Acoustic response and ambient pressure sensitivity characterization of SonoVue for noninvasive pressure estimation.

Subharmonic aided pressure estimation (SHAPE) is a noninvasive pressure measurement technique based on the pressure dependent subharmonic signal from contrast microbubbles. Here, SonoVue microbubble with a sulfur hexafluoride (SF6) core, was investigated for use in SHAPE. The study uses excitations of 25-700 kPa peak negative pressure (PNP) and 3 MHz frequency over eight pressurization cycles between atmospheric pressure and overpressures, ranging from 0 to 25 kPa (0 to 186 mm Hg). The SonoVue subharmonic response was characterized into two types. Unlike other microbubbles, SonoVue showed significant subharmonic signals at low excitations (PNPs, 25-400 kPa), denoted here as type I subharmonic. It linearly decreased with increasing overpressure (-0.52 dB/kPa at 100 kPa PNP). However, over multiple pressurization-depressurization cycles, type I subharmonic changed; its value at atmospheric pressure decreased over multiple cycles, and at later cycles, it recorded an increase in amplitude with overpressure (highest, +13 dB at 50 kPa PNP and 10 kPa overpressure). The subharmonic at higher excitations (PNP > 400 kPa), denoted here as type II subharmonic, showed a consistent decrease with the ambient pressure increase with strongest sensitivity of -0.4 dB/kPa at 500 kPa PNP.

Optimizing the accuracy of viscoelastic characterization with AFM force-distance experiments in the time and frequency domains.

Atomic Force Microscopy (AFM) force-distance (FD) experiments have emerged as an attractive alternative to traditional micro-rheology measurement techniques owing to their versatility of use in materials of a wide range of mechanical properties. Here, we show that the range of time dependent behaviour which can reliably be resolved from the typical method of FD inversion (fitting constitutive FD relations to FD data) is inherently restricted by the experimental parameters: sampling frequency, experiment length, and strain rate. Specifically, we demonstrate that violating these restrictions can result in errors in the values of the parameters of the complex modulus. In the case of complex materials, such as cells, whose behaviour is not specifically understood a priori, the physical sensibility of these parameters cannot be assessed and may lead to falsely attributing a physical phenomenon to an artifact of the violation of these restrictions. We use arguments from information theory to understand the nature of these inconsistencies as well as devise limits on the range of mechanical parameters which can be reliably obtained from FD experiments. The results further demonstrate that the nature of these restrictions depends on the domain (time or frequency) used in the inversion process, with the time domain being far more restrictive than the frequency domain. Finally, we demonstrate how to use these restrictions to better design FD experiments to target specific timescales of a material's behaviour through our analysis of a polydimethylsiloxane (PDMS) polymer sample.

Improved Sensitivity of Ultrasound-Based Subharmonic Aided Pressure Estimation Using Monodisperse Microbubbles.

OBJECTIVES: Subharmonic aided pressure estimation (SHAPE) has been shown effective for noninvasively measuring hydrostatic fluid pressures in a variety of clinical applications. The objective of this study was to explore potential improvements in SHAPE sensitivity using monodisperse microbubbles. METHODS: Populations of monodisperse microbubbles were created using a commercially available microfluidics device (Solstice Pharmaceuticals). Size distributions were assessed using a Coulter Counter and stability of the distribution following fabrication was evaluated over 24 hours. Attenuation of the microbubble populations from 1 to 10 MHz was then quantified using single element transducers to identify each formulation's resonance frequency. Frequency spectra over increasing driving amplitudes were investigated to determine the nonlinear phases of subharmonic signal generation. SHAPE sensitivity was evaluated in a hydrostatic pressure-controlled water bath using a Logiq E10 scanner (GE Healthcare). RESULTS: Monodisperse lipid microbubble suspensions ranging from 2.4 to 5.3 µm in diameter were successfully created and they showed no discernable change in size distribution over 24 hours following activation. Calculated resonance frequencies ranged from 2.1 to 6.3 MHz and showed excellent correlation with microbubble diameter (R2 > 0.99). When investigating microbubble frequency response, subharmonic signal occurrence was shown to begin at 150 kPa peak negative pressure, grow up to 225 kPa, and saturate at approximately 250 kPa. Using the Logiq E10, monodisperse bubbles demonstrated a SHAPE sensitivity of -0.17 dB/mmHg, which was nearly twice the sensitivity of the commercial polydisperse microbubble currently being used in clinical trials. CONCLUSIONS: Monodisperse microbubbles have the potential to greatly improve the sensitivity of SHAPE for the noninvasive measurement of hydrostatic pressures.

