Controlled vesicle self-assembly in microfluidic channels with hydrodynamic focusing

Traditional liposome preparation methods are based on mixing of bulk phases, leading to inhomogeneous chemical and/or mechanical conditions during formation; hence liposomes are often polydisperse in size and lamellarity. Here we show the formation of liposomes that encapsulate reagents in a continuous two-phase flow microfluidic network with precision control of size from 100 to 300 nm by manipulation of liquid flow rates. We demonstrate that by creating a solvent-aqueous interfacial region in a microfluidic format that is homogeneous and controllable on the length scale of a liposome, we can facilitate the fine control of liposome size and polydispersity.     

Stable polymeric nanoballoons: lyophilization and rehydration of cross-linked liposomes

The cross-linking of supramolecular assemblies of hydrated lipids is an effective method to stabilize these assemblies to disruption by surfactants or aqueous alcohol. The heterobifunctional lipids, Acryl/DenPC(16,18) and Sorb/DenPC(18,21), are examples of a new class of polymerizable lipid designed for the creation of cross-linked lipid structures. The robust nature of cross-linked liposomes was demonstrated by lyophilization of the liposomes followed by their essentially complete redispersion in water. The resulting liposomes were compared to the original sample by quasi-elastic light scattering and transmission electron microscopy. There was no major change in the size or structure of the cross-linked liposomes after rehydration of the freeze-dried powder of liposomes. Moreover, the rehydrated cross-linked liposomes continued to be resistant to surfactant solubilization. Neutral cross-linked liposomes were predominantly redispersed after freeze-drying with the aid of bath sonication. The small amount of residual liposome aggregation observed with neutral liposomes could be prevented by incorporating a surface charge into the liposome or attaching hydrophilic polymers, for example, poly(ethylene glycol), onto the liposome.     

Scattering studies of hydrophobic monomers in liposomal bilayers: an expanding shell model of monomer distribution

Hydrophobic monomers partially phase separate from saturated lipids when loaded into lipid bilayers in amounts exceeding a 1:1 monomer/lipid molar ratio. This conclusion is based on the agreement between two independent methods of examining the structure of monomer-loaded bilayers. Complete phase separation of monomers from lipids would result in an increase in bilayer thickness and a slight increase in the diameter of liposomes. A homogeneous distribution of monomers within the bilayer would not change the bilayer thickness and would lead to an increase in the liposome diameter. The increase in bilayer thickness, measured by the combination of small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS), was approximately half of what was predicted for complete phase separation. The increase in liposome diameter, measured by dynamic light scattering (DLS), was intermediate between values predicted for a homogeneous distribution and complete phase separation. Combined SANS, SAXS, and DLS data suggest that at a 1.2 monomer/lipid ratio approximately half of the monomers are located in an interstitial layer sandwiched between lipid sheets. These results expand our understanding of using self-assembled bilayers as scaffolds for the directed covalent assembly of organic nanomaterials. In particular, the partial phase separation of monomers from lipids corroborates the successful creation of nanothin polymer materials with uniform imprinted nanopores. Pore-forming templates do not need to span the lipid bilayer to create a pore in the bilayer-templated films.     

Spherical phospholipid polymer hydrogels for cell encapsulation prepared with a flow-focusing microfluidic channel device

To prepare spherical polymer hydrogels, we used a flow-focusing microfluidic channel device for mixing aqueous solutions of two water-soluble polymers. Continuous encapsulation of cells in the hydrogels was also examined. The polymers were bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenyl boronic acid groups (PMBV) and poly(vinyl alcohol) (PVA), which spontaneously form a hydrogel in aqueous medium via specific molecular complexation upon mixing, even when they were in cell culture medium. The microfluidic device was prepared with polydimethylsiloxan, and the surface of the channel was treated with fluoroalkyl compound to prevent sticking of the polymers on the surface. The microfluidic channel process could control the diameter of the spherical hydrogels in the range of 30-90 μm and generated highly monodispersed diameter spherical hydrogels. We found that the polymer distribution in the hydrogel was influenced by the PVA concentration and that the hydrogel could be dissociated by the addition of d-sorbitol to the suspension. The single cells could be encapsulated and remain viable in the hydrogels. The localized distribution of polymers in the hydrogel may provide an environment for modulating cell function. It is concluded that the spontaneous hydrogel formation between PMBV and PVA in the flow-focusing microfluidic channel device is applicable for continuous preparation of a spherical hydrogel-encapsulating living cell.     

Liposome solubilization induced by surfactant molecules in a microchip

The dynamics of liposome solubilization was monitored by dynamic light scattering and optical microscopy. A newly designed Y-shape microchannel connected to a room was incorporated into a microchip and the reaction processes of the liposome suspension and surfactant solution were observed in the room after mixing the two fluids and stopping the flow. By using this microchip, we succeeded in real-time monitoring of liposome solubilization and the following dynamic processes of solubilization were proposed: 1) Deformed liposomes become spherical. 2) The liposome size increases until the surfactant/liposome ratio in the liposome membrane reaches a threshold value. 3) Mixed micelles of surfactants and phospholipids are released and the liposomes collapse.     

