Experimental and Computational Investigation of Microbubble Formation in a Single Capillary Embedded T-junction Microfluidic Device.

In recent years, there has been a notable increase in the interest toward microfluidic devices for microbubble synthesis. The upsurge can be primarily attributed to the exceptional control these devices offer in terms of both the size and the size distribution of microbubbles. Among various microfluidic devices available, capillary-embedded T-junction microfluidic (CETM) devices have been extensively used for the synthesis of microbubbles. One distinguishing feature of CETM devices from conventional T-junction devices is the existence of a wall at the right-most end, which causes a backflow of the continuous phase at the mixing zone during microbubble formation. The back flow at the mixing zone can have several implications during microbubble formation. It can possibly affect the local velocity and shearing force at the mixing zone, which in turn can affect the size and production rate of the microbubbles. Therefore, in this work, we experimentally and computationally understand the process of microbubble formation in CETM devices. The process is modeled using computational fluid dynamics (CFD) with the volume-of-fluid approach, which solves the Navier-Stokes equations for both the gas and liquid phases. Three scenarios with a constant liquid velocity of 0.053 m/s with varying gas velocity and three with a constant gas velocity of 0.049 m/s at different liquid velocities were explored. Increase in the liquid and gas velocity during microbubble formation was found to enhance production rates in both experiments and simulations. Additionally, the change in microbubble size with the change in liquid velocity was found to agree closely with the findings of the simulation with a coefficient of variation of 10%. When plotted against the time required for microbubble generation, the fluctuations in the pressure showed recurrent crests and troughs throughout the microbubble formation process. The understanding of microbubble formation in CETM devices in the presence of backflow will allow improvement in size reduction of microbubbles.

Effect of temperature on the acoustic response and stability of size-isolated protein-shelled ultrasound contrast agents and SonoVue.

Limited work has been reported on the acoustic and physical characterization of protein-shelled UCAs. This study characterized bovine serum albumin (BSA)-shelled microbubbles filled with perfluorobutane gas, along with SonoVue, a clinically approved contrast agent. Broadband attenuation spectroscopy was performed at room (23 ± 0.5 °C) and physiological (37 ± 0.5 °C) temperatures over the period of 20 min for these agents. Three size distributions of BSA-shelled microbubbles, with mean sizes of 1.86 µm (BSA1), 3.54 µm (BSA2), and 4.24 µm (BSA3) used. Viscous and elastic coefficients for the microbubble shell were assessed by fitting de Jong model to the measured attenuation spectra. Stable cavitation thresholds (SCT) and inertial cavitation thresholds (ICT) were assessed at room and physiological temperatures. At 37 °C, a shift in resonance frequency was observed, and the attenuation coefficient was increased relative to the measurement at room temperature. At physiological temperature, SCT and ICT were lower than the room temperature measurement. The ICT was observed to be higher than SCT at both temperatures. These results enhance our understanding of temperature-dependent properties of protein-shelled UCAs. These findings study may guide the rational design of protein-shelled microbubbles and help choose suitable acoustic parameters for applications in imaging and therapy.

Combining Ultrasound and Capillary-Embedded T-Junction Microfluidic Devices to Scale Up the Production of Narrow-Sized Microbubbles through Acoustic Fragmentation.

Microbubbles are tiny gas-filled bubbles that have a variety of applications in ultrasound imaging and therapeutic drug delivery. Microbubbles can be synthesized using a number of techniques including sonication, amalgamation, and saline shaking. These approaches can produce highly concentrated microbubble suspensions but offer minimal control over the size and polydispersity of the microbubbles. One of the simplest and effective methods for producing monodisperse microbubbles is capillary-embedded T-junction microfluidic devices, which offer great control over the microbubble size. However, lower production rates (∼200 bubbles/s) and large microbubble sizes (∼300 µm) limit the applicability of such devices for biomedical applications. To overcome the limitations of these technologies, we demonstrate in this work an alternative approach to combine a capillary-embedded T-junction device with ultrasound to enhance the generation of narrow-sized microbubbles in aqueous suspensions. Two T-junction microfluidic devices were connected in parallel and combined with an ultrasonic horn to produce lipid-coated SF6 core microbubbles in the size range of 1-8 µm. The rate of microbubble production was found to increase from 180 microbubbles/s in the absence of ultrasound to (6.5 ± 1.2) × 106 bubble/s in the presence of ultrasound (100% ultrasound amplitude). When stored in a closed environment, the microbubbles were observed to be stable for up to 30 days, with the concentration of the microbubble suspension decreasing from ∼2.81 × 109/mL to ∼2.3 × 106/mL and the size changing from 1.73 ± 0.2 to 1.45 ± 0.3 µm at the end of 30 days. The acoustic response of these microbubbles was examined using broadband attenuation spectroscopy, and flow phantom imaging was performed to determine the ability of these microbubble suspensions to enhance the contrast relative to the surrounding tissue. Overall, this approach of coupling ultrasound with microfluidic parallelization enabled the continuous production of stable microbubbles at high production rates and low polydispersity using simple T-junction devices.

