Controlled Tempering of Lipid Concentration and Microbubble Shrinkage as a Possible Mechanism for Fine-Tuning Microbubble Size and Shell Properties.

The acoustic response of microbubbles (MBs) depends on their resonance frequency, which is dependent on the MB size and shell properties. Monodisperse MBs with tunable shell properties are thus desirable for optimizing and controlling the MB behavior in acoustics applications. By utilizing a novel microfluidic method that uses lipid concentration to control MB shrinkage, we generated monodisperse MBs of four different initial diameters at three lipid concentrations (5.6, 10.0, and 16.0 mg/mL) in the aqueous phase. Following shrinkage, we measured the MB resonance frequency and determined its shell stiffness and viscosity. The study demonstrates that we can generate monodisperse MBs of specific sizes and tunable shell properties by controlling the MB initial diameter and aqueous phase lipid concentration. Our results indicate that the resonance frequency increases by 180-210% with increasing lipid concentration (from 5.6 to 16.0 mg/mL), while the bubble diameter is kept constant. Additionally, we find that the resonance frequency decreases by 260-300% with an increasing MB final diameter (from 5 to 12 µm), while the lipid concentration is held constant. For example, our results depict that the resonance frequency increases by ∼195% with increasing lipid concentration from 5.6 to 16.0 mg/mL, for ∼11 µm final diameter MBs. Additionally, we find that the resonance frequency decreases by ∼275% with increasing MB final diameter from 5 to 12 µm when we use a lipid concentration of 5.6 mg/mL. We also determine that MB shell viscosity and stiffness increase with increasing lipid concentration and MB final diameter, and the level of change depends on the degree of shrinkage experienced by the MB. Specifically, we find that by increasing the concentration of lipids from 5.6 to 16.0 mg/mL, the shell stiffness and viscosity of ∼11 µm final diameter MBs increase by ∼400 and ∼200%, respectively. This study demonstrates the feasibility of fine-tuning the MB acoustic response to ultrasound by tailoring the MB initial diameter and lipid concentration.

Microfluidic nanobubbles: observations of a sudden contraction of microbubbles into nanobubbles.

Microfluidic devices are often utilized to generate uniform-size microbubbles. In most microfluidic bubble generation experiments, once the bubbles are formed the gas inside the bubbles begin to dissolve into the surrounding aqueous environment. The bubbles shrink until they attain an equilibrium size dictated by the concentration and type of amphiphilic molecules stabilizing the gas-liquid interface. Here, we exploit this shrinkage mechanism, and control the solution lipid concentration and microfluidic geometry, to make monodisperse bulk nanobubbles. Interestingly, we make the surprising observation of a critical microbubble diameter above and below which the scale of bubble shrinkage dramatically changes. Namely, microbubbles generated with an initial diameter larger than the critical diameter shrinks to a stable diameter that is consistent with previous literature. However, microbubbles that are initially smaller than the critical diameter experience a sudden contraction into nanobubbles whose size is at least an order-of-magnitude below expectations. We apply electron microscopy and resonance mass measurement methods to quantify the size and uniformity of the nanobubbles, and probe the dependence of the critical bubble diameter on the lipid concentration. We anticipate that further analysis of this unexpected microbubble sudden contraction regime can lead to more robust technologies for making monodisperse nanobubbles.

Controlled Shrinkage of Microfluidically Generated Microbubbles by Tuning Lipid Concentration.

Monodisperse microbubbles with diameters less than 10 µm are desirable in several ultrasound imaging and therapeutic delivery applications. However, conventional approaches to synthesize microbubbles, which are usually agitation-based, produce polydisperse bubbles that are less desirable because of their heterogeneous response when exposed to an ultrasound field. Microfluidics technology has the unique advantage of generating size-controlled monodisperse microbubbles, and it is now well established that the diameter of microfluidically made microbubbles can be tuned by varying the liquid flow rate, gas pressure, and dimensions of the microfluidic channel. It is also observed that once the microbubbles form, the bubbles shrink and eventually stabilize to a quasi-equilibrium diameter, and that the rate of stabilization is related to the lipid solution. However, how the lipid solution concentration affects the degree of bubble shrinkage, and the stable size of microbubbles, has not been thoroughly examined. Here, we investigate whether and how the lipid concentration affects the degree of microbubble shrinkage. Namely, we utilize a flow-focusing microfluidic geometry to generate monodisperse bubbles, and observe the effect of gas composition (2.5, 1.42, and 0.17 wt % octafluoropropane in nitrogen) and lipid concentration (1-16 mg/mL) on the degree of microbubble shrinkage. For the lipid system and gas utilized in these experiments, we observe a monotonic increase in the degree of microbubble shrinkage with decreasing lipid concentration, and no dependency on the gas composition. We hypothesize that the degree of shrinkage is related to lipid concentration by the self-assembly of lipids on the gas-liquid interface during bubble generation and subsequent lipid packing on the interface during shrinkage, which is arrested when a maximum packing density is achieved. We anticipate that this approach for creating and tuning the size of monodisperse microbubbles will find utility in biomedical applications, such as contrast-enhanced ultrasound imaging and ultrasound-triggered gene delivery.

Biomedical nanobubbles and opportunities for microfluidics.

The use of bulk nanobubbles in biomedicine is increasing in recent years, which is attributable to the array of therapeutic and diagnostic tools promised by developing bulk nanobubble technologies. From cancer drug delivery and ultrasound contrast enhancement to malaria detection and the diagnosis of acute donor tissue rejection, the potential applications of bulk nanobubbles are broad and diverse. Developing these technologies to the point of clinical use may significantly impact the quality of patient care. This review compiles and summarizes a representative collection of the current applications, fabrication techniques, and characterization methods of bulk nanobubbles in biomedicine. Current state-of-the-art generation methods are not designed to create nanobubbles of high concentration and low polydispersity, both characteristics of which are important for several bulk nanobubble applications. To date, microfluidics has not been widely considered as a tool for generating nanobubbles, even though the small-scale precision and real-time control offered by microfluidics may overcome the challenges mentioned above. We suggest possible uses of microfluidics for improving the quality of bulk nanobubble populations and propose ways of leveraging existing microfluidic technologies, such as organ-on-a-chip platforms, to expand the experimental toolbox of researchers working to develop biomedical nanobubbles.