Microfluidic light scattering as a tool to study the structure of aqueous polymer solutions

A small-angle light scattering (SALS) apparatus, coupled with a specially designed microfluidic device is shown to monitor the formation and subsequent size distribution of giant multilamellar vesicles of a diblock copolymer in aqueous solution. The closed-face design, fabricated between glass slides using a UV-curable optical adhesive, incorporates multiple inlets, a mixing system, and a viewing window to perform on-line SALS. The mixing of each component is tested using polystyrene latex microspheres. Vesicles of the block copolymer, EO6BO11 in aqueous solution are formed on the SALS chip and the pair distance distribution function determined using an inverse Fourier transformation of the scattered intensity to quantify the population and distribution for a range of vesicle sizes. These experiments provide demonstrations of how SALS on a microfluidic device can be used as a rapid screening tool to optimize processing conditions for a range of polymer solutions.     

Liposomes Prepared from Inverse Micelles

A method for the preparation of liposome is introduced, which contains two experimental steps: (a) inverse micelles of lecithin are formed in water-in-oil system by sonication; (b) the micelles are spread on the water surface, passed Through the oil-water interface, and transformed into liposomes in the water phase. The main advantage of this method is that the inner aqueous solution encapsulated by liposomes could be different from their enviromental medium. The liposome size is less than 0.5 μm in diameter by atomic force microscope. Comparison of activities of urease with and without liposome encapsulation suggested that urease was well entraped into liposomes.     

Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing

Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.     

Efficient nanoparticle focusing utilizing cascade AC electroosmotic flow

This study presents on-chip continuous accumulation and concentration of nanoscale samples using a cascade alternating current electroosmosis (cACEO) flow. ACEO can generate flow motion caused by ion movement due to interactions between the AC electric field and the induced charge layer on the electrode surface, with the potential to accumulate particles, especially in low-conductive liquid. However, the intrinsic particle diffusive motion, which is sensitive to particle size, is an essential element influencing accumulation efficiency. In this study, an electrode combining chevron and double-gap geometry embedded in a microfluidic channel was developed to perform efficient three-dimensional (3D) nanoparticle focusing using ACEO. The chevron electrode pattern was introduced upstream of the focusing zone to overcome particle accumulation in scattering zones near the channel sidewall. To demonstrate the efficiency of the proposed device for particle accumulation, three nanoparticle types were used: latex, metal, and biomaterial. Continuous 3D concentration of 50-nm polystyrene particles was confirmed. The concentration factor, determined based on image processing, became quite high when 50-nm gold nanoparticles were used. Moreover, nanoparticles with a 20-nm diameter were accumulated using cACEO. Finally, we used the concentrator chip to accumulate 50-nm liposome particles, confirming that the device could also successfully concentrate biomaterials.     

Controllable preparation of nanoparticle-coated chitosan microspheres in a co-axial microfluidic device

In this work, we describe a novel and simple microfluidic method for fabricating nanoparticle-coated chitosan microspheres. Uniform droplets of aqueous chitosan solution were dispersed into an oil phase containing partially hydrophilic nanoparticles via a co-axial microfluidic device. Recirculating flow in the continuous phase in the area between drops enhanced mixing and allowed the nanoparticles to coat the surface of the droplets as they passed through the channel. The chitosan droplets were then crosslinked with glutaraldehyde and nanoparticle-coated microspheres were obtained. SEM characterization shows that the microspheres are monodispersed with uniform nanoparticle distribution on the surface. The dispersity, size and composition of the microspheres could all easily be controlled by changing the microfluidic flow parameters and three different types of nanoparticles were successfully used to synthesize hybrid microspheres to demonstrate the method's versatility.     

Arraying of intact liposomes into chemically functionalized microwells

Here, we describe a protocol to bind individual, intact phospholipid bilayer liposomes, which are on the order of 1 microm in diameter, in microwells etched in a regular array on a silicon oxide substrate. The diameter of the wells is on the order of the liposome diameter, so only one liposome is located in each well. The background of the silicon oxide surface is functionalized with a PEG oligomer using the contact printing of a PEG silane to present a surface that resists the adsorption of proteins, lipid material, and liposomes. The interiors of the wells are functionalized with an aminosilane to facilitate the conjugation of biotin, which is then bound to Neutravidin. The avidin-coated well interiors bind the liposomes whose surfaces contain biotinylated lipids. The specific binding of the liposomes to the surface using the biotin-avidin linkage, together with the resistant nature of the background and the physical confinement of the wells, allows the liposomes to remain intact and to not unravel, rupture, and fuse onto the surface. We demonstrate this intact arraying using confocal laser scanning microscopy of fluorophores specifically tagging the microwells, the lipid bilayer, and the aqueous interior of the liposome.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