Generating Lifetime-Enhanced Microbubbles by Decorating Shells with Silicon Quantum Nano-Dots Using a 3-Series T-Junction Microfluidic Device.

Long-term stability of microbubbles is crucial to their effectiveness. Using a new microfluidic device connecting three T-junction channels of 100 µm in series, stable monodisperse SiQD-loaded bovine serum albumin (BSA) protein microbubbles down to 22.8 ± 1.4 µm in diameter were generated. Fluorescence microscopy confirmed the integration of SiQD on the microbubble surface, which retained the same morphology as those without SiQD. The microbubble diameter and stability in air were manipulated through appropriate selection of T-junction numbers, capillary diameter, liquid flow rate, and BSA and SiQD concentrations. A predictive computational model was developed from the experimental data, and the number of T-junctions was incorporated into this model as one of the variables. It was illustrated that the diameter of the monodisperse microbubbles generated can be tailored by combining up to three T-junctions in series, while the operating parameters were kept constant. Computational modeling of microbubble diameter and stability agreed with experimental data. The lifetime of microbubbles increased with increasing T-junction number and higher concentrations of BSA and SiQD. The present research sheds light on a potential new route employing SiQD and triple T-junctions to form stable, monodisperse, multi-layered, and well-characterized protein and quantum dot-loaded protein microbubbles with enhanced stability for the first time.

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.

Effectiveness of Oil-Layered Albumin Microbubbles Produced Using Microfluidic T-Junctions in Series for In Vitro Inhibition of Tumor Cells.

This work focuses on the synthesis of oil-layered microbubbles using two microfluidic T-junctions in series and evaluation of the effectiveness of these microbubbles loaded with doxorubicin and curcumin for cell invasion arrest from 3D spheroid models of triple negative breast cancer (TNBC), MDA-MB-231 cell line. Albumin microbubbles coated in the drug-laden oil layer were synthesized using a new method of connecting two microfluidic T-mixers in series. Double-layered microbubbles thus produced consist of an innermost core of nitrogen gas encapsulated in an aqueous layer of bovine serum albumin (BSA) which in turn, is coated with an outer layer of silicone oil. In order to identify the process conditions leading to the formation of double-layered microbubbles, a regime map was constructed based on capillary numbers for aqueous and oil phases. The microbubble formation regime transitions from double-layered to single layer microbubbles and then to formation of single oil droplets upon gradual change in flow rates of aqueous and oil phases. In vitro dissolution studies of double-layered microbubbles in an air-saturated environment indicated that a complete dissolution of such bubbles produces an oil droplet devoid of a gas bubble. Incorporation of doxorubicin and curcumin was found to produce a synergistic effect, which resulted in higher cell deaths in 2D monolayers of TNBC cells and inhibition of cell proliferation from 3D spheroid models of TNBC cells compared to the control.

Kinetics of albumin microbubble dissolution in aqueous media.

The effectiveness of microbubbles as ultrasound contrast agents and targeted drug delivery vehicles depends on their persistence in blood. It is therefore necessary to understand the dissolution behavior of microbubbles in an aqueous medium. While there are several reports available in the literature on the dissolution of lipid microbubbles, there are no reports available on the dissolution kinetics of protein microbubbles. Moreover, shell parameters such as interfacial tension, shell resistance and shell elasticity/stiffness which characterize microbubble shells, have been reported for lipid shells but no such data are available for protein shells. Accordingly, this work was focused on capturing the dissolution behavior of protein microbubbles and estimation of shell parameters such as surface tension, shell resistance and shell elasticity. Bovine serum albumin (BSA) was used as a model protein and microbubbles were synthesized using sonication. During dissolution, a large portion of the protein shell was found to disengage from the gas-liquid interface after a stagnant dissolution phase, leading to a sudden disappearance of the microbubbles due to complete dissolution. In order to estimate shell parameters, microbubble dissolution kinetic data (radius vs. time) was fit numerically to a mass transfer model describing a microbubble dissolution process. Analysis of the results shows that the interfacial tension increases drastically and the shell resistance reduces significantly, as protein molecules leave the gas-liquid interface. Furthermore, the effect of processing conditions such as preheating temperature, microbubble size, and core gas and shell composition on the protein shell parameters was also evaluated.