Stability and rheological behavior of concentrated monodisperse food emulsifier coated microbubble suspensions

Nearly monodispersed populations of microbubbles were produced using flow focusing with a food grade emulsifier. The microbubbles produced by this technique have diameters in the range of 120-200 microm. The flow focusing device uses metered streams of air and liquid to produce a jet that periodically pinches to make individual microbubbles. The size of the microbubbles can be controlled by changing the relative flow rates of the gas and the liquid. The emulsifier consists of a mixture of monoglycerides, diglycerides and sodium stearoyl lactylate in combination with polyethylene glycol (PEG)-40 stearate. The emulsifier forms a thin shell that stabilizes the microbubbles. The microbubbles are stable over time with their sizes remaining roughly constant over 2 h. Such stability allows suspensions of microbubbles to be formed and their rheological properties tested. The sizes of the microbubbles are also monitored off-line while testing, examining the effect of shearing on the bubble sizes, as well as their stability over time. These results show that the microbubble suspensions are viscoelastic and exhibit power law behavior. The relationship between the air fraction of the suspension and fluid viscosity is determined.     

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated 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.     

Tunable diacetylene polymerized shell microbubbles as ultrasound contrast agents

Monodisperse gas microbubbles, encapsulated with a shell of photopolymerizable diacetylene lipids and phospholipids, were produced by microfluidic flow focusing, for use as ultrasound contrast agents. The stability of the polymerized shell microbubbles against both aggregation and gas dissolution under physiological conditions was studied. Polyethylene glycol (PEG) 5000, which was attached to the diacetylene lipids, was predicted by molecular theory to provide more steric hindrance against aggregation than PEG 2000, and this was confirmed experimentally. The polymerized shell microbubbles were found to have higher shell-resistance than nonpolymerizable shell microbubbles and commercially available microbubbles (Vevo MicroMarker). The acoustic stability under 7.5 MHz ultrasound insonation was significantly greater than that for the two comparison microbubbles. The acoustic stability was tunable by varying the amount of diacetylene lipid. Thus, our polymerized shell microbubbles are a promising platform for ultrasound contrast agents.     

Maintaining monodispersity in a microbubble population formed by flow-focusing

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.     

On-chip generation of microbubbles as a practical technology for manufacturing contrast agents for ultrasonic imaging

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.     

Microbubble size isolation by differential centrifugation

Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential centrifugation was used to successfully isolate the 1-2 and 4-5 mum diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (<1 vol%) were susceptible to Ostwald ripening. For example, 4-5 mum microbubbles disintegrated into 1-2 mum microbubbles. This latter observation indicated the existence of an optimally stable diameter in the 1-2 mum range for these lipid-coated microbubbles. Overall, differential centrifugation provided a rapid and robust means for size selection and reduced polydispersity of lipid-coated microbubbles.     

双重乳液/溶剂蒸发法制备超声造影微泡

通过水包油包水(W1/O/W2)双重乳液的油相溶剂蒸发过程, 制备了聚左旋乳酸(PLLA)微泡, 结合扫描电子显微镜(SEM)、激光共聚焦显微镜(LCSM)和粒度分析(PSA)等表征手段, 研究了外水相乳化剂的种类、浓度、两次乳化的水油比、均质机转速等参数对微泡性能的影响. 研究结果表明, 聚乙烯醇(PVA)是该体系外水相有效的乳化剂; 通过调节PVA水溶液的浓度或第二次乳化时均质机转速, 能有效地控制微泡的平均粒径(1~10 μm); 第一次乳化的水油比是微泡空心率的重要影响因素. 对微泡负压充气后, 进行体外超声显影检测, 证明该微泡具有较好的超声造影增强效果.     