Surface enhanced Raman spectroscopy for microfluidic pillar arrayed separation chips

Numerous studies have addressed the challenges of implementing miniaturized microfluidic platforms for chemical and biological separation applications. However, the integration of real time detection schemes capable of providing valuable sample information under continuous, ultra low volume flow regimes has not fully been addressed. In this report we present a chip based chromatography system comprising of a pillar array separation column followed by a reagent channel for passive mixing of a silver colloidal solution into the eluent stream to enable surface enhanced Raman spectroscopy (SERS) detection. Our design is the first integrated chip based microfluidic device to combine pressure driven separation capability with real time SERS detection. With this approach we demonstrate the ability to collect distinctive SERS spectra with or without complete resolution of chromatographic bands. Computational fluidic dynamic (CFD) simulations are used to model the diffusive mixing behaviour and velocity profiles of the two confluent streams in the microfluidic channels. We evaluate the SERS spectral band intensity and chromatographic efficiency of model analytes with respect to kinetic factors as well as signal acquisition rates. Additionally, we discuss the use of a pluronic modified silver colloidal solution as a means of eliminating contamination generally caused by nanoparticle adhesion to channel surfaces.     

High-efficiency electrokinetic micromixing through symmetric sequential injection and expansion

Rapid electric field switching is an established microfluidic mixing strategy for electrokinetic flows. Many such microfluidic mixers are variations on the T- or Y-form channel geometry. In these configurations, rapid switching of the electric field can greatly improve initial mixing over that achieved with static-field mixing. Due to a fundamental lack of symmetry, however, these strategies produce lingering cross-channel concentration gradients which delay complete mixing of the fluid stream. In this paper, a field switching microfluidic mixing strategy which utilizes a symmetric sequential injection geometry with an expansion chamber to achieve high efficiency microfluidic mixing is demonstrated experimentally. A three-inlet injector sequentially interlaces two dissimilar incoming solutions. Downstream of the injector, the sequence enters an expansion chamber resulting in a dramatic (two orders of magnitude) decrease in Peclet number and rapid axial diffusive mixing. The outlet concentration may be accurately varied over the full spectrum by tuning the duty cycle of the field switching waveform. The chips are designed with input from a previous numerical study, manufactured in poly(dimethylsiloxane) using soft-lithography based microfabrication, and tested using fluorescence microscopy. In the context of on-chip chemical processing for analytical operations, the demonstrated mixing strategy has several features: high mixing efficiency (99%), compact axial length (2.3 mm), steady outflow velocity, and readily variable outlet concentration (0.15 < c* < 0.95).     

Singlet-Oxygen Generation from Liposomes: A Comparison of 6β-Cholesterol Hydroperoxide Formation with Predictions from a One-Dimensional Model of Singlet-Oxygen Diffusion and Quenching

Abstract— We measured 6β-cholesterol hydroperoxide (6β-CHP), a specific singlet-oxygen (O₂(δg)) product, during irradiation of unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes containing cholesterol and zinc phthalocyanine (ZnPC). The effects of liposome size, the hydrophobic (O₂(¹δ_g)) quencher, β-carotene, and hydrophilic O₂(¹δ_g) quenchers upon the amount of 6β-CHP formed were determined and interpreted in terms of a one dimensional model of ₂(¹δ_g) quenching and diffusion. The model correctly predicted (1) that the amount of 6β-CHP was increased with increasing liposome size, (2) that P-carotene was more effective at reducing 6β-CHP formation in 400 nm diameter liposomes than 100 nm diameter liposomes and (3) that the hydrophilic quencher, water, was also more effective in large liposomes than in small liposomes.
The hydrophobic quencher, β-carotene, was more effective at reducing the formation of 6β-CHP than at reducing the 1270 nm O₂(¹δ_g) emission. This difference was found to be due to the size distribution present in the liposome preparations because the difference between the 6β-CHP data and the 1270 nm emission data was much smaller in liposome preparations with a narrow size distribution. When a significant size distribution was present, the 6β-CHP data were weighted more heavily with large-diameter liposomes, while the 1270 nm emission data were weighted more heavily with small-diameter liposomes.     

Analysis of liposomes using asymmetrical flow field-flow fractionation: Separation conditions and drug/lipid recovery

Liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol were analyzed by asymmetrical flow field-flow fractionation coupled with multi-angle laser light scattering. In addition to evaluation of fractionation conditions (flow conditions, sample mass, carrier liquid), radiolabeled drug-loaded liposomes were used to determine the liposome recovery and a potential loss of incorporated drug during fractionation. Neither sample concentration nor the cross-flow gradient distinctly affected the size results but at very low sample concentration (injected mass 5 μg) the fraction of larger vesicles was underestimated. Imbalance in the osmolality between the inner and outer aqueous phase resulted in liposome swelling after dilution in hypoosmotic carrier liquids. In contrast, liposome shrinking under hyperosmotic conditions was barely visible. The liposomes themselves eluted completely (lipid recoveries were close to 100%) but there was a loss of incorporated drugs during separation with a strong dependence on the octanol-water partition coefficient of the drug. Whereas corticosterone (partition coefficient ～2) was washed out more or less completely (recovery about 2%), loss of temoporfin (partition coefficient ～9) was only minor (recovery about 80%). All fractionations were well repeatable under the experimental conditions applied in the present study.     

Structure of small actin-containing liposomes probed by atomic force microscopy: effect of actin concentration & liposome size

Actin-containing liposomes were prepared via extrusion through 400 and 600 nm pore diameter membranes at different monomeric actin concentrations in low ionic strength buffer (G-buffer). After subjecting the liposome dispersions to high ionic strength polymerization buffer (F-buffer), topological changes in liposome structure were studied using atomic force microscopy (AFM). Paired dumbbell, horseshoelike, and disklike assemblies were observed for actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes. The topology of actin-containing liposomes was found to be highly dependent on both liposome size and actin concentration. At 1 mg/mL actin, the actin-containing liposomes transformed into a disklike shape, whereas, at 5 mg/mL actin, the actin-containing liposomes retained a spherical shape. On the basis of these observations, we hypothesize that actin could either polymerize on the surface of the inner leaflet of the liposome membrane or polymerize in the aqueous core of the liposome. We explain the associated shape changes induced in actin-containing liposomes on the basis of the hypothesized mechanism of actin polymerization inside the liposomes. At higher actin concentrations (5 mg/mL), we observed membrane-induced actin self-assembly in G-buffer, which implies that G-actin is able to interact directly with lipid bilayers at sufficiently high concentrations.     

Formation and shear-induced processing of quantum dot colloidal assemblies in a multiphase microfluidic chip

The controlled self-assembly of polymer-stabilized quantum dots (QDs) into mesoscale aqueous spherical assemblies termed quantum dot compound micelles (QDCMs) using a two-phase gas-segmented microfluidic reactor is described. Self-assembly is initiated by the fast mixing of water (approximately 1 s) with a blend solution of polystyrene-coated QDs and amphiphilic polystyrene-block-poly(acrylic acid) stabilizing chains via chaotic advection within liquid plugs moving through a sinusoidal channel. Subsequent recirculating flow within a post-formation channel subjects the dynamic QDCMs to shear-induced processing, controlled via the flow rate and channel length, before a final quench into pure water. During processing, larger QDCMs within the initial population undergo breakup into smaller particles, resulting in smaller mean particle sizes, smaller relative standard deviations, and more skewed distribution shapes, as the overall shear exposure is increased. For these cases, shear-induced size reduction is sufficient to dominate surface tension-driven growth.     

Preparative size exclusion chromatography combined with detergent removal as a versatile tool to prepare unilamellar and spherical liposomes of highly uniform size distribution

Detergent removal from mixed micelles was combined with preparative size exclusion chromatography (SEC) on Sephacryl S 500 HR to prepare unilamellar and spherical liposomes of defined sizes between 50 and 100 nm with a very narrow size distribution (RSD of vesicle diameter between 13% and 25%). For neutral phosphatidylcholine and negatively charged phosphatidylcholine/phosphatidylglycerol liposome preparations, efficient sizing at the preparative scale was demonstrated by analyzing isolated SEC peak fractions with cryo-transmission electron microscopy and dynamic light scattering. The number-weighted average vesicle diameters obtained using both methods are in very good agreement for fractions of low polydispersity.     

Hydrodynamic focusing—a versatile tool

The control of hydrodynamic focusing in a microchannel has inspired new approaches for microfluidic mixing, separations, sensors, cell analysis, and microfabrication. Achieving a flat interface between the focusing and focused fluids is dependent on Reynolds number and device geometry, and many hydrodynamic focusing systems can benefit from this understanding. For applications where a specific cross-sectional shape is desired for the focused flow, advection generated by grooved structures in the channel walls can be used to define the shape of the focused flow. Relative flow rates of the focused flow and focusing streams can be manipulated to control the cross-sectional area of the focused flows. This paper discusses the principles for defining the shape of the interface between the focused and focusing fluids and provides examples from our lab that use hydrodynamic focusing for impedance-based sensors, flow cytometry, and microfabrication to illustrate the breadth of opportunities for introducing new capabilities into microfluidic systems. We evaluate each example for the advantages and limitations integral to utilization of hydrodynamic focusing for that particular application.