided delivery systems for ultrasound-mediated cancer treatment, and for cell labelling and imaging. (1) M. P. Krafft and J. G. Riess, Adv. Colloid Interface Sci. 294, 102407 (2021).

Enhancing In Vitro Stability of Albumin Microbubbles Produced Using Microfluidic T-Junction Device.

Microfluidics is an efficient technique for continuous synthesis of monodispersed microbubbles. However, microbubbles produced using microfluidic devices possess lower stability due to quick dissolution of core gas when exposed to an aqueous environment. This work aims at generating highly stable monodispersed albumin microbubbles using microfluidic T-junction devices. Microbubble generation was facilitated by an aqueous phase consisting of bovine serum albumin (BSA) as a model protein and nitrogen (N2) gas. Microbubbles were chemically cross-linked using dilute glutaraldehyde (0.75% v/v) solution and thermally cross-linked by collecting microbubbles in hot water maintained at 368 (±2) K. These microbubbles were then subjected to in vitro dissolution in an air-saturated water. Microbubbles cross-linked with a combined treatment of thermal and chemical cross-linking (TC & CC) had longer dissolution time compared to microbubbles chemically cross-linked (CC) alone, thermally cross-linked (TC) alone, and non-cross-linked microbubbles. Circular dichroism (CD) spectroscopy analysis revealed that percent reduction in alpha-helices of BSA was higher for the combined treatment of TC & CC when compared to other treatments. In contrast to non-cross-linked microbubbles where microbubble shell dissolved completely, a significant shell detachment was observed during the final phase of the dissolution for cross-linked microbubbles captured using high speed camera, depending upon the extent of cross-linking of the microbubble shell. SEM micrographs of the microbubble shell revealed the shell thickness of microbubbles treated with TC & CC to be highest compared to only thermally or only chemically cross-linked microbubbles. Comparison of microbubble dissolution data to a mass transfer model showed that shell resistance to gas permeation was highest for microbubbles subjected to a combined treatment of TC & CC.

Experimental and numerical evidence of intensified non-linearity at the microscale: The lipid coated acoustic bubble

A lipid coated bubble (LCB) oscillator is a very interesting non-smooth oscillator with many important applications ranging from industry and chemistry to medicine. However, due to the complex behavior of the coating intermixed with the nonlinear behavior of the bubble itself, the dynamics of the LCB are not well understood. In this work, lipid coated Definity® microbubbles (MBs) were sonicated with 25 MHz 30 cycle pulses with pressure amplitudes between 70 and 300 kPa. Here, we report higher order subharmonics in the scattered signals of single MBs at low-amplitude high-frequency ultrasound excitations. Experimental observations reveal the generation of period 2, period 3, and two different period 4 oscillations at low excitation amplitudes. Despite the reduced damping of the uncoated bubble system, such enhanced nonlinear oscillations have not been observed and cannot be theoretically explained for the uncoated bubble. To investigate the mechanism of the enhanced non-linearity, the bifurcation structure of the lipid coated MBs is studied for a wide range of MBs sizes and shell parameters. Consistent with the experimental results, we show that this unique oscillator can exhibit chaotic oscillations and higher order subharmonics at excitation amplitudes considerably below those predicted by the uncoated oscillator. Buckling or rupture of the shell and the dynamic variation of the shell elasticity cause the intensified non-linearity at low excitation pressure amplitudes. The simulated scattered pressure by single MBs is in good agreement with the experimental signals.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Multivariable Dependence of Acoustic Contrast of Fluorocarbon and Xenon Microbubbles under Flow

Microbubbles (MBs) are 1 to 10 µm gas particles stabilized by an amphiphilic shell capable of responding to biomedical ultrasound with strong acoustic signals, allowing them to be commonly used in ultrasound imaging and therapy. The composition of both the shell and the core determines their stability and acoustic properties. While there has been extensive characterization of the dissolution, oscillation, cavitation, collapse and therefore, ultrasound contrast of MBs under static conditions, few reports have examined such behavior under hydrodynamic flow. In this study, we evaluate the interplay of ultrasound parameters (five different mechanical indices [MIs]), MB shell parameter (shell stiffness), type of gas (perfluorocarbon for diagnostic imaging and xenon as a therapeutic gas), and a flow parameter (flow rate) on the ultrasound signal of phospholipid-stabilized MBs flowing through a latex tube embedded in a tissue-mimicking phantom. We find that the contrast gradient (CG), a metric of the rate of decay of contrast along the length of the tube, and the contrast peak (CP), the location where the maximum contrast is reached, depend on the conditions of flow, imaging, and MB material. For instance, while the contrast near the flow inlet of the field of view is highest for a softer shell (dipalmitoylphosphatidylcholine [DPPC], C16) than for stiffer shells (distearoylphosphatidylcholine [DSPC], C18, and dibehenoylphosphatidylcholine [DBPC], C22), the contrast decay is also faster; stiffer shells provide more resistance and hence lead to slower MB dissolution/destruction. At higher flow rates, the CG is low for a fixed length of time because each MB is exposed to ultrasound for a shorter period. The CG becomes high for low flow rates, especially at high incident pressures (high MI), causing more MB destruction closer to the inlet of the field of view. Also, the CP shifts toward the inlet at low flow rates, high MIs, and low shell stiffness. We also report the first demonstration of sustained ultrasound flow imaging of a water-soluble, therapeutic gas MB (xenon). We find that an increased MB concentration is necessary for obtaining the same signal magnitude for xenon MBs. In summary, this study builds a framework depicting how multiple variables simultaneously affect the evolution of MB ultrasound contrast under flow. Depending on the MB composition, imaging conditions, transducer positioning, and image processing, building on such a framework could potentially allow for extraction of additional diagnostic information than is commonly analyzed for physiological flow.

