Non-covalent reconfigurable microgel colloidosomes with a well-defined bilayer shell

Microgels are extremely interfacially active and are widely used to stabilize emulsions. However, they are commonly used to stabilize oil-in-water emulsions due to their intrinsic hydrophilicity and initially dispersed in water. In addition, there have been no attempts to control microgel structural layers that are formed at the interface and as a result it limits applications of microgel in advanced materials. Here, we show that by introducing octanol into poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAM-co-MAA) microgels, octanol-swollen microgels can rapidly diffuse from the initially dispersed oil phase onto the water droplet surface. This facilitates the formation of microgel-laden interfacial layers with strong elastic responses and also generates stable inverse water-in-oil Pickering emulsions. These emulsions can be used as templates to produce microgel colloidosomes, herein termed ‘microgelsomes’, with shells that can be fine-tuned from a particle monolayer to a well-defined bilayer. The microgelsomes can then be used to encapsulate and/or anchor nanoparticles, proteins, vitamin C, bio-based nanocrystals or enzymes. Moreover, the programmed release of these substances can be achieved by using ethanol as a trigger to mediate shell permeability. Thus, these reconfigurable microgelsomes with a microgel-bilayer shell can respond to external stimuli and demonstrate tailored properties, which offers novel insights into microgels and promise wider application of Pickering emulsions stabilized by soft colloids.

Table. S1 System composition of hydrogen bond systems. 6 Table. S2 System composition of oil-water interface systems. The CLSM image and (c) size distribution of PNIPAM-co-MAA microgels dispersed in octanol. The size of microgels in octanol is around 800 nm which is slightly smaller than dispersed in water.

Experimental Procedures
Materials.

Synthesis of fluorescent PNIPAM-co-MAA microgels.
Typically, 2.4 g monomer (NIPAM) were firstly dissolved into 30 mL deionized water and then transferred to a 250 mL three-neck bottle flask. Then, 0.064 g crosslinker (MBA), 600 μL co-monomer (MAA) and 1 mL PolyFluor® 570 solution [1 mg/mL, 10%(v/v) ethanol solution] were added into monomer solution followed by nitrogen gas bubbling for 30 mins. During the de-gas process, the temperature of water bath increased from room temperature to 60°C. After that, 2 mL initiator solution containing 0.11 g KPS dissolved in deionized water was injected into the reaction mixture to initiate polymerization. Subsequently, the reaction solution turned from transparent to translucent in few mins and then to totally opaque in 1 h. The complete synthesis of PNIPAM-co-MAA microgels lasted for about 5 h.
To endow green fluorescence to PNIPAM-co-MAA microgels. 10 mL non-stained microgels with COOH groups (0.5 wt%) were firstly activated by 15 mg NHS and 20 mg EDC for 20 min at pH 5. Then TEA was added into the reaction solution to adjust the pH value around 7.4 followed by adding 0.25 mg fluoresceinamine to react with COOH groups in microgels. The reaction would last for 12 h to obtain the green fluorescent PNIPAMco-MAA microgels

Synthesis of fluorescent PDEAEMA microgels.
Specifically, 0.5 g VBTMAC was firstly dissolved into 100 mL deionized water in a 250 mL three-neck round flask. Then, 10 mL DEAEMA and 100 μL EGDMA were introduced in the flask with 1mL PolyFluor® 570 solution [1 mg/mL, 10%(v/v) ethanol solution]. The monomer solution was de-gassed by bubbling nitrogen gas for 30 mins. After increasing water bath temperature up to 60°C, 3 mL initiator solution containing 100 mg AIBA was added in the reactant system to initiate polymerization. The polymerization reaction was carried on for 24 h under magnetic stirring (450 rpm) before the collection and purification of products.

Synthesis of fluorescent PS-co-MAA latex particles.
For the synthesis of PS latex particles with COOH functional groups, 5.5 mL styrene, 0.1 g DVB, 250 μL MAA and 5mL PolyFluor® 570 solution [1 mg/mL, 10%(v/v) ethanol solution were dissolved in 140 mL deionized water and then transferred into a 250 mL two-necked round flask with nitrogen gas bubbling inlet and outlet for 1 h. The reactant solution kept stirring at 400 rpm by using a magnetic stir bar. After increasing the temperature of water bath from room temperature to 70°C, 10 mL initiator solution containing 0.15 g KPS was injected into the reaction medium to initiate the reaction. The reaction was conducted in 70°C for 24 h to finish the polymerization.
The purification of synthetic fluorescent PNIPAM-co-MAA microgels and PS-co-MAA latex particles.
All synthesized microgels and PS latex particles were collected and purified by high spped centrifugation to remove the unreacted monomers, oligomers, unreacted initiator and fluorescent dyes. Specifically, PNIPAM-co-MAA microgels were purified by centrifucation at 10000 rpm for 20 min. The centrifugation and re-dispersion would conducted for at least five times to get the purified PNIPAM-co-MAA microgel dispersion. Finally, the purified microgel dispersion was freeze-drying to get the lyophilized microgels for following experiments. Similarly, the dispersion of PS-co-MAA latex particles was centrifuged at 12000 rpm for 10 min. The whole centrifugation and re-dispersion process would repeat for four times to get the particle dispersion.