The size of solid lipid nanoparticles: an interpretation from experimental design

This study aimed to investigate the role of different factors affecting the size of solid lipid nanoparticles (SLN), prepared by the emulsification-solvent evaporation method. A double factorial design was conducted so as to cover a wide range of sizes, highlighting zones with different behaviour with respect to changes in the controlled variables: lipid concentration, solvent:lipid ratio and emulsifier concentration. The solvent:lipid ratio constituted the main factor influencing particle size. Increasing the amount of solvent induced a decrease in the size. This was a general trend, essentially independent from solvent and lipid type. The amount of emulsifier had a non-trivial impact on size, depending on whether systems were located below, above or close to the optimal surface coverage. The amount of lipid had a limited influence upon particle size, being more relevant for lower lipid concentrations. An optimal formulation was selected for intermediate levels of the three variables. Sonication reduced both particle size and polydispersity. These particles were also tested as drug carriers using simvastatin as a model of lipophilic drug. SLN were able to entrap a high amount of simvastatin, with little effect upon size and zeta potential, constituting a promising carrier for lipophilic drugs.     

Influence of the dissolution rate on the collapse and shedding behavior of monostearin/monopalmitin-rich coated microbubbles

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.     

Diffusion of nanoparticles in monolayers is modulated by domain size

Langmuir monolayers are often used as simple models for biological membranes. The possibility to change their composition and phase state in a very controlled manner as well as access to a large observation area makes them a versatile tool for the investigation of membrane-related interactions. Inspired by experiments in our group, we investigate the interaction of single, partially charged nanoparticles with lipid microdomains by Monte Carlo simulations. Condensed domains in inhomogeneous Langmuir monolayers exhibit an electric dipole field interacting attractively with the nanoparticle's dipole moment. With increasing domain size, the resulting electric field changes from single dipole to semi-infinite domain characteristics, significantly influencing the motion of the particle. Small immobile domains (R = 1 microm) confine the movement of the tracer to the boundary of the domain whereas for large domains (R > or = 10 microm) its motion is only temporarily hindered. This suggests a powerful mechanism for controlling diffusive transport in lipid membranes.     

Solid-state characterization of spray-dried ice cream mixes

The main physicochemical properties of spray-dried ice cream mixes (i.e. surface composition, wettability, flowability and microstructure) were analyzed. Emulsions contained 19-44% milk fat on a dry basis and included mixes with no added emulsifier and/or sucrose. The time necessary for complete wetting of the powders correlated with the amount of surface free-fat measured by means of solvent extraction. Non-micellar casein (sodium caseinate) showed to be a better co-encapsulant than micellar casein (skim milk) as demonstrated by surface fat coverage measured by electron spectroscopy for chemical analysis (ESCA). Emulsifiers influenced the fat surface composition of the powders by reducing the amount of surface protein due to their lower interfacial tension. Surface fat caused an initial overestimation of the particle size of the powders due to fat-related caking. Powders showed no flow before and after surface fat extraction which was attributed to fat-related caking and very small particle size (<80 microm), respectively.     

Collapse and shedding transitions in binary lipid monolayers coating microbubbles

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.     

Free-radical generation from collapsing microbubbles in the absence of a dynamic stimulus

Free radicals are generated by the collapse of ultrasound-induced cavitation bubbles when they are forcefully compressed by dynamic stimuli. Radical generation occurs as a result of the extremely high temperatures induced by adiabatic compression during the violent collapse process. It is generally believed that extreme conditions are required for this type of radical generation. However, we have demonstrated free-radical generation from the collapse of microbubbles (diameter = <50 microm) in the absence of a harsh dynamic stimulus. In contrast to ultrasound-induced cavitation bubbles, which collapse violently after microseconds, the microbubbles collapsed softly under water after several minutes. Electron spin-resonance spectroscopy confirmed free-radical generation by the collapsing microbubbles. The increase of the surface charges (zeta potentials) of the microbubbles, which were measured during their collapse, supported the hypothesis that the significant increase in ion concentration around the shrinking gas-water interface provided the mechanism for radical generation. This technique of radical generation from collapsing microbubbles could be employed in numerous engineering applications, including wastewater treatment.     