Gemcitabine-loaded microbubble system for ultrasound imaging and therapy

Ultrasound imaging presents many positive attributes, including safety, real-time imaging, universal accessibility, and cost. However, inherent difficulties in discrimination between soft tissues and tumors prompted development of stabilized microbubble contrast agents. This presents the opportunity to develop agents in which drug is entrapped in the microbubble shell. We describe preparation and characterization of theranostic poly(lactide) (PLA) and pegylated PLA (PEG-PLA) shelled microbubbles that entrap gemcitabine, a commonly used drug for pancreatic cancer (PDAC). Entrapping 6 wt% gemcitabine did not significantly affect drug activity, microbubble morphology, or ultrasound contrast activity compared with unmodified microbubbles. In vitro microbubble concentrations yielding ≥ 500nM entrapped gemcitabine were needed for complete cell death in MIA PaCa-2 PDAC drug sensitivity assays, compared with 62.5 nM free gemcitabine. In vivo administration of gemcitabine-loaded microbubbles to xenograft MIA PaCa-2 PDAC tumors in athymic mice was well tolerated and provided substantial tumoral image enhancement before and after destructive ultrasound pulses. However, no significant differences in tumor growth were observed among treatment groups, in keeping with the in vitro observation that much higher doses of gemcitabine are required to mirror free gemcitabine activity. Statement of significanceThe preliminary results shown here are encouraging and support further investigation into increased gemcitabine loading. Encapsulation of gemcitabine within polylactic acid (PLA) microbubbles does not damage its activity towards pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) cells. Excellent imaging and evidence of penetration into the highly desmoplastic PDAC tumors is demonstrated. Microbubble destruction was confirmed in vivo, showing that elevated mechanical index shatters the microbubbles for enhanced delivery. The potential to slow PDAC growth in vivo is shown, but higher gemcitabine concentrations are required. Current efforts are directed at increasing drug loading by inclusion of drug-carrying nanoparticles for effective in vivo treatment.

Expert Consensus Statement from the American Society of Echocardiography on Hypersensitivity Reactions to Ultrasound Enhancing Agents in Patients with Allergy to Polyethylene Glycol

Expert Consensus Statement from the American Society of Echocardiography on Hypersensitivity Reactions to Ultrasound Enhancing Agents in Patients with Allergy to Polyethylene Glycol

Structural Coloration by Internal Reflection and Interference in Hydrogel Microbubbles and Their Precursors

AbstractCompound microbubbles with engineered shells, fabricated by microfluidic techniques, have drawn considerable interest for interdisciplinary research, including ultrasound imaging agents, chemical micromotors, and optical microcavities. Meanwhile, though vastly investigated, dynamic shell thickness variations of microbubbles can be hardly calculated via conventional techniques. Here the fabrication of colorful microbubbles encapsulated in hydrogel precursor using glass‐capillary microfluidic methods is demonstrated. The proposed optical geometrical model elucidating the structural coloration is used for thickness assessment. Thin‐film interference occurring upon illumination of the microbubble shell is found to be responsible for the coloration. The interfered lights travel along with the shell interfaces through total internal reflection in microbubbles with buoyancy‐induced asymmetric shell. A concise thickness evaluation methodology provided by the established model predicts the thickness evolution of microbubbles. These results demonstrate tunable optical properties of shin‐shell compound microbubbles for potential applications in displays, sensing, and anti‐counterfeiting materials.

Contrast-enhanced ultrasound molecular imaging of activated platelets in the progression of atherosclerosis using microbubbles bearing the von Willebrand factor A1 domain.