Rayleigh limit extended: Scattering from a fluid sphere.

The Reflections series takes a look back on historical articles from The Journal of the Acoustical Society of America that have had a significant impact on the science and practice of acoustics.

Shear-induced gradient diffusivity of a red blood cell suspension: effects of cell dynamics from tumbling to tank-treading.

Hydrodynamic interactions generate a diffusive motion in particulates in a shear flow, which plays seminal roles in overall particulate rheology and its microstructure. Here we investigate the shear induced diffusion in a red-blood cell (RBC) suspension using a numerical simulation resolving individual motion and deformation of RBCs. The non-spherical resting shape of RBCs gives rise to qualitatively different regimes of cell dynamics in a shear flow such as tank-treading, breathing, tumbling and swinging, depending on the cell flexibility determined by the elastic capillary number. We show that the transition from tumbling to tank-treading causes a reduction in the gradient diffusivity. The diffusivity is computed using a continuum approach from the evolution of a randomly packed cell-layer width with time as well as by the dynamic structure factor of the suspension. Both approaches, although operationally different, match and show that for intermediate capillary numbers RBCs cease tumbling accompanied by a drop in the coefficient of gradient diffusivity. A further increase of capillary number increases the diffusivity due to increased deformation. The effects of bending modulus and viscosity ratio variations are also briefly investigated. The computed shear induced diffusivity was compared with values in the literature. Apart from its effects in margination of cells in blood flow and use in medical diagnostics, the phenomenon broadly offers important insights into suspensions of deformable particles with non-spherical equilibrium shapes, which also could play a critical role in using particle flexibility for applications such as label free separation or material processing.

Inhibition of Human Breast Cancer Cell Proliferation by Low-Intensity Ultrasound Stimulation.

OBJECTIVES: Cancer is characterized by uncontrolled cell proliferation, which makes novel therapies highly desired. In this study, the effects of near-field low-intensity pulsed ultrasound (LIPUS) stimulation on T47D human breast cancer cell and healthy immortalized MCF-12A breast epithelial cell proliferation were investigated in monolayer cultures. METHODS: A customized ultrasound (US) exposure setup was used for the variation of key US parameters: intensity, excitation duration, and duty cycle. Cell proliferation was quantified by 5-bromo-2'-deoxyuridine and alamarBlue assays after LIPUS excitation. RESULTS: At a 20% duty cycle and 10-minute excitation period, we varied LIPUS intensity from to 100 mW/cm2 (spatial-average temporal-average) to find a gradual decrease in T47D cell proliferation, the decrease being strongest at 100 mW/cm2 . In contrast, healthy MCF-12A breast cells showed an increase in proliferation when exposed to the same conditions. Above a 60% duty cycle, T47D cell proliferation decreased drastically. Effects of continuous wave US stimulation were further explored by varying the intensity and excitation period. CONCLUSIONS: These experiments concluded that, irrespective of the waveform (pulsed or continuous), LIPUS stimulation could inhibit the proliferation of T47D breast cancer cells, whereas the same behavior was not observed in healthy cells. The study demonstrates the beneficial bioeffects of LIPUS on breast cancer cells and offers the possibility of developing novel US-mediated cancer therapy.

Exosomes as Drug Carriers for Cancer Therapy.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30-120 nm in size, exosomes are secreted from cells and isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity-all critical to the success of any nanoparticle drug delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid tumors.

Mechanisms of spontaneous chain formation and subsequent microstructural evolution in shear-driven strongly confined drop monolayers.

It was experimentally demonstrated by Migler and his collaborators [Phys. Rev. Lett., 2001, 86, 1023; Langmuir, 2003, 19, 8667] that a strongly confined drop monolayer sheared between two parallel plates can spontaneously develop a flow-oriented drop-chain morphology. Here we show that the formation of the chain-like microstructure is driven by far-field Hele-Shaw quadrupolar interactions between drops, and that drop spacing within chains is controlled by the effective drop repulsion associated with the existence of confinement-induced reversing streamlines, i.e., the swapping trajectory effect. Using direct numerical simulations and an accurate quasi-2D model that incorporates quadrupolar and swapping-trajectory contributions, we analyze microstructural evolution in a monodisperse drop monolayer. Consistent with experimental observations, we find that drop spacing within individual chains is usually uniform. Further analysis shows that at low area fractions all chains have the same spacing, but at higher area fractions there is a large spacing variation from chain to chain. These findings are explained in terms of uncompressed and compressed chains. At low area fractions most chains are uncompressed (spacing equals lst, which is the stable separation of an isolated pair). At higher area fractions compressed chains (with tighter spacing) are formed in a process of chain zipping along y-shaped structural defects. We also discuss the relevance of our findings to other shear-driven systems, such as suspensions of spheres in non-Newtonian fluids.