Open tubular capillary electrochromatography: a new technique for in situ enzymatic modification of low density lipoprotein particles and their protein-free derivatives

Electrochromatography with open tubular capillaries coated with human low density lipoprotein (LDL) particles and their protein-free derivatives was studied as a method for their in situ enzymatic modification. LDL particles as monolayers or their protein-free derivatives (lipid microemulsions) were coated on 50 microm i.d. capillaries, which resemble tiny human blood vessels in size, the arterioles. The immobilized LDL particles were exposed to sphingomyelinase, phospholipase A2 or alpha-chymotrypsin at 25 and 37 degrees C. The mobility of the electro-osmotic flow was employed as a surface charge indicator, and the retention factors of steroids were used as hydrophobicity indicators. Moreover, the capillaries were, for the first time, coated with lipid microemulsions containing either LDL-derived or commercial lipids, and the immobilized microemulsions were treated with sphingomyelinase in capillary. The results demonstrate that open tubular capillaries provide a good microreactor for the in situ modification of LDL particles and lipid microemulsions. The technique only requires extremely low quantities of LDL particles, lipid microemulsions, and enzymes. It allows quick and easy alteration of the reaction conditions, and the enzymes can be collected and reused. Asymmetrical flow field flow fractionation provides useful information on the size of the enzymatically modified LDL particles.     

Influence of droplet characteristics on the formation of oil-in-water emulsions stabilized by surfactant-chitosan layers

The objective of this study was to establish the optimum conditions for preparing stable oil-in-water emulsions containing droplets surrounded by surfactant-chitosan layers. A primary emulsion containing small droplets (d32 approximately = 0.3 microm) was prepared by homogenizing 20 wt% corn oil with 80 wt% emulsifier solution (20 mM SDS, 100 mM acetate buffer, pH 3) using a high-pressure valve homogenizer. The primary emulsion was diluted with chitosan solutions to produce secondary emulsions with a range of oil and chitosan concentrations (0.5-10 wt% corn oil, 0-1 wt% chitosan, pH 3). The secondary emulsions were sonicated to help disrupt any droplet aggregates formed during the mixing process. The electrical charge, particle size, and amount of free chitosan in the emulsions were then measured. The droplet charge changed from negative to positive as the amount of chitosan in the emulsions was increased, reaching a relatively constant value (approximately +50 mV) above a critical chitosan concentration (C(Sat)), which indicated that saturation of the droplet surfaces with chitosan occurred. Extremely large droplet aggregates were formed at chitosan concentrations below C(Sat), but stable emulsions could be formed above C(Sat) provided the droplet concentration was not high enough for depletion flocculation to occur. Interestingly, we found that stable multilayer emulsions could also be formed by mixing chitosan with an emulsion stabilized by a nonionic surfactant (Tween 20) due to the fact the initial droplets had some negative charge. The information obtained from this study is useful for preparing emulsions stabilized by multilayer interfacial layers.     

Control of particle alignment in water by an alternating electric field

We attempted to align a large number of silica particles dispersed in aqueous solution by controlling the alternating electric field between the two electrodes (400 microm apart). Relatively large particles (9.9 microm) were found to align forming strings in the direction parallel to the electric field while relatively small particles (2.0 and 4.9 microm) were observed to align making stripes in the direction perpendicular to the field. The number of stripes formed by particles between the electrodes increased with increasing frequency of the alternating field. This peculiar perpendicular particle alignment appeared when the contribution to particle alignment of electroosmotic flow exceeded that of dielectric polarization and the osmotic flow was found to be stronger around the particles than in the vicinity of the electrode surface.     

