Encapsulation of multiple emulsions in a single droplet
The objective of this application note is to describe the encapsulation of “core” multiple aqueous droplets inside a single oil droplet called a “shell”. To perform encapsulation of multiple emulsions, we used two Raydrop devices, and a capillary-based microfluidic device equipped with a 3D printed injection nozzle, developed and manufactured by Secoya. The two devices, installed in the series on the , allow the encapsulation of multiple emulsions in a single droplet with a fine tuning of size and number of encapsulated cores by varying the flow-rates.
Introduction to encapsulation of multiple emulsions
Traditionally, multiple emulsions are produced using batch methods, with a 2-step mixing of immiscible phases: a vigorous mixing followed by a gentle one. This method is straightforward and enables the production of large batches, but is limited by a wide size distribution. This leads to numerous particle sizes and a low encapsulation rate of Active Pharmaceutical Ingredient (API) or double emulsion production (core-shell particles)1.
Microfluidics emerged in the 2000’s as a powerful tool to overcome the limitations of batch methods as it allows a fine control over the size, monodispersity, and structure of droplets2. In particular, microfluidics is helpful when it comes to generating complex emulsions such as water-in-oil-in-water (W/O/W) or oil-in-water-in-oil (O/W/O) emulsions.
Based on the work of Li, E. Q. et al 3, a new tool called the Raydrop that had the ability to make controlled encapsulations of multiple emulsions was demonstrated by the scientific experts at Secoya technologies.These devices, which enable multiple applications that create single and double emulsions, are of particular interest for the creation of solid microcapsules used for triggered release1, or to co-encapsulate incompatible and cross-reactive solutions4.
We demonstrate in this application note that we can use two Raydrops placed in series to encapsulate multiple aqueous core droplets inside oil shell droplets with a precise control over droplet number and size.
Materials and methods for encapsulation of multiple emulsions
- 2% Tween20 (Sigma-Aldrich) in water
- Mineral oil light containing 2% ABIL EM 90 (Evonik) and 0.08% Bromocresol Purple (Sigma-Aldrich).
- Solution of 70% wt glycerol (>99.5%, Sigma-Aldrich) in water with 2% Tween20 (Sigma-Aldrich)
Complex emulsion production platform
A fully integrated platform, developed by Secoya, was used to make the encapsulation of multiple emulsions. Mechanic, fluidic, and optic modules are gathered to allow a straightforward, user-friendly and controlled production of simple and double emulsions.
It is composed of a displacement stage (in x,y,z) allowing focus adjustment and the correct positioning of the RayDrop observation window.
Three Flow-EZ pressure-based flow controllers with the required tubing and valves and connected to pressurized reservoirs perform the automated fluidic injection. Emulsions are generated in the RayDrop. (a filter placed after each reservoir prevents impurities from reaching the RayDrop).
A color USB 3.0 camera with a LED source is connected to a computer to observe the droplet formation in live, control the stability of the emulsion and measure the size of droplets core and shell.
The experimental setup used for the encapsulation of multiple emulsion generation is schematically represented in Figure 2. Here, a second RayDrop (R2) is placed next to the first RayDrop (R1).
|F1||50||Continuous||70% glycerol + 30 water + 2% Tween 20|
|F2||25||Shell (priming & cleaning)||Not used in this application note|
|F3||25||Shell||Mineral oil light + 2% ABIL 90 + 0.08% Bromocresol|
|F4||25||Core (priming & cleaning)||Not used in this application note|
|F5||25||Core||Water + 2% Tween 20|
The RayDrop is a microfluidic droplet generator based on the alignment of two glass capillaries: the first one is terminated by a 3D printed nozzle and injects the droplet phase in the second one. At the junction of both capillaries, the continuous phase filling the cavity pinches the jet of the droplet phase, leading to the formation of droplets with a high monodispersity.
In this case, two RayDrop systems are placed in a series in order to perform an encapsulation of multiple emulsions. A first device R1 is mounted on the platform as done for simple emulsion generation, and a second Raydrop R2 is added immediately after (see figure 2). To efficiently control the formation of droplets inside the second RayDrop, an additional optical module (LED + camera) is placed on the platform.
The oil phase, containing the aqueous “core” droplets encapsulated in the R1 device, is transported as the “shell” phase (see Figure 4) towards the R2 device. Here, the oil phase is pinched by the continuous phase, which leads to the formation of monodisperse oil droplets, each containing multiple aqueous cores. The number of cores in each droplet can be controlled by tuning the flow-rates.
The range of droplet sizes formed depends on the dimension of both the injection nozzle and the collection capillary. The dimensions used for the encapsulation of multiple emulsions in this application note are reported below.
|Part||RayDrop||Nozzle size (µm)||Collection capillary size (µm)|
Multiple emulsion generation
Oily double emulsions with multiple cores are formed. To generate droplets, the protocol below can be followed:
1. Set the valve V2 on the reservoir F3 containing the shell phase
2. Set the valve V3 on the reservoir F5 containing the core phase
3. Fill RayDrop R1 with the shell phase solution (refer to the user guide for more details about how to fill the RayDrop)
4. Fill RayDrop R2 with the continuous phase solution
5. Connect the two filled RayDrops together with the tubing
6. Carefully set the continuous phase to a low flow rate (for example, Qcontinuous= 40µl/min) and check that there is no backflow in the first RayDrop.
7. Set the shell phase to a low flow rate (for example Qshell= 7µl/min with Qshell<Qcontinuous) to create a simple oil in water emulsion visible in the second RayDrop.
8. Set the core phase to a low flow rate (for example Qcore= 1µl/min) to generate simple emulsions in R1, which leads to the generation of multiple emulsions in R2.
9. The encapsulation of multiple emulsions in a single droplet is now ongoing. The dripping mode provides a high stability for droplet formation. Droplets show high monodispersity. It is possible to vary the flow rates in order to change the size of the emulsion as well as the number of cores (see the following section for more details).
Partial results of the encapsulation of multiple emulsions
The number of cores in a single droplet depends on the flow rate of the core phase.
During the formation of the multiple emulsions in RayDrop R2, the flow rates of the shell and of the continuous phases are kept fixed. Thus, only the flow rate of the core phase is changed. By increasing the core flow rate, the number of encapsulated core droplets increases. For a core flow rate of 0.5µl/min, most droplets contain one core, for a core flow rate of 2.3µl/min, most droplets contain four cores.
Conclusion & Perspectives
The possibility to make an encapsulation of multiple emulsions in a single shell by using two Raydrop devices in series on the complex emulsion production platform was demonstrated. By playing with the flow-rates of the core phase, one can control the number of encapsulated cores. By combining the “two chips in series” platform structure with the use of a double emulsion Raydrop device, even more complex emulsions could be achieved, such as the multiple encapsulation of double emulsions in a single droplet.
 Vladisavljević, G., al Nuumani, R. & Nabavi, S. Microfluidic Production of Multiple Emulsions. Micromachines (Basel) 8, 75 (2017).
 Lee, T. Y., Choi, T. M., Shim, T. S., Frijns, R. A. M. & Kim, S.-H. Microfluidic production of multiple emulsions and functional microcapsules. Lab on a Chip 16, 3415–3440 (2016).
 Li, E. Q., Zhang, J. M. & Thoroddsen, S. T. Simple and inexpensive microfluidic devices for the generation of monodisperse multiple emulsions. Journal of Micromechanics and Microengineering 24, 015019 (2014).
 Wang, W. et al. Controllable microfluidic production of multicomponent multiple emulsions. Lab on a Chip11, 1587 (2011).
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