Effects of droplet size and perfluorocarbon boiling point on the frequency dependence of acoustic vaporization threshold.

Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenic microbubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are used for therapeutic and diagnostic applications. This study systematically investigated the effect of excitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds of lipid-coated PFC droplets of three different liquid cores-perfluoropentane (PFP), perfluorohexane (PFH), and perfluorooctyl bromide (PFOB)-and of two different sizes-average diameters smaller than 3 µm and larger than 10 µm-in a tubeless setup. This study found that the ADV threshold increases with frequency for the lowest boiling point liquid, PFP, for both large and small size droplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization for small size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The large PFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFH droplets, ADV threshold decreases with frequency that could possibly be due to the superharmonic focusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresholds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholds indicating that phase transition precedes inertial cavitation.

Tissue-Penetrating, Hypoxia-Responsive Echogenic Polymersomes For Drug Delivery To Solid Tumors.

Hypoxia in solid tumors facilitates the progression of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anticancer drugs to solid tumors. The polymersomes are composed of a hypoxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In-vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying pore geometries (square and hexagonal channels) were investigated. The scaffold with square pores resulted in higher hMSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz were found to be 100 mW/cm2 and 20% duty cycle. LIPUS stimulation increased proliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilage biomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen and total collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% for scaffolds with square and hexagonal patterns, respectively, after 2 weeks. Additionally, type II collagen production increased by 60% and 40% for the same patterns, respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUS stimulation, which has already been approved by FDA for treatment of bone fracture, could be a highly efficient tool for tissue engineering in combination with 3D printing and hMSCs to regenerate damaged cartilage tissues.

Nucleus-Targeted, Echogenic Polymersomes for Delivering a Cancer Stemness Inhibitor to Pancreatic Cancer Cells.

Chemotherapeutic agents for treating cancers show considerable side effects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport and subsequently release the encapsulated anticancer drugs within the nuclei of pancreatic cancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3-polyethylene glycol (PEG)-polylactic acid (PLA) copolymer employing the Cu2+ catalyzed "Click" reaction. We prepared polymersomes from the dexamethasone-PEG-PLA conjugate along with a synthesized stimuli-responsive polymer PEG-S-S-PLA. The dexamethasone group dilates the nuclear pore complexes and transports the vesicles to the nuclei. We designed the polymersomes to release the encapsulated drugs in the presence of a high concentration of reducing agents in the nuclei of pancreatic cancer cells. We observed that the nucleus-targeted, stimuli-responsive polymersomes released 70% of encapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulated the cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608 encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. The polymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medical ultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have the potential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Acoustic vaporization threshold of lipid-coated perfluoropentane droplets.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing microbubbles as contrast agents in situ as well as higher stability and the possibility of achieving smaller sizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with a perfluoropentane (PFP) core (diameter 400-3000 nm) is acoustically measured as a function of the excitation frequency in a tubeless setup at room temperature. The changes in scattered responses-fundamental, sub-, and second harmonic-are investigated, a quantitative criterion is used to determine the ADV phenomenon, and findings are discussed. The average threshold obtained using three different scattered components increases with frequency-1.05 ± 0.28 MPa at 2.25 MHz, 1.89 ± 0.57 MPa at 5 MHz, and 2.34 ± 0.014 MPa at 10 MHz. The scattered response from vaporized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV threshold value.

4D printing of polymeric materials for tissue and organ regeneration.

