M5904

The preparation of the lipid-in-oil mixture is based on published protocols^{27 (link),71 (link)}. We use POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids, Inc.) with 1% DSPE-PEG(2000) biotin (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], Avanti Polar Lipids, Inc.; both 25 mg/ml in chloroform) and give 77 µl thereof in a 10 ml vial with 600 µl chloroform. In experiments with labeled membranes, 3 µl DOPE-ATTO655 (0.1 mg/ml in chloroform) is added. While being mixed on a vortex mixer, 10 ml of a silicon oil and mineral oil (Sigma Aldrich, M5904) mix (4:1 ratio) is slowly added to the lipid solution. Since the lipids are not fully soluble in this mix of silicon oil, mineral oil, and chloroform, the resulting liquid is cloudy.

For the synthesis of the core-shell nanoparticles, iron oxide nanoparticles were firstly synthesized within the droplets of the reactor, as previously described^{24 (link),34 (link)}. Briefly, through the first two fused silica capillaries of the reactor, two aqueous solutions were injected at a flow rate of 10 µL/min. The first solution was 0.06 M of FeCl₃·6H₂O (Alfa Aesar, USA) and 0.03 M of FeCl₂∙4H₂O (Sigma-Aldrich, USA) dissolved in DI water, and the second solution was 4 M ammonia (Alfa Aesar, USA). As a continuous phase mineral oil (M5904, Sigma-Aldrich USA) with 0.075 vol % Triton X-100 (Samchun Chemical, Korea) and 1.75 vol % Abil EM 90 (Evonik Industrial, Germany) was used, the flowrate injected through the central Tygon tubing was 10 µL/min. For the synthesis of the gold shell around the iron cores, a gold precursor solution was injected into the existing droplets through the single capillaries at 100, 130, and 160 cm. The gold precursor solution consisted of 0.03 M HAuCl₄ (Sigma-Aldrich USA) dissolved in DI water. For all three injections, the same flowrate was used and flowrate was determined by a self-optimizing algorithm based on the transmission of the droplets and two initial guesses for the flowrate. To quench the reaction, core-shell nanoparticles were collected in a vial filled with water, once they left the capillary droplet reactor.

Originally, the experiment would have alginates from Sigma Aldrich (W201502) of various concentrations between 0 to 5%. However, any concentration above 2% proved detrimental to the droplet’s stability and some trials showed no formation of droplets. To prepare the alginate in water solutions of 1% and 2% w/w, two steps were conducted. Firstly, a mixture of 0.5–1.0 g alginate with 50 mL of distilled water was prepared in a beaker glass. Secondly, stirring with a magnetic bar was performed for 1 day at room temperature. Then, the alginate solution was ready to use. The solution was stored in a refrigerator at a temperature of around 6–7 °C. The oil phase used was from Sigma Aldrich (St. Louis, MO, USA) (M-5904). Properties for fluids in this experiment are presented in Table 1.

Deionized water was used as the dispersed phase, and 97 wt % mineral oil (M5904, Sigma Aldrich, St. Louis, MO, USA) and 3 wt % SPAN^®80 (S6760, Sigma Aldrich, USA) were used as the continuous phase. SPAN^®80 was used to prevent merging with each microdroplet in the microchannel before coming out from the device. The dispersed and continuous phases were injected into the fabricated microfluidic device using syringe pumps (Pump 11 Elite & Pico Plus, Harvard Apparatus, Holliston, MA, USA). Microdroplets in the microchannel were inspected using an optical inverted microscope (CSB-IH5, Samwon Scientific Ind. Co., Ltd., Seoul, Korea) as shown in Figure 1c. The diameter of microdroplets was analyzed using the Image J program with at least 30 samples. The microdroplet production rate was measured using a high-speed camera (Motionxtra N3, IDT, Tallahasse, FL, USA).

GUVs solution were incubated in the dark at temperatures of 4 °C, 25 °C, 37 °C, and 45 °C. Samples were taken on day 0, day 1, day 3, day 5, and day 7 of incubation, respectively. Before sampling, the homogenous solution was thoroughly mixed. Then, 10 μL of the GUVs solution was dropped into the glass bottom cell culture dish (801001, NEST, China), sealed with mineral oil (M5904, Sigma, USA), and allowed to settle for 3 h in the dark. The samples were viewed using an inverted fluorescence microscope (RVL-100-G, ECHO, San Diego, CA, USA) or a confocal laser scanning microscope (CLSM, A1R; Nikon, Tokyo, Japan), and the center of the GUVs solution and eight directions around it were photographed and recorded. Experiments were repeated three or more times and used for subsequent data analysis. Individual GUVs or aggregates were observed with a high-power lens and the Z-axis layer sweep recombination technique of CLSM (thickness is 0.5 μm). The excitation and emission wavelengths of DiO are 488 nm and 500–520 nm, and the excitation and emission wavelengths of DiI are 551 nm and 569 nm, respectively.