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Saturated diacyl (disaturated) phosphatidylcholine (PC) mixed with the lipopolymer distearoylphosphatidylethanolamine (DSPE)-polyethyleneglycol molecular weight 2000 (PEG2000) self-assemble as a monolayer at the air-water interface of air-in-water micrometer-scale bubbles (microbubbles), similar to coatings (shells) on leading medical ultrasound contrast agents (UCAs). This system is characterized here to study the impact of the DSPE-PEG2000 species and PC chain-length on the monolayer coating phase behavior, collapse, shedding, and air transport resistance and microbubble dissolution rate and surface contour. Using fluorescence microscopy of dissolving microbubbles, we found that film microstructure and collapse behavior for all chain lengths (n = 14-20) was indicative of primarily condensed phase monolayers, unlike similar coatings containing polyethyleneglycol 40 stearate (PEG40S) that are either expanded phase or coexisting expanded-condensed phase monolayers. Additionally, we observed a new surface buckling type of behavior with all chain lengths, by bright field microscopy, where the air-water interface continuously appears rough (rather than cyclically rough and smooth), with this behavior most frequently observed for n = 16. In correlating the statistical frequency of this behavior with the monolayer microstructure, we propose that it arises from a slowed nucleation rate of collapse structures at condensed-condensed phase interfaces, not present in systems containing PEG40S. By modeling the dissolution (radius vs time) data, we obtained, for each chain length, the film air transport resistance (R(shell)) that was then fit to a chain-length-dependent energy barrier model. Importantly, the pre-exponential factor was approximately 10 x higher and the microbubbles persisted approximately 4 x longer (from 15 microm at a fixed dissolved oxygen content) in comparison to previously studied films containing PEG40S. We attribute the unique stability properties of microbubble coatings containing DSPE-PEG2000 to the propensity of this molecule to form a condensed-phase monolayer, such that the monolayer coatings approach the properties of one continuous condensed domain.

A low cost food grade emulsifier (a mixture of monoglycerides, diglycerides, and sodium stearoyl lactylate) in combination with polyethylene glycol-40 stearate (PEG-40S) was used as an alternative to pure saturated phospholipids to form the thin shell of a microbubble. To investigate the stability of these microbubbles in a water system over time, their dissolution behavior was studied at various degas factors and at two percentages of PEG-40S. It was found that the favored shell collapse/shedding mechanism switched, as the dissolution rate increased (degas factor decreased), from folding with a smooth surface contour to buckling accompanied by surface folding/expulsion with a cyclic buckled-smooth surface contour. The compositional change that we made played a more minor role, mainly controlling the resistance to mass transfer of the microbubble shell and again modifying the mechanism-determinant dissolution rate. The shell resistance behavior for these microbubbles varied from that of previous lipid/PEG-40S-coated microbubbles by the presence of a maximum in shell resistance during dissolution. We hypothesize that the dominance of one collapse mechanism over another for these compositions is related to the time scales of two competing processes, fold nucleation and area compression. For these mixtures at room temperature, we estimate that the maximum area compression rate for folding as the major collapse mechanism is approximately 0.2 s (-1), a rate unattainable in a traditional Langmuir trough but achievable by the use of a dissolving microbubble.

The most common type of ultrasound contrast agents are encapsulated microbubbles, typically 1 to 5 microns in diameter. These microbubbles are injected into the bloodstream to provide image enhancement during an ultrasound examination. Because of their compressibility, these microbubbles are inherently sensitive to changes in pressure. For imaging, this is beneficial in that these microbubbles oscillate in an acoustic field and allow imaging systems to detect their response uniquely from tissue. However, this sensitivity also means that microbubbles can be readily destroyed by significant hydrostatic pressure. Injection of these microbubbles through a small-gauge catheter, such as is sometimes performed in small animal imaging studies, can result in microbubble destruction. In this manuscript, the effects of microbubble injection through catheters of varying diameter are examined. Our results indicate that the concentration and size distribution of microbubbles can be substantially altered in cases of rapid injection through small-gauge needles.

The dynamic processes impacting the size distributions of lipid-encapsulated microbubbles formed by flow-focusing were observed by video optical microscopy. Parameters studied included the filling gas, gas saturating the surrounding solution, and microbubble size (initial size 2-12 microm) to simulate typical laboratory conditions. Typically, dissolution or growth, followed by Ostwald ripening at a collection cover glass, were observed and quantified. However, in the case of small nitrogen-filled microbubbles surrounded by an air-saturated solution, Ostwald ripening was avoided for at least 9 h. These bubbles had a final size distribution of 1.5 +/- 0.1 microm. This work suggests that lipid-encapsulated microbubbles formed by flow-focusing should be given sufficient time to reach a terminal size before coming into contact with each other. These long-lived mondisperse microbubbles should be of interest in ultrasound contrast agents, microfabrication, food, and research applications.