Four dimensional (4D) printing is an emerging technology with great capacity for fabricating complex, stimuli-responsive 3D structures, providing great potential for tissue and organ engineering applications. Although the 4D concept was first highlighted in 2013, extensive research has rapidly developed, along with more-in-depth understanding and assertions regarding the definition of 4D. In this review, we begin by establishing the criteria of 4D printing, followed by an extensive summary of state-of-the-art technological advances in the field. Both transformation-preprogrammed 4D printing and 4D printing of shape memory polymers are intensively surveyed. Afterwards we will explore and discuss the applications of 4D printing in tissue and organ regeneration, such as developing synthetic tissues and implantable scaffolds, as well as future perspectives and conclusions.

Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics and imaging. Although multiple experiments have established their echogenicity, the underlying mechanism has remained unknown. However, freeze-drying in the presence of mannitol during ELIP preparation has proved critical to ensuring echogenicity. Here, the role of this key component in the preparation protocol was investigated by measuring scattering from freshly prepared freeze-dried aqueous solution of mannitol-and a number of other excipients commonly used in lyophilization-directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol, glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bubbles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, and xylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystalline substances, if thawed before being introduced into the scattering volume, did not produce echogenicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in a degassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. The bubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in making ELIPs echogenic.

Effects of ambient hydrostatic pressure on the material properties of the encapsulation of an ultrasound contrast microbubble.

Ultrasound contrast microbubbles experience widely varying ambient blood pressure in different organs, which can also change due to diseases. Pressure change can alter the material properties of the encapsulation of these microbubbles. Here the characteristic rheological parameters of contrast agent Definity are determined by varying the ambient pressure (in a physiologically relevant range 0-200 mm Hg). Four different interfacial rheological models are used to characterize the microbubbles. Effects of gas diffusion under excess ambient pressure are investigated in detail accounting for size decrease of contrast microbubbles. Definity contrast agent show a change in their interfacial dilatational viscosity (3.6 × 10(-8) Ns/m at 0 mm Hg to 4.45 × 10(-8) Ns/m at 200 mm Hg) and interfacial dilatational elasticity (0.86 N/m at 0 mm Hg to 1.06 N/m at 200 mm Hg) with ambient pressure increase. The increase results from material consolidation, similar to such enhancement in bulk properties under pressure. The model that accounts for enhancement in material properties with increasing ambient pressure matches with experimentally measured subharmonic response as a function of ambient pressure, while assuming constant material parameters does not.

Interpreting attenuation at different excitation amplitudes to estimate strain-dependent interfacial rheological properties of lipid-coated monodisperse microbubbles.

Broadband attenuation of ultrasound measured at different excitation pressures being different raises a serious theoretical concern, because the underlying assumption of linear and independent propagation of different frequency components nominally requires attenuation to be independent of excitation. Here, this issue is investigated by examining ultrasound attenuation through a monodisperse lipid-coated microbubble suspension measured at four different acoustic excitation amplitudes. The attenuation data are used to determine interfacial rheological properties (surface tension, surface dilatational elasticity, and surface dilatational viscosity) of the encapsulation according to three different models. Although different models result in similar rheological properties, attenuation measured at different excitation levels (4-110 kPa) leads to different values for them; the dilatation elasticity (0.56 to 0.18 N/m) and viscosity (2.4 × 10(-8) to 1.52 × 10(-8) Ns/m) both decrease with increasing pressure. Numerically simulating the scattered response, nonlinear energy transfer between frequencies are shown to be negligible, thereby demonstrating the linearity in propagation and validating the attenuation analysis. There is a second concern to the characterization arising from shell properties being dependent on excitation amplitude, which is not a proper constitutive variable. It is resolved by arriving at a strain-dependent rheology for the encapsulation. The limitations of the underlying analysis are discussed.

pH-triggered echogenicity and contents release from liposomes.

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).

Polymer-coated echogenic lipid nanoparticles with dual release triggers.

Although lipid nanoparticles are promising drug delivery vehicles, passive release of encapsulated contents at the target site is often slow. Herein, we report contents release from targeted, polymer-coated, echogenic lipid nanoparticles in the cell cytoplasm by redox trigger and simultaneously enhanced by diagnostic frequency ultrasound. The lipid nanoparticles were polymerized on the external leaflet using a disulfide cross-linker. In the presence of cytosolic concentrations of glutathione, the lipid nanoparticles released 76% of encapsulated contents. Plasma concentrations of glutathione failed to release the encapsulated contents. Application of 3 MHz ultrasound for 2 min simultaneously with the reducing agent enhanced the release to 96%. Folic acid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressing the folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be used as multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: This study indicates the possible development of novel and improved SHAPE methodologies.