Platelet-endothelial interactions have been linked to increased inflammatory activation and a prothrombotic state in atherosclerosis. The interaction between von Willebrand factor (vWF)-A1 domain and platelet glycoprotein (GP) Ib/IX plays a significant role in mediating the adhesion of platelets to the injured endothelium. In the present study, contrast-enhanced ultrasound (CEU) molecular imaging with microbubbles bearing the vWF-A1 domain was performed to non-invasively monitor activated platelets on the vascular endothelium in the procession of atherosclerosis. A targeted CEU contrast agent was prepared by attaching the vWF-A1 domain to the shell of microbubbles (MbA1). Rat isotype control antibody was used to produce control (Mbctrl) microbubbles. The binding of MbA1 and Mbctrl to activated platelets was assessed in in vitro flow chamber experiments. Apolipoprotein E (ApoE-/-) deficient mice were studied as a model of atherosclerosis. At 8, 16 and 32 weeks of age, CEU molecular imaging of the proximal aorta with MbA1 and Mbctrl was performed and the imaging signals from microbubbles were quantified. Atherosclerotic lesion severity and platelets on the endothelial surface were assessed by histology and immunohistochemistry. In in vitro flow chamber studies, attachment of MbA1 to activated platelets on culture dishes was significantly greater than that of Mbctrl across a range of shear stresses (P<0.05). The attachment of Mbctrl was sparse and not related to the aggregated platelets. As lesion development progressed in the ApoE-/- mice, molecular imaging of activated platelets demonstrated selective signal enhancement of MbA1 (P<0.05 vs. Mbctrl) at all ages. Selective signal enhancement from MbA1 increased from 8 to 32 weeks of age. Immunohistochemistry for GPIIb revealed the presence of platelets on the endothelial cell surface in each group of ApoE-/- mice and that the degree of platelet deposits was age-dependent. The results of the present study indicated that non-invasive CEU molecular imaging with targeted microbubbles bearing the vWF-A1 domain could not only detect activated platelets on the vascular endothelium but also indicate lesion severity in atherosclerosis.

Fluorescence intensity changes depending on viscoelasticity of lipid shell coating microbubbles labeled with an indocyanine green derivative

Lipid-coated microbubbles (MBs) with an indocyanine green (ICG) derivative were fabricated for ultrasound and near-infrared (NIR) fluorescence dual imaging. We characterized the NIR-fluorescence intensity, stability and viscoelastic properties of the encapsulating lipid shell, focusing on the influence of the ICG derivative and lipid compositions. In terms of the NIR fluorescence intensity, the fluorescence intensity of the MBs (with the ICG derivative) was significantly affected by the lipid composition of the MB shell. Regarding the contrast agent used for ultrasound imaging, the stability of the MBs and viscoelastic properties of shell also depended on the lipid compositions, while the incorporation of the ICG derivative into the MB shells had a negligible effect. The performance of this contrast agent for ultrasound and NIR fluorescence dual-imaging exhibited a significant trade-off relationship for the lipid composition.

Free-Radical Generation from Bulk Nanobubbles in Aqueous Electrolyte Solutions: ESR Spin-Trap Observation of Microbubble-Treated Water.

Microbubbles are very fine bubbles that shrink and collapse underwater within several minutes, leading to the generation of free radicals. Electron spin resonance spectroscopy (ESR) confirmed the generation of hydroxyl radicals under strongly acidic conditions. The drastic environmental change caused by the collapse of the microbubbles may trigger radical generation via the dispersion of the elevated chemical potential that had accumulated around the gas-water interface. The present study also confirmed the generation of ESR signals from the microbubble-treated waters even after several months had elapsed following the dispersion of the microbubbles. Bulk nanobubbles were expected to be the source of the spin-adducts of hydroxyl radicals. Such microbubble stabilization and conversion might be caused by the formation of solid microbubble shells generated by iron ions in the condensed ionic cloud around the microbubble. Therefore, the addition of a strong acid might cause drastic changes in the environment and destroy the stabilized condition. This would restart the collapsing process, leading to hydroxyl radical generation.

First quantitative assessment of the adsorption of a fluorocarbon gas on phospholipid monolayers at the air/water interface

HypothesisFluorocarbon gases introduced above monolayers of phospholipids at the air/water interface were recently found to promote the adsorption of diverse molecular compounds, with potential application in drug-loaded microbubble design. Quantitative determination of the fluorocarbon present in the monolayers is strongly needed for the development of such applications. We hypothesized that neutron reflectometry (NR) and ellipsometry experiments would allow quantification of the fluorocarbon trapped in the monolayers. ExperimentsWe report the first quantitative determination of the extents of adsorption of perfluorohexane (F-hexane) on different phospholipid monolayers with respect to both their phase and isotopic form. To this aim, we applied an approach based on co-modeling the data obtained from NR and ellipsometry. FindingsWe found that F-hexane adsorbs strongly in monolayers of dipalmitoylphosphatidylcholine (DPPC) when they are both in the liquid expanded (LE) and liquid condensed (LC) phases, but to different extents according to the isotopic form of the phospholipid. Kinetic resolution of the interfacial composition from data on both isotopic contrasts (assuming chemical identicality) was therefore not possible using NR alone, so an alternative NR/ellipsometry co-modeling treatment was applied to data from each isotopic contrast. F-hexane adsorbs more abundantly on monolayers of hydrogenous DPPC than chain-deuterated DPPC when they are in the LE phase, whilst the opposite was observed when they monolayers are in the LC phase. The extents of adsorption of F-hexane in monolayers of dimyristoylphosphatidylcholine (DMPC, LE phase) and distearoylphosphatidylcholine (DSPC, LC phase) concurs with the strong dependence of those with phospholipids of different isotopic contrasts according to the monolayer phase. This new methodology can lead to advances in the novel characterization of fluorocarbons interacting with phospholipid monolayers of relevance to applications such as in the shells of fluorocarbon-stabilized medically-oriented microbubbles.