This paper presents a new manufacturing method to generate monodisperse microbubble contrast agents with polydispersity index (sigma) values of <2% through microfluidic flow-focusing. Micron-sized lipid shell-based perfluorocarbon (PFC) gas microbubbles for use as ultrasound contrast agents were produced using this method. The poly(dimethylsiloxane) (PDMS)-based devices feature expanding nozzle geometry with a 7 microm orifice width, and are robust enough for consistent production of microbubbles with runtimes lasting several hours. With high-speed imaging, we characterized relationships between channel geometry, liquid flow rate Q, and gas pressure P in controlling bubble sizes. By a simple optimization of the channel geometry and Q and P, bubbles with a mean diameter of <5 microm can be obtained, ideal for various ultrasonic imaging applications. This method demonstrates the potential of microfluidics as an efficient means for custom-designing ultrasound contrast agents with precise size distributions, different gas compositions and new shell materials for stabilization, and for future targeted imaging and therapeutic applications.

In this letter, the long-term stabilization of monodisperse microbubbles produced by flow focusing is demonstrated using lipid encapsulation. Fluorescence microscopy, high-speed camera imaging, and particle size analysis were used to investigate the roles of lipid phase behavior, dissolution, Ostwald ripening, and coalescence in the stability of microbubbles formed by flow focusing. It was found that these behaviors were controlled through compositional changes with respect to lipid, emulsifier, and viscosity agents. Microbubbles coated with lipid and PEG emulsifier in a viscous solution were found to contain an extremely narrow size distribution (diameter(av) = 51 microm, standard deviation = 4 microm), which was maintained for up to several months.

We report on a fluorescence microscopy study of the monolayer collapse and shedding behavior due to shell compression during the dissolution of air-filled, lipid-coated microbubbles in degassed media. The monolayer shell was comprised of saturated diacyl phosphatidylcholine (C12:0 to C22:0) and an emulsifier, poly(ethylene glycol)-40 stearate. The morphologies of monolayer collapse structures and shed particles were monitored as a function of phospholipid acyl chain length (n) and temperature. The two components formed a single miscible phase when the phospholipid was near or above its main phase transition temperature, and collapse occurred via suboptical particles to vesicles (both were shed) and tubes as chain length increased. Conversely, two-phase coexistence was observed when the lipid was below its main phase transition temperature. For these bubbles, a transition from primary collapse to secondary collapse was observed. Primary collapse was observed as a loss of expanded phase due to vesiculation. Secondary collapse involved the rapid propagation of monolayer folds and simultaneous deformation. For very rigid monolayers, we observed substantial surface buckling with simultaneous nucleation and growth of folds. The folds merged at a single point or region, providing a conduit for the entire excess lipid to shed in a single event, and the bubble smoothed and became more spherical. These results are discussed in the context of general binary phospholipid collapse behavior, microbubble dissolution behavior, medical applications, and the dissolution behavior of natural microbubbles.

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating-cooling followed by compression-expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.

Encapsulated microbubble contrast agents incorporating an adhesion ligand in the microbubble shell are used for molecular imaging with ultrasound. Currently available microbubble agents are produced with techniques that result in a large size variance. Detection of these contrast agents depends on properties related to the microbubble diameter such as resonant frequency, and current ultrasound imaging systems have bandwidth limits that reduce their sensitivity to a polydisperse contrast agent population. For ultrasonic molecular imaging, in which only a limited number of targeted contrast agents may be retained at the site of pathology, it is important to optimize the sensitivity of the imaging system to the entire population of contrast agent. This article presents contrast agents with a narrow size distribution that are targeted for molecular imaging applications. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters that influence the size distribution and demonstrate initial acoustic testing. [ABSTRACT FROM AUTHOR]

Abstract: The stability and presence of micron-scale bubbles (microbubbles) is of considerable interest in environmental, biomedical, and food sciences. Here we show that microbubbles can be formed and stabilized in a solution of low cost food-grade emulsifier (a mixture of saturated long-chain monoglycerides, diglycerides and sodium stearoyl-2-lactylate) in combination with polyethylene glycol (PEG)-40 stearate. Langmuir trough methods and fluorescence microscopy were combined to investigate the surface tension, interfacial elastic modulus, phase behavior and microstructure of monolayer shells coating these microbubbles. Our results strongly suggest that although the PEG40S is necessary to form microbubbles this component, as well as sodium stearoyl-2-lactylate, are “squeezed out” in the form of collapse aggregates. This process leaves a microbubble shell, composed of a condensed-phase low surface tension mono- and diglycerides mixure with some of the PEG40S and SSL2 remaining trapped between the condensed-phase domains. We find that other commercially available emulsifiers, containing unsaturated or bulky components unable to form condensed phases, do not to form or stabilize a microbubble layer, although they may form a foam, a finding that we relate to differences in surface tension. [Copyright &y& Elsevier]