Preparation of conventional O/W Pickering emulsions.
For the preparation of conventional O/W Pickering emulsions. The concentration of PNIPAM-co-MAA microgels or PDEAEMA microgels in water phase was adjusted to 1 wt% before use. After that, same volume of oil phase (e.g., toluene and dodecane) was added into the emulsification system. Then the two immisable liquids were mixed together by vortexing at 2700 rpm for 1 min to form conventional O/W Pickering emulsions.

Preparation of inverse W/O Pickering emulsions solely stabilized by PNIPAM-co-MAA microgels.
The preparation of inverse W/O Pickering emulsions involved the special treatment of PNIPAM-co-MAA microgels. In detail, PNIPAM-co-MAA microgels were firstly freeze-drying under vacume to prepare lyopilized microgels. The in situ modification of microgels can be achieved by two strategies. The first one is using a slight of octanol to swollen microgels powders before dispersing into an apolar oil phase such as dodecane and toluene. Another strategy is directly adding microgel powders into apolar solvents followed by adding a low concentration of octanol. Both of the two methods can facilitate the hydrophobilized modification of PNIPAM-co-MAA microgels. Typically, PNIPAM-co-MAA microgels (1 wt%) and octanol (10 vol%) were added into the oil phase followed by ultrasonification till microgels were well modified and dispersed in oil phase. Then, same volume of deionized water was added and mixed with the oil phase which was stained by perylene. The final W/O Pickering emulsion was obtained by vortexing at 2700 rpm for 1 min.
In situ preparation of microgelsomes co-stabilized by binary microgels based on electrostatic attraction.
The fabrication of microgelsomes was based on the preparation of inverse W/O Pickering emulsions (W:O = 1:1, v/v) by voetxing at 2700 rpm. Binary microgels were introduced in a special Pickering emulsion system which was utilized as the template for further microgelsomes fabrication. Typically, the oil phase contained 0.5 wt% PNIPAM-co-MAA microgels with 10 vol% octanol, and the water phase contained 0.5 wt% PDEAEMA microgels with opposite charges. After emulsifying these two immiscible liquids by vortexing, two type of microgels with opposite charges would like to self-assemble onto the interface thus constructing ordered interfacial bilayer structure based on electrostatic attraction. The strong connection between microgels can help fabricate microgelsomes based on reversable physical bonding.

Encapsulation and anchoring of nanoparticles, bio-based nanocrystals and enzyme in inverse W/O Pickering emulsion systems.
Different kinds of substances including PNIPAM-co-MAA microgels and PS latex particles, starch nanocrystals and lipase were used to investigate the encapsulation and anchoring effect of inverse W/O Pickering emulsions. Generally, 0.5 wt% negatively charged PNIPAM-co-MAA microgels were dispersed in the perylene stained oil phase initially and another substance was added into the water phase before emulsification. After emulsification by vorteing at 2700 rpm at neutral condition, like-charged substance would encapsulated inside emulsion droplets because of electrostatic repulsion. On the other hand, oppositely charged substance would anchor on the interface because of electrostatic attraction.

The effect of interfacial structure of microgelsomes on their encapsulation and controlled release performances.
The encapsulation and controlled release performances of microgelsomes were conducted by using phycocyanin and vitamin C as model components. Microgelsomes with particle bilayer were prepared by using 0.5 wt% PNIPAM-co-MAA microgels in oil phase and 0.5 wt% PDEAEMA in water phase. 1 wt% phycocyanin and 0.045 wt% vitamin C were dispersed or dissolved in water phase initially. The emulsification involved mixing water phase with oil phase together (oil:water = 3:1, v/v) by vortexing at 2700 rpm for 1 min. Then, the corresponding microgelsomes floated on the surface of oil phase (DCM), followed by the careful addition of 4 mL of deionized water. To aviod the oxidation, the pH value of deionized water was adjusted to 3 for the release detection of vitamin C. For ethanol induced programmed release of vitamin C, 4 mL ethanol was used to substitude water as the receptor phase. The glass vials containing microgelsomes were kept at 25 °C in a shaking chamber, and 100 μL of the supernatants was periodically extracted for the subsequent UV−visible measurement.

Dynamic interfacial tension, interfacial rheology measurement and contact angle measurement.
The dynamic interfacial tension (IFT) and interfacial rheology between oil and water as well as water contact angle of different microgels were measured by an all-purpose contact angle measuring & contour analysis system (OCA 25, Dataphysics) at room temperature (25 °C) and analyzed with SCA 20 software. Briefly, "pendent drop" method was applied to measure the IFT and interfacial rheology. For example, a 10 µL of water drop was injected at 15 µL/s from the syringe and suspended in the oil containing 0.1 wt% PNIPAM-co-MAA microgels and 0.5 vol% octanol for continuous measurement. The shape of droplet was captured and analyzed by Young-Laplace equation to get the dynamic IFT. Based on the recorded instantaneous IFT and surface area, the interfacial dilational rheology of microgel self-assembled oil-water interface was measured by calculating dilational moduli. The interface was measured at surface area (A) of 20 mm 2 , deformation amplitude (dA/A) of 0.07 or 0.05, frequency (ω) of 0.05 Hz for at least 2 h. The dilational modulus |E*| was determined through the equation below: Where A represents the surface area of the droplet, dA and dγ are the surface area change and interfacial tension change during the measurement, respectively. The interface was measured within the viscoelastic range with neglectable inertia under the measurement parameters.
"Sessile drop" method was used to obtain the water contact angle between pure water and microgel surface in both air and oil. Before the contact angle measurement, the concentration of PNIPAM-co-MAA microgels and PDEAEMA microgels in dispersions was diluted to 1 wt%. Then microgel dispersions was dripped on the surface of silicon wafer and dried at room temperature for 4 h to evaporate water. After dripping and drying, a multi-layer of microgels was deposited on the substrates. For water contact angle in the air, a 5 µL water drop at different pH values was dripped and stayed on the wafer surface in the air. To obtain the three-phase contact angle of microgels, the silicon wafer coated with microgel multilayers were soaked in the oil solution containing different concentration of octanol before measurement. Afterwards, 5 µL water drop was dripped on the wafer surface in the oil. The shape of water drop was captured, and the water contact angle can be automatically calculated by SCA 20 software. All contact angle measurement was repeated 3 times on the same substrates. The error bar comes from the difference between the left and right contact angle by computer calculation.

Characterization.
The optical microscopy images of the prepared emulsions were obtained with an optical microscope (OLYMPUS, Japan). The confocal micrographs were taken with a Nikon Eclipse Ti inverted microscope (Nikon, Japan). The confocal laser scanning microscope (CLSM) was equipped with a 40×oil immersion objective. Perylene, FITC, and rhodamine B were excited by a laser with wavelength 408, 488 and 543 nm, respectively. The emulsions were placed on the cover glasses and a series of x/y layers was scanned. Dynamic light scattering (DLS) and ζ potentials were done by Zetasizer Nano ZS90 (Malvern). For SEM observation, microgels and particles were fully dried and then sputter-coated with a thin layer of Au prior to imaging using a Quanta 400F (FEI Company) scanning electron microscope equipped with a field emission electron gun at 10 kV. The concentrations of octanol as a function of microgels amount were determined using GC-2010 Plus (Shimadzu, Japan) equipped with a flame ionization detector (FID) on a column (AT-WAX column, 30.0 m length, 0.53 mm inner diameter, 1.00 μm film thickness). The encapsulation and release experiments were measured by using a UV−visible spectrometer (Alpha-1, Shanghai Lab-spectrum Instruments Co., Ltd.) to calculate the absorbance at 615 nm and 260 nm, respectively.

Simulation details.
We carried out all the simulations using the GPU-accelerated OpenMM (7.5.0) simulation package 1, 2 and the CHARMM Generalized Force Field (CGenFF) 3 . Langevin Integrator was applied and the temperature was maintained at 298 K. The time step was 2 fs. The Lennard-Jones interactions were smoothly switched off between 10 and 12 Å by a forced-based switching function. Long-range electrostatic interactions were calculated using the particle-mesh Ewald (PME) method 4 with an error tolerance of 10^(-5). The polymer parameters were generated by the CHARMM-GUI polymer builder 5 and the starting structure was built with the Packmol package 6 .
For systems investigating hydrogen bonds, a polymer was solvated in the oil box (9nm×9nm×9nm) and the system compositions are shown in Table S1. The isotropic simulation cells were maintained by the Monte Carlo barostat at 1 bar. The first 10 ns simulation was discarded in the analysis process. For labelling the polymers, the two numbers in the parentheses represent the numbers of the two types of monomers in the diblock copolymer, respectively. If there is only one number in the parentheses, it is then the number of that only monomer type.
In the oil/water interface simulations, the size of the box was kept at 4.44nm×4.44nm×8.21nm and the interfaces located at around z = 0 nm and z = 3 nm. The system compositions are shown in Table S2. In each simulation, different numbers of octanol were added to the system, shown in Fig. 2f3. The number of toluene molecules was adjusted to maintain a similar volume. During equilibration, the polymers were restrained at the center of the oil region in the z-axis (perpendicular to the interface) with a force constant of 1000 kJ/mol -1 under constant external pressure at 1 bar for 3 ns. In the production run, constant NVT dynamics was applied without a barostat, and the restraints on the polymers were removed. 30 simulations runs were carried out for each system and 3 polymers were included in each simulation. In Fig. 2f3, we considered each polymer separately in a certain simulation, even though polymers may form aggregates and affect the dynamics on each other before reaching the interface. Therefore, each point in Fig. 2f3 was computed based on the 90 polymers in 30 runs (3 interacting polymers in each run).