A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells

Abstract In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1140/epje/s10189-024-00428-5.


Interfacial Tension Measurements 1.Interfacial Tensions Between I-M and M-O Phases
The interfacial tensions between the inner and middle (I-M) and middle and outer (M-O) phases are determined by an inverted pendant drop method using an Attention Theta (OneAttention) tensiometer.The interfacial tension value is recorded until the pendant drop detaches from the dispenser.Although interfacial tension measurements for the I-M and M-O without F-68 samples are recorded for >5 min, the interfacial tension rapidly decreases in the M-O with F-68 sample for ≈1 min before the drop detaches from the dispensing needle.To systematize our reported measurements, a time period of 1 min is chosen (Figure S2).

Interfacial Tension Between I-O Phases
Since directly measuring the interfacial tension of the bilayer membrane between two water phases is not feasible, the method described involves estimating the interfacial tension of a monolayer that forms a bilayer between two water droplets (I and O) in an oil phase (M) [1][2][3].This is achieved by conducting adhesion experiments with two polymer-stabilized water drops dispersed in a continuous oil phase.The interfacial tensions are calculated based on the balance of forces in the system.The primary formula used in this process is the Neumann triangle equation: where γ IO represents the interfacial tension of the monolayer forming the bilayer, and γ IM and γ M O are the interfacial tensions between the water and oil phases.The angles, θ 1 and θ 2 , are the angles formed at the point of contact between the water droplets and the oil phase.Essentially, θ 1 corresponds to the angle at the interface between the oil phase and water droplet I, and θ 2 corresponds to the angle at the interface between the oil phase and water droplet O.These angles range between π/2 and π.
The adhesion energy (∆F) of the system is given by the following equation: The interfacial tensions between the oil-water phases (γ IM and γ M O ) are determined using the pendant drop method, and γ IM is calculated using Equation S1.The method also considers two boundary conditions based on the behavior of the droplets: Case 1: When the angles θ 1 and θ 2 equal π, there is no adhesion between the drops, leading to γ IO = γ IM + γ M O and ∆F = 0.This indicates the absence of an adhesion force, although the possibility of γ IO being greater than γ IM + γ M O (∆F < 0) is also possible.For simplicity of the calculation, we only consider Case 2: When the angles θ 1 and θ 2 are π/2, the doublet forms a spherical shape.In this scenario, γ IO = 0, and ∆F = γ IM + γ M O > 0, suggesting that polymersomes composed of this bilayer would have a zero interfacial tension.
For the adhesion experiment, I and O drops are independently formed at the ends of two separate 20 µm capillaries submerged in M (2 mg/mL PEO 30 -b-PBD 46 in 30:70 chloroform:hexane).These drops are brought into contact and their configurations are recorded (Videos S3 and S4) on an optical microscope (Nikon Diaphot 300) for subsequent analysis.

Figure S1
Figure S1The surface tension over time between I-M and M-O, with and without F-68.Each interfacial tension measurement is taken three times and averaged.