Structure and acoustical properties control of magnetite/PLA composite microbubbles

Poly(lactic acid) (PLA) microbubbles and magnetite/PLA composite microbubbles with various structures and controllable average size were prepared by a modified double emulsion–solvent evaporation process, and sound attenuation spectrum was performed to investigate the influence of size, composite structure, and magnetite loading on the acoustical properties of microbubbles. With the increase of time or energy of the emulsification, the inner structure of composite microbubbles changed continuously from honeycomb to solid structure, accompanied by the change of surface morphology and the decrease of average particle size. The sound attenuation and resonance frequency of concentric microbubbles were higher than those of other structures, while microbubbles with multicavities had the lowest sound attenuation and resonance frequency. The difference in the acoustical properties was related to their different structures and water permeability. In vitro ultrasonography of composite microbubbles showed a higher video intensity than that of PLA microbubbles, which was consistent with their sound attenuation spectra.     

DNA and polylysine adsorption and multilayer construction onto cationic lipid-coated microbubbles

We report on a novel application of the layer-by-layer (LbL) assembly technique to attach multiple layers of DNA and poly-l-lysine (PLL) onto preformed lipid-coated microbubbles to increase the DNA loading capacity. We first measured the effects of the cationic lipid fraction and salt concentration on the microbubble stability. Microbubble production and stability were robust up to a cationic lipid fraction of 40 mol % in 10 mM NaCl. DNA adsorption was heterogeneous over the microbubble shell and occurred primarily on the condensed phase domains. The amount of adsorbed DNA, and subsequently adsorbed PLL, increased linearly with the fraction of cationic lipid in the shell. DNA loading was further enhanced by the LbL assembly method to construct polyelectrolyte multilayers (PEMs) of DNA and PLL. PEM buildup was demonstrated by experimental results from zeta potential analysis, fluorescence microscopy, UV spectroscopy, and flow cytometry. The PEMs exhibited two growth stages and were heterogeneously distributed over the microbubble surface. The DNA loading capacity onto the microbubbles was enhanced by over 10-fold by using five paired layers. However, the PEM shell did not prevent oscillation or destruction during ultrasound insonification. These results suggest that the surface can be compartmentalized to make multifunctional, high-payload ultrasound contrast agents for targeted gene therapy.     

Controllable gas/liquid/liquid double emulsions in a dual-coaxial microfluidic device

This article presents a simple and novel approach to prepare monodispersed gas-in-oil-in-water (G/O/W) and gas-in-water-in-oil (G/W/O) double-emulsions in the same dual-coaxial microfluidic device. The effects of three phase flow rates on the sizes of microbubbles and droplets and the number of the encapsulated microbubbles were systematically studied. We successfully synthesized two different types of gas/liquid/liquid (G/L/L) double emulsions with different inner structures in the same geometry by adjusting the flow rates sequentially. Mathematical models were developed to predict the size and structures of the double emulsions. This simple approach gives a new idea for preparing hollow and porous microspheres with microbubbles as the direct core/pores templates.     

Premix Membrane Emulsification: Preparation and Stability of Medium-Chain Triglyceride Emulsions with Droplet Sizes below 100 nm

Premix membrane emulsification is a promising method for the production of colloidal oil-in-water emulsions as drug carrier systems for intravenous administration. The present study investigated the possibility of preparing medium-chain triglyceride emulsions with a mean particle size below 100 nm and a narrow particle size distribution using sucrose laurate as an emulsifier. To manufacture the emulsions, a coarse pre-emulsion was repeatedly extruded through alumina membranes (Anodisc^™) of 200 nm, 100 nm and 20 nm nominal pore size. When Anodisc^™ membranes with 20 nm pore size were employed, nanoemulsions with z-average diameters of about 50 nm to 90 nm and polydispersity indices smaller than 0.08 could be obtained. Particle growth due to Ostwald ripening was observed over 18 weeks of storage. The Ostwald ripening rate linearly depended on the emulsifier concentration and the concentration of free emulsifier, indicating that micelles in the aqueous phase accelerated the Ostwald ripening process. Long-term stability of the nanoemulsions could be achieved by using a minimised emulsifier concentration or by osmotic stabilisation with soybean oil added in a mass ratio of 1:1 to the lipid phase.