One‐Step Synthesis of Hydrogen‐Bonded Microcapsules for pH‐Triggered Protein Release

One‐step microfluidic approach in which interfacial complexation of hydrogen bonding polymers is utilized to prepare pH‐responsive microcapsules is presented. Using water‐in‐oil‐in‐oil‐in‐water (W1/O1/O2/W2) triple‐emulsion droplets as templates, it is shown that polymeric microcapsules with an aqueous core and a shell that consists of two complementary hydrogen bonding polymers, poly(propylene oxide)(PPO) and poly(methacrylic acid)(PMAA), can be prepared in a robust manner. The presence of a buffer layer enables controlled interfacial complexation of PMAA and PPO at the water/oil interface, followed by spontaneous dewetting of the oil droplet to result in hydrogen‐bonded microcapsules dispersed in aqueous media. In addition, it is demonstrated that the permeability of the PPO/PMAA membrane can be readily tuned by varying the molecular weight of PMAA. Furthermore, it is shown that the resulting PPO/PMAA microcapsules are stable at a high salt concentration (>1 m NaCl) unlike analogous capsules prepared through electrostatic complexation while they release the encapsulated protein above the critical pH of which the PMAA ionizes and results in disassembly of the shell membrane.


Introduction
Smart microcapsules are core-shell-structured microparticles with a membrane that effectively protects the sensitive and valuable actives loaded in the inner aqueous core from the surrounding environment and allow on-demand release of these actives by responding to diverse external stimuli including temperature, [1] salt, [2] chemical agents, [3] and mechanical stress. [4]These stimuli-responsive nature of microcapsules and the ability to precisely tune the release behavior have great potential in various fields such as food, medicine, and pharmaceutics.Among various stimuli explored, pH is one of the most widely investigated cues in the oral delivery of nutrients and therapeutics due to the distinctive pH environments in the gastrointestinal (GI) tract, [5] which enables localized delivery of the encapsulated active by appropriate design of the capsule shell membrane. [6]Moreover, the local pH is often affected by infection, inflammation, and cancer development, all of which make pH-responsive microcapsules suitable for the targeted release of actives in therapeutic applications. [7]H-responsive microcapsules are commonly prepared by photo-or thermally initiated polymerization of monomers that leads to the formation of a polymeric network consisting of charged functional groups such as acrylamide and carboxylic acids in which the degree of ionization and thus charge density is sensitive to a local change in pH.[8] However, due to the polar and water-soluble nature of the monomers, it is often challenging to directly prepare microcapsules with an aqueous core.While hydrophobic anhydride monomer and subsequent posthydrolysis of the shell membrane were recently exploited to fabricate pH-responsive poly(acid)-based microcapsules with an aqueous core, the additional hydrolysis procedure limits the usage of sensitive bioactive and thus demands postloading.[9] Alternatively, pH-responsive microcapsules with a membrane that forms through the complexation of two oppositely charged polyelectrolytes, polycation and polyanion, have been presented.[10] Depending on the nature of the polyelectrolyte used, whether they are strong or weak, they either completely dissociate within a reasonable pH ranging from 2 to 10 or exhibit fractional charge depending on the dissociation constant (pK a or pK b ) as well as the solution pH, ionic strength, counterion, and concentration, respectively.[11] As a result, the net electrostatic interaction among the polyelectrolytes can be