Double emulsion for encapsulation using microfluidics

Introduction to double emulsion using microfluidics

Double emulsions (DEs) are colloidal structures that co-localize amphipathic properties. They can be water-in-oil-in-water (W/O/W) or oil-in-water-in-oil (O/W/O) droplets. The use of immiscible liquids prevents transport across inner and outer environments, providing DEs with great protective capabilities for cargo [1]. 

The most common method for producing DEs is two-step agitation in bulk [1,2], resulting in a highly polydisperse population with low encapsulation efficiency, which can prove limiting for high precision applications such as pharmaceuticals. 

Pressure-driven flow-controlled droplet-based microfluidics is known for its high monodispersity and encapsulation efficiency [1], being well-positioned to address these limitations. This application note focuses on the production of W/O/W double emulsion droplets via microfluidics.

Applications

Double emulsions are versatile structures that can be used in several industries:

• Pharmaceutics: given the amphipathic and protective nature of double emulsions, they have great potential as drug delivery systems. Also, hydrophilic and hydrophobic drugs can be co-encapsulated in the inner (aqueous) and intermediate (oil) phases simultaneously, allowing for seemingly incompatible drug combinations [3,4]. 
• Food: emulsions are widely used in the food industry. Drawing from their protective and co-localization capabilities, double emulsions can be loaded with compounds that are otherwise easily degraded, such as phytochemicals [5,6].

Cosmetics: besides their previously mentioned advantages that can be also applied to cosmetics, double emulsions can be used as templates for microparticles [1].

Double emulsion microfluidic setup

Flow controller OB1 Mk3+

Tubings, fittings and reservoirs

Homemade PDMS microfluidic chip

Hardware

• OB1 flow controller with, at least, 3 channels (1 x 0/2000 mbar & 2 x -1000/+1000 mbar)
• Kit starter pack Luer Lock + 1/32”OD tubing with sleeves
• 1 x 50 mL Falcon reservoir with 4 ports for surface coating 
• 2 x 15 mL Falcon reservoirs for surface coating
• 3 x 1.5 mL Eppendorfs reservoirs for double emulsion generation
• Homemade PDMS Microfluidic chip (Petit et al, 2016. [7])
• Microscope for observation
• Vacuum pump
• High-speed camera for imaging

Chemicals

• Polyvinyl alcohol (PVA), 87-90% hydrolyzed, average mol wt 31,000-50,000 (Sigma Aldrich) for the hydrophilic treatment
• 1-octanol, anhydrous, ≥99% (Sigma Aldrich) as the intermediate/oil phase
• 1,2-dioleoyl-sn-glycero-3-phosphocholine, chloroform (DOPC) (Sigma Aldrich) as intermediate phase surfactant
• Poloxamer 188 (P188) 10% (wt/vol) solution (Sigma Aldrich) as outer phase surfactant
• Glycerol (Carlo Erba, Dutscher) for increased viscosity and stability
• Ethanol absolute (Carlo Erba, Dutscher) for the lipid stock solution

Design of the chip

The homemade PDMS chip used for this protocol is based on the published design of Petit et al, 2016 [7].
This design has a double junction, allowing for increased control of formation of each droplet (W/O and then W/O/W, for example).

Figure 1: (a) Overview of the Petit et al.,2016 [7] microfluidic chip design, representing the three different inlets and one outlet. (b) Specifications of the chip, (c) Picture of the double junction during double emulsion generation.

Surface coating for double emulsion production

A major part of a successful double emulsion production relies on surface interactions. Thus, the surface treatment is a key part of the process. To form W/O/W double emulsions, the chip needs to be hydrophobic in the first junction and hydrophilic in the second.

Figure 2: (a) Schematics of the regions with different surface interaction needs, hydrophobic on the left side and hydrophilic on the right side. (b) Representative image of a chip during surface coating treatment.

PDMS already presents hydrophobic properties, so PVA is used to turn the post-junction channel hydrophilic. In order to avoid getting PVA into the channels that should remain hydrophobic, positive air pressure is applied in the inner and intermediate channels, while vacuum is applied at the outlet, as shown in the schematics below.

Figure 3: Schematic of the microfluidic setup used for surface coating.

TIP: For optimal surface treatment with PVA, bind the PDMS chips to PDMS-covered glass slides, so the channel walls are all made from the same material.

TIP: For better results, wait overnight after plasma binding to do the surface coating. That will ensure that the PDMS is hydrophobic again and avoid attracting the PVA to the wrong channels, thus improving fluid control.

Quick start guide for double emulsion encapsulation using microfluidics

INSTRUMENT CONNECTION

• Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using a USB cable. For detailed instructions on OB1 pressure controller setup, please read the “OB1 user guide”.
• Turn on the OB1 by pressing the power switch.
• Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the “ESI user guide”. Please refer to the guide for a detailed description.
• Press Add instrument \ choose OB1 \ set as MK3+, set pressure channels if needed, give a name to the instrument and press OK to save changes. Your OB1 should now be on the list of recognized devices.
• OB1 calibration is required for the first use. Please refer to the “OB1 user guide”.
• Open the OB1 Window. 

SOLUTIONS PREPARATION

This protocol is based on Deshpande et al, 2018 [8].

• Hydrophilic surface treatment solution: prepare a 2,5% (wt/v) PVA solution in water. Place a beaker with distilled water with a clean magnetic stirrer over a hot plate stirrer and start stirring (~500 rpm). Gently add the respective weight of PVA to it. Gradually increase the temperature to 85°C and leave it under agitation until there are no more clumps of PVA (usually around 1,5 hour). Wait for the solution to cool down and readjust the volume to account for the evaporation.

TIP: 10 mL of solution is convenient to prepare and use in several chips and several experiments over time. Be careful because the PVA solution is prone to contamination. Always filter the solution with a syringe filter before storage to avoid clogging the chip channels.

• Inner Aqueous (IA) solution: Prepare a 15% (v/v) solution of glycerol in distilled water. Mix well. This solution can be stored at room temperature for as long as there are no aggregates. A typical experiment will use around 0,5 mL of solution, so a 10 mL solution is convenient to prepare for several experiments.

TIP: Cut the tip of the pipette tip to facilitate the pipetting of glycerol, which is very viscous. Release the pipette very slowly to give enough time for the glycerol to enter the tip. When releasing it into the solution, wait until all the glycerol that is stuck to the walls of the pipette tip accumulates at the bottom and press the pipette again. This might need to be repeated a couple of times for the tip to be completely empty.

• Outer Aqueous (OA) solution: Prepare a 15% (v/v) and 0,5% (v/v) of P188 solution of glycerol in distilled water. This solution can be stored at room temperature for as long as there are no aggregates. A typical experiment will use around 2 mL, so a 20 mL solution is convenient to prepare for several experiments.
• Lipid Stock solution: Prepare a 10% (wt/v) DOPC in ethanol. Dispense the respective amount of DOPC in chloroform with a glass syringe into a round bottom flask and evaporate the chloroform under a gentle stream of nitrogen to form a lipid film at the bottom of the flask. To ensure that all chloroform is evaporated, put the flask under partial vacuum in a desiccator for at least 2 hours. Add ethanol to form a 10% (wt/vol) solution and close the flask with parafilm. Dissolve the lipids in the ethanol as well as possible. Pipette the solution into a glass vial, replace the atmosphere of the vial with a gentle stream of nitrogen, seal the vial with Parafilm and place it at -20°C. The solution can be used as long as there are no aggregates or changes in color. 250 µL is convenient to prepare and use in several experiments.

TIP: If the lipids don’t dissolve well in ethanol, add equal parts of ethanol and chloroform up to 5% to 10% (wt/v). DOPC is easily oxidized, so avoid leaving it too exposed to the atmosphere and work with inert gases as much as possible.

• Lipid-oil (LO) solution: Prepare a 6,5 mM of DOPC solution in octanol. A typical experiment will use around 150 µL of LO solution for a half day experiment, and 250 µL for a full day experiment.

TIP: Prepare this solution right before use. To ensure the correct concentration of lipids, fill the pipette tip once with the DOPC in ethanol solution, empty it back into the stock and fill it up again.

CHIP SURFACE COATING

• Following the schematic on figure 3, connect a 15 mL Falcon tube (called PVA) containing 1 to 2 mL of 2,5% PVA solution to one of the vacuum channels of the OB1. Then, connect an empty 50 mL Falcon tube with the 4 ports (called Air) to the 2000 mbar channel (for the positive air pressure). And finally, connect an empty 15 mL Falcon tube (called Outlet) to the second vacuum channel (it will act as a waste). For more details, refer to “Elveflow microfluidic reservoirs assembly instructions”.

TIP: Use a 15 mL Falcon tube for the PVA solution because PVA foams under vacuum (applied during the chip cleaning step) and can easily enter the pneumatic tubing and potentially damage the OB1.

• Connect the tubings of the Air reservoir to the LO and IA inlets (inlets 2 and 3), and the Outlet reservoir to the outlet. For more details, refer to “reservoirs installation”.
• Set a pressure of 40 mbar for the Air reservoir (air starts flowing), and a vacuum of -50 mbar for the Outlet reservoir.
• Set a pressure 20 mbar to the PVA reservoir until the solution starts dripping out of the tubing and then connect the tubing to the OA inlet (inlet 1).
• Slowly decrease the pressure of the Air reservoir and increase the pressure of the PVA reservoir so that the PVA uniformly covers all of the post-junction channel and let it flow for 3 to 5 minutes.

TIP: The PVA solution should not enter the IA channel otherwise the chip will not be able to form double emulsions due to the wetting of the inner solution to the walls. If that happens, the chip is destroyed. It is good practice to have a “half-moon” of air pushing the PVA away from the walls of the inner channel, as shown in Figure 1.

• Increase the pressure of the Air reservoir to 2000 mbar while applying -1000 mbar of vacuum to both PVA  and Outlet reservoirs to clear the PVA from the chip.
  

TIP: For an instantaneous switch of these pressures, use the Editor Mode of the OB1 in the ESI. To avoid the PVA solution entering the pneumatic tubing of the 15mL Falcon tube and potentially damaging the OB1, turn off the vacuum on this channel and disconnect the tubing from the chip as soon as bubbles start to form inside the reservoir. Be sure to remove all the PVA from the chip before disconnecting the remaining tubings.

• Place the chip into an oven at 120 °C for, at least, 15 minutes.

DOUBLE EMULSION GENERATION

• Add [1.5 – 2] mL of IA and OA solutions into two separate Eppendorf tubes. Add [125 – 250] µL of LO solution into another Eppendorf tube. Connect the tubing to the three Eppendorfs. For more details, refer to “reservoirs installation”.
• Plug the three reservoirs to the corresponding OB1 pressure controller outlet, as represented in the setup figure. For more details, refer to “Elveflow microfluidic reservoirs assembly instructions”.
• Set a low pressure (10 mbar) of the OA solution until the solution starts dripping out of the tubing and then connect the tubing to the inlet 1. Fill the microfluidic chip with the OA solution.

TIP: Ensure that the tubing is all the way down in the inlet and there are no air bubbles.

• Repeat Step 2 for the LO (inlet 2) and the IA (inlet 3) solutions.

TIP: The order of the introduction of the solutions is important because the IA solution must not touch the walls before the LO solution to avoid it wetting the surface.

• The double emulsions can be collected into an eppendorf tube connected at the outlet. The resulting solution will have two phases: an aqueous phase at the bottom and an oil phase at the top. The double emulsions can be found at the interface of both phases.

TIP: Alternatively, double emulsions can be collected into other chips connected to the outlet of the production chip, like an observational chamber, for example. Regardless of the collection vial, the double emulsions take time to arrive because they float and usually get stuck at the highest curvature of the collection tubing. Eventually, they reach a critical number and flow through, reaching the desired location.

• Apply a pressure of 70 mbar for the OA solution and start increasing the pressure of the LO solution until oil droplets start to form. Then, increase the pressure for the IA solution until it reaches the second junction. Following, there is an example of pressure range to generate double emulsion:

TIP: It takes a few minutes for the production to stabilize and, in the beginning, it is common that the IA wets the surface and disrupts production. That might mean that the IA pressure is too strong in comparison to the LO pressure, so small changes (1 to 5 mbar) in one or the other might solve the problem. Make very gentle changes and observe if the production stability improves. When the production is going well, double emulsions can be formed until the solutions run out (usually the OA finishes first) without interference.

Double emulsion results

The produced double emulsions were tested in terms of monodispersity, encapsulation efficiency and stability over time.

The double emulsions were highly monodisperse (CV~5%) and presented an encapsulation efficiency of over 80%, as illustrated in Figure 4. The stability assays demonstrate that about 50% of the DEs remain stable after 5 days at RT, as illustrated in figure 5.

Figure 4: Encapsulation efficiency. Double emulsions with the intermediate phase stained with DiI, lipid-specific fluorescent dye (red, a), encapsulated calcein (green, b). c) Double emulsions encapsulating liposomes (POPC, 100 nm) stained with DiI.

Figure 5: Stability at RT. Percentage of remaining DEs and Size STDEV through time at RT. Double emulsions remain stable at RT for at least 72 hours, with over 50% of them still present after 5 days. Orange line, DEs radius in µm. 

This application note successfully demonstrated the reproducible production of stable double emulsions using droplet-based microfluidics technology and Elveflow instruments.

The double emulsions are highly monodisperse, present a high encapsulation efficiency of cargo and remain stable after several days at physiological temperature, thus addressing the limitations of the bulk methods, such as lack of reproducibility, high reagent consumption and low encapsulation efficiency.

References