Microchannel Emulsification Research Articles

Mammalian cell encapsulation in monodisperse chitosan beads using microchannel emulsification

Microchannel emulsification can produce droplets of uniform size at high throughput for wide-ranging applications in the food and drug industry. Mammalian cell encapsulation using emulsion-based systems is of significant interest for immobilized culture or transplantation applications. Microchannel emulsification has recently been applied to mammalian cell encapsulation using alginate. Here, we extend this approach to thermoresponsive hydrogels using chitosan as a model. The performance of the microchannel emulsification process was compared to a previously established stirred emulsification method for mesenchymal stromal cell encapsulation. Chitosan microbeads with controlled sizes ranging from 600 µm to 1500 µm in diameter at a coefficient of variation less than 10 % were obtained after adapting the microchannel system design, channel geometries and operating parameters that affect the capillary number. In a single pass through a 21 G syringe needle, the fraction of ruptured beads was significantly reduced for monodisperse microchannel-generated vs polydisperse stirred emulsion-generated beads of matching De Brouckere mean diameter. The viability of the immobilized cells immediately after the process was 95 % ± 2 % and no significant difference in cell survival and growth factor secretion was observed between microchannel and stirred emulsion-generated beads over 3 days of culture. Microchannel emulsification is a promising and scalable approach to microencapsulate mammalian cells in hydrogels that undergo temperature-dependent gelation.

Impact of polyelectrolyte complex layer on the stability of palm oil multiple emulsions encapsulating a water-soluble compound during heating, cooling, and storage processes

The effect of an interfacial polyelectrolyte complex layer on the stability of multiple water-in-oil-in-water (W/O/W) emulsions containing palm oil (oil phase) was investigated. Monodisperse W/O/W emulsions prepared by primary ultrasonic emulsification using polyglycerin polycondensed ricinoleic acid ester as an oil-soluble emulsifier followed by secondary microchannel emulsification contained multiple sodium caseinate-stabilized droplets with a mean diameter of 44 μm. The multiple droplets containing palm oil maintained their diameter at 60 °C for a minimum of 24 h, whereas significant aggregation of the multiple droplets, with a partially crystallized palm oil phase, was observed below its crystallization temperature (∼27 °C). The formation of a chitosan-sodium caseinate complex layer by the electrostatic interaction of positively charged chitosan with negatively charged sodium caseinate at the oil–water interface could suppress the aggregation of the multiple droplets, even below the crystallization temperature of palm oil. Calcein, a water-soluble fluorescent dye, was stably encapsulated for a month with a high entrapment yield at 25 °C with a chitosan-sodium caseinate complex layer, although without the complex layer, it leaked to the external water phase within a day. The W/O/W emulsion with the polyelectrolyte complex layer improved both the droplet dispersibility and encapsulation efficiency of the multiple droplets during heating at 80 °C, followed by cooling at 25 °C. Polarized microscopic observations indicated that the chitosan-sodium caseinate complex layer affected the crystallization of palm oil in multiple droplets: partial crystallization of palm oil in the multiple droplets may be delayed by the presence of the polyelectrolyte complex layer at the oil–water interface.

Enhancing the Formation and Stability of Oil-In-Water Emulsions Prepared by Microchannels Using Mixed Protein Emulsifiers.

Although natural emulsifiers often have many drawbacks when used alone, their emulsifying ability and stability can usually be improved unexpectedly when used in combination. In this study, monodisperse emulsions stabilized by combining two natural protein emulsifiers, i.e., whey protein isolate (WPI) and sodium caseinate (SC), in different proportions were prepared using microchannel (MC) emulsification. The influences of temperature, pH, ionic strength, and storage time on the microstructure and stability of the emulsions were examined. Analysis of the microstructure and droplet size distribution revealed that the WPI-, SC-, and mixed protein-stabilized emulsions exhibited uniform droplet distribution. The droplet size and ξ-potential of the MC emulsions stabilized by mixed protein emulsifiers were higher than those of the emulsions stabilized by WPI or SC separately. The emulsions stabilized by the two types of proteins and mixed emulsifiers had better stability under high salt concentrations than the synthetic emulsifier Tween 20. WPI-SC-stabilized emulsions were more resistant to high temperatures (70–90°C) and exhibited excellent stabilization than those stabilized by WPI and SC, which was attributed to the more sufficient coverage provided by the two types of protein emulsifier layers and better protein adsorption at the oil-water interface. These results indicate that WPI-SC is a potential stabilizer for MC emulsion requirements. This study provides a basis for the formulation of monodisperse and stable natural emulsion systems.

Ultrasonic enhanced emulsification process in 3D printed microfluidic device to encapsulate active pharmaceutical ingredients

A new method to prepare polymer encapsulated repaglinide nanoparticles through ultrasonic enhanced microchannel emulsification technique was explored. Using the concept of 3D printing, three different shaped micromixers (T-type, Y-type, and F-type) followed by a serpentine microchannel was fabricated using SS-316. Parametric study was performed on all three fabricated micromixers. The best results were obtained for the Y-microchannel in a microfluidic system alone, which showed a minimum particle size of 513.6 nm with 75.4% encapsulation efficiency (EE). In the selected microchannel, to further reduce the drug particle size and to increase% EE, convective mixing between immiscible fluids was enhanced by implementing ultrasound. Compared to the microfluidic system, particle size and EE were significantly improved in the ultrasonic microfluidic system. The experimental results revealed that the minimum particle size of 75.4 ± 1.3 nm with 82.9 ± 0.2% EE was achieved using an ultrasonic enhanced microfluidic system. The zeta potential of + 29.5 mV was obtained for emulsion prepared using the ultrasonic microfluidic system, whereas + 22 mV was prepared using a microfluidic system. Moreover, a backscattering measurement was performed to predict the stability of prepared emulsions. Integrating the ultrasound with a microfluidic system has proven beneficial for drug encapsulation.

Improved Stability of the Palm Oil-in-Water Emulsion via the Surface Modification of Droplets with a Chitosan-Casein Complex Layer

The stabilization effect of a biopolymer complex layer consisting of sodium caseinate (SC) and chitosan (CHI) formed via electrostatic interaction was investigated using an oil-in-water (O/W) emulsion containing palm oil droplets as the dispersed phase. O/W emulsions containing palm oil droplets with a narrow droplet diameter distribution were successfully prepared using two microchannel emulsification devices using SC as an emulsifier at 60℃. Surface modification of the palm oil droplets was conducted using different CHI/SC ratios. The surface charge of the droplets was negative and positive at CHI/SC ratios of less than 0.025 and more than 2.5, respectively. At an intermediate CHI/SC ratio between 0.025 and 2.5, the droplets formed large aggregates and became polydisperse. The adsorption of CHI on the surface of palm oil droplets at a high CHI/SC ratio was confirmed by fluorescent microscopy and ζ-potential measurement. Improved stability of the O/W emulsions with the CHI-SC complex layer was demonstrated during heating (60℃) and cooling (20℃) treatment, and long-term (7 months) storage at room temperature (20-25℃). Interestingly, separation of the large oil phase on the top of the emulsion samples and the formation of large crystals of palm oil were suppressed in the emulsion samples with the CHI-SC complex layer.

Formulation characteristics of monodisperse structured lipid microparticles using microchannel emulsification

Structured lipid microparticles (StLMs) composed of oil and solid lipid in specific ratios are drawing significant attention, owing to their stability in loading and controllability in releasing bioactive compounds. In this study, we formulated monodisperse StLMs with palmitic acid or tripalmitin mixed with soybean oil in different ratios using microchannel emulsification (MCE) at ∼70 °C and following crystallization by cooling. Based on the physicochemical properties of the different dispersed phases, the formulation characteristics were observed and investigated using a microscope video system connected to the MCE system. For each ratio and composition at ∼70 °C, the lipid droplets presented average diameter and coefficient of variation (CV) values in respective ranges of 15.5–19.0 μm and 3.3–8.4%. Following droplet crystallization by cooling, the StLMs exhibited morphological changes but negligible changes in average size and CV. From the internal structure of each lipid molecular and the fundamental information supplied by differential scanning calorimetry (DSC) experiments, we outline the assumptions for morphology variation, which can be used as reference for encapsulation research of StLMs.

Protein-Stabilized Palm-Oil-in-Water Emulsification Using Microchannel Array Devices under Controlled Temperature.

Microchannel (MC) emulsification for the preparation of monodisperse oil-in-water (O/W) and water-in-oil-in-water (W/O/W) emulsions containing palm oil as the oil phase was investigated for application as basic material solid/semi-solid lipid microspheres for delivery carriers of nutrients and drugs. Emulsification was characterized by direct observation of droplet generation under various operation conditions, as such, the effects of type and concentration of emulsifiers, emulsification temperature, MC structure, and flow rate of to-be-dispersed phase on droplet generation via MC were investigated. Sodium caseinate (SC) was confirmed as the most suitable emulsifier among the examined emulsifiers, and monodisperse O/W and W/O/W emulsions stabilized by it were successfully obtained with 20 to 40 µm mean diameter (dm) using different types of MCs.

Comparative study of oil-in-water emulsions encapsulating fucoxanthin formulated by microchannel emulsification and high-pressure homogenization

In this study, we investigated the formulation and stability characteristics of monodisperse oil-in-water (O/W) emulsions encapsulating fucoxanthin using straight-through microchannel emulsification (MCE). Monodisperse O/W emulsions stabilized by hydrophilically modified lecithin (ML), whey protein isolate (WPI), or polyoxyethylene (20) sorbitan monolaurate (Tw20) were stably formulated using straight-through MCE. The resulting emulsions maintained the droplet sizes steadily at 30.3 ± 0.2 μm (ML), 32.3 ± 0.1 μm (WPI) and 29.0 ± 0.8 μm (Tw20) and the relative span factor (RSF) < 0.5 after 30 days storage at 25 °C. The fucoxanthin retention (RTf) in the collected emulsions, decreased from 100% to 44.3 ± 0.1% (ML), 90.7 ± 5.4% (WPI) and 51.4 ± 10.6% (Tw20) after storage. Subsequently, we compared the characteristics of O/W emulsions which were formulated by 1 wt% Tw20 using high-pressure homogenization (HPH) and MCE under the same storage and digestion conditions. After 15 days storage at 4, 25 and 50 °C, all emulsions revealed good physical stability, and there was no drastic change in the droplet sizes. In terms of chemical stability, MCE was pronouncedly better than HPH at the same level of storage temperature. In contrast, the free fatty acids (FFAs) release and fucoxanthin bioaccessibility in emulsions using HPH were significantly higher than MCE. The findings of our study give useful information for food and pharmaceutical industries which express more concern about the natural bioactive compounds for usage.

Lipid Vesicle Preparation Using W/O/W Multiple Emulsions Via Solvent Evaporation: The Effect of Emulsifiers on the Entrapment Yield of Hydrophilic Materials

AbstractWe studied the effects of emulsifiers on the entrapment yields of a hydrophilic material, calcein, in lipid vesicles formed using the multiple emulsion method. The primary emulsion (W1/O) was prepared via sonication while the secondary emulsification that produced the W1/O/W2 multiple emulsions was achieved using the microchannel (MC) emulsification technique. The emulsifiers used in the continuous (W2) phase were Tween® 80, Pluronic® F68, and bovine serum albumin (BSA). Lipid vesicles formed via solvent evaporation of the multiple emulsion droplets had an average diameter of about 180 nm, similar to the size of the water droplets in the primary W1/O emulsions. The entrapment yields of calcein increased with decreasing concentrations of Tween® 80 but decreased with decreasing concentrations of Pluronic® F68 and BSA. The effects of type and concentration of emulsifier were considered in relation to three possible mechanisms: (i) destabilization/solubilization of lipid bilayers by emulsifiers, (ii) reversed‐micellar transport of W1 contents from internal to external water phases through the O phase, and (iii) release of inner water (W1) contents into the continuous (W2) phase via the instability of W1/O/W2 and leakage of W1 contents. Using the food grade emulsifier Tween® 80 at a low concentration of about 0.1 or 0.05 wt%, calcein as a model hydrophilic material could be efficiently entrapped (ca. 80%) in homogenously dispersed lipid vesicles.

Preparation of monodisperse O/W emulsions using a crude surface-active extract from argan by-products in microchannel emulsification

In this study, we evaluated the performance of a mildly derived extract from argan by-products in microchannel emulsification (MCE). Our aim is to produce stable monodisperse O/W emulsions using this extract as a sole emulsifier. Preliminary investigations about the characteristics of argan extract indicated the potential interaction between surface-active components (i.e. saponins, proteins), in bulk phase prior to adsorption at the oil/water interface, resulting in the formation of biogenic complexes with strong interfacial properties. This is important for successful MCE as this technique depends exclusively on dynamic interfacial tension reduction for droplet formation. Nevertheless, upon performance of emulsification experiments, we also found that the complex composition of this extract could counteract its emulsifying efficiency, by creating a hydrophobic, or slightly hydrophilic, layer on the MC array plate surface. This resulted in unsuccessful emulsification using short MCs but did not affect the emulsification behavior in longer ones. Using longer MCs, we could produce stable monodisperse O/W emulsions, with similar droplet size (∼ 36 μm) and droplet size distribution (Relative span factor <0.25) to those obtained using Tween 80, and for up to 10 h of continuous emulsification.

Development of a microchannel emulsification process for pancreatic beta cell encapsulation.

In this study, we developed a high‐throughput microchannel emulsification process to encapsulate pancreatic beta cells in monodisperse alginate beads. The process builds on a stirred emulsification and internal gelation method previously adapted to pancreatic cell encapsulation. Alginate bead production was achieved by flowing a 0.5–2.5% alginate solution with cells and CaCO3 across a 1‐mm thick polytetrafluoroethylene plate with 700 × 200 μm rectangular straight‐through channels. Alginate beads ranging from 1.5–3 mm in diameter were obtained at production rates exceeding 140 mL/hr per microchannel. Compared to the stirred emulsification process, the microchannel emulsification beads had a narrower size distribution and demonstrated enhanced compressive burst strength. Both microchannel and stirred emulsification beads exhibited homogeneous profiles of 0.7% alginate concentration using an initial alginate solution concentration of 1.5%. Encapsulated beta cell viability of 89 ± 2% based on live/dead staining was achieved by minimizing the bead residence time in the acidified organic phase fluid. Microchannel emulsification is a promising method for clinical‐scale pancreatic beta cell encapsulation as well as other applications in the pharmaceutical, food, and cosmetic industries.

A versatile and robust microfluidic device for capillary-sized simple or multiple emulsions production.

Ultrasound-vaporizable microdroplets can be exploited for targeted drug delivery. However, it requires customized microfluidic techniques able to produce monodisperse, capillary-sized and biocompatible multiple emulsions. Recent development of microfluidic devices led to the optimization of microdroplet production with high yields, low polydispersity and well-defined diameters. So far, only few were shown to be efficient for simple droplets or multiple emulsions production below 5μm in diameter, which is required to prevent microembolism after intravenous injection. Here, we present a versatile microchip for both simple and multiple emulsion production. This parallelized system based on microchannel emulsification was designed to produce perfluorocarbon in water or water within perfluorocarbon in water emulsions with capillary sizes (<5μm) and polydispersity index down to 5% for in vivo applications such as spatiotemporally-triggered drug delivery using Ultrasound. We show that droplet production at this scale is mainly controlled by interfacial tension forces, how capillary and viscosity ratios influence droplet characteristics and how different production regimes may take place. The better understanding of droplet formation and its relation to applied pressures is supported by observations with a high-speed camera. Compared to previous microchips, this device opens perspectives to produce injectable and biocompatible droplets with a reasonable yield in order to realize preclinical studies in mice.

Effect of polysaccharide emulsifiers on the fabrication of monodisperse oil-in-water emulsions using the microchannel emulsification method

The microchannel emulsification method was used to prepare monodisperse soybean oil-in-water emulsions, with polysaccharides as the sole emulsifier. The effects of different types (sodium alginate, carboxymethyl cellulose, pectin, gum arabic) and concentrations (0.1, 0.5, 1.0%) of polysaccharide on the droplet size, droplet size distribution and stability of the emulsions were investigated. The droplet diameters of the resulting emulsions were in the range of 35 ± 2 to 47 ± 3 μm, with coefficient of variations (CV) of below 6.6%. Generally, the droplet size increased as the concentration of the polysaccharides increased. The stability of emulsions prepared using 0.5% of sodium alginate, carboxymethyl cellulose and gum arabic was also evaluated in situ. Sodium alginate- and gum arabic-stabilized emulsions were stable for at least 6 h.

Microencapsulation of betanin in monodisperse W/O/W emulsions

Betanin is the main pigment of the food color beetroot red (E162). Due to the fair heat and light stability of E162, this pigment is mainly used in minimally processed packaged food products. Encapsulation increases the stability of betanin, but detailing on the effect of different sources of betanin on the properties and stability of multiple emulsions are scarce. Here we describe the encapsulation of E162, spray-dried beetroot juice and betanin in a monodisperse food-grade water-in-oil-in-water (W/O/W) emulsions by using microchannel emulsification. We compare the tinctorial strength of the encapsulated pigments and investigate the effect of temperature, storage period and pigment concentration on emulsion stability and color. Betanin increases the overall stability of the W/O/W emulsion, reduce the oil droplet size and improve size distribution when compared to the negative control without pigment and to emulsions containing betanin from other sources.

Critical review of encapsulation methods for stabilization and delivery of astaxanthin

Astaxanthin is an important carotenoid pigment and potent antioxidant with proven health benefits. It is primarily obtained from algae or bacteria and is the natural colorant in crustaceans and salmonids. However, this pigment readily degrades in the presence of light and oxygen and has low absorption rates in the human digestive system. Different encapsulation strategies have been adopted to improve astaxanthin physical stability and bioavailability for nutritional use. This review summarizes and compares different encapsulation techniques and matrices applied to astaxanthin. Typical methods for encapsulation include high pressure homogenization, nano dispersions, liposomes and nanoparticles. More recently low energy methods like membrane and microchannel emulsification have been used to improve astaxanthin stability and delivery. Despites the use of numerous delivery methods, improved delivery systems for concentrated astaxanthin formulations are still required. In addition, not all methods have undergone comprehensive in vivo studies that are required to assure the bioavailability of encapsulated astaxanthin.

Numerical Study of Surfactant Dynamics during Emulsification in a T-Junction Microchannel.

Microchannel emulsification requires large amounts of surfactant to prevent coalescence and improve emulsions lifetime. However, most numerical studies have considered surfactant-free mixtures as models for droplet formation in microchannels, without taking into account the distribution of surfactant on the droplet surface. In this paper, we investigate the effects of nonuniform surfactant coverage on the microfluidic flow pattern using an extended lattice-Boltzmann model. This numerical study, supported by micro-particle image velocimetry experiments, reveals the likelihood of uneven distribution of surfactant during the droplet formation and the appearance of a stagnant cap. The Marangoni effect affects the droplet breakup by increasing the shear rate. According to our results, surfactant-free and surfactant-rich droplet formation processes are qualitatively different, such that both the capillary number and the Damköhler number should be considered when modeling the droplet generation in microfluidic devices. The limitations of traditional volume and pressure estimation methods for determining the dynamic interfacial tension are also discussed on the basis of the simulation results.

Preparation of monodisperse W/O emulsions using a stainless-steel microchannel emulsification chip

ABSTRACTIn this study, hydrophobically treated stainless-steel microchannel (MC) array chips were used for preparing monodisperse W/O emulsions. A water-saturated decane containing 5 wt% tetraglycerin monolaurate condensed ricinoleic acid ester was used as the continuous phase. A Milli-Q water containing 5 wt% polyethylene glycol (molecular weight 20,000) and 5 wt% NaCl was used as the dispersed phase. The stainless-steel MC array chips used for MC emulsification had a sufficiently high contact angle of the dispersed phase to their surface in the continuous phase. The resultant uniform-sized aqueous droplets with a coefficient of variation of <5% had average diameters of 100–300 µm, depending on the MC cross-sectional size. The maximum productivity of uniform-sized aqueous droplets reached higher than 1 mL h−1. The difference in the critical capillary number of the dispersed phase that flows in a 100-µm depth MC was 1.5 times greater than that in a 30-µm depth MC.

Mass spectrometric imaging of localization of fat molecules in water-in-oil emulsions containing semi-solid fat

Firstly, we report the localization analysis of the lipid components of a water-in-oil (W/O) semi-solid emulsion by mass spectrometry imaging (MSI). Uniform emulsion droplets were prepared using microchannel emulsification devices with lecithin, stearic acid-binding monoglyceride (St-MAG), and polyglycerol polyricinoleate (PGPR) as emulsifiers. The mass image gives us the localization of phosphatidylcholine (PC) in lecithin, St-MAG, tripalmitin (PPP), medium-chain triglyceride (MCT), and high-melting-point triglyceride tristearin (C18-TAG). PC, St-MAG, and PPP were localized at the interface with the dispersed water droplets. PC and PPP took the same localized position, suggesting an interaction between PC and PPP at the interface. Conversely, PC existed in other regions with St-MAG. MSI revealed multiple target molecules in fat products in a single measurement, and it is expected to reveal fat crystallization at the emulsion interfaces, which will clarify the mechanisms related to the physical properties of high-fat products such as fat spread and butter.

Long-term stability of droplet production by microchannel (step) emulsification in microfluidic silicon chips with large number of terraced microchannels

A long-term production stability of oil-in-water emulsions by microchannel (step) emulsification was investigated using two cross flow silicon chips consisting of 540 or 1850 microchannels fabricated on 10 parallel terraces. Each terrace was 9.54mm long and consisted of 54 channels with a depth of 5μm and a width of 18μm (chip 1) or 185 channels with a depth of 4μm and a width of 8μm (chip 2). The dispersed phase was a mixture of 2wt% polycaprolactone (PCL) and 1wt% poly(d,l-lactic) acid (PLA) dissolved in dichloromethane and the continuous phase was 2wt% poly(vinyl alcohol). After solvent evaporation, the droplets were transformed into highly uniform composite polymer particles with an average diameter of 8.8 or 4.9μm. The percentages of active channels, droplet sizes and droplet generation frequencies at individual terraces were investigated in both chips to reveal any flow maldistribution. After 6h of production in chip 1, overall 95% of the channels produced droplets and the percentage of active channels on each terrace was at least 91%. The percentage of active channels decreased by decreasing the size of the channels. The mean droplet size varied negligibly across individual terraces over 7h. Slightly higher droplet generation frequencies and smaller percentages of active channels were observed at central terraces. The droplet generation frequencies at the channels located close to each other were similar, indicating that droplet formation dynamics was coupled. The droplet size was not affected by the dispersed phase flow rate.

Asymmetrical Microchannel Emulsification Plates for Production of Small‐Sized Monodispersed Emulsion Droplets

AbstractMonodispersed emulsion droplets have promising advantages in food, pharmaceutical, and chemical industries. Previous microchannel emulsification (MCE) plates having the capacity of generating small‐sized droplets exhibited very low droplet productivity since these plates operate at a low dispersed‐phase flow rate and with a relatively small number of microchannels. An innovative MCE plate with 176 176 circular asymmetric through‐holes was manufactured, which is comprised of a series of discrete outlets on each microchannel line with an effective cross‐sectional area of 1 cm2. The newly fabricated MCE plate is capable to produce monodispersed emulsion droplets with small Sauter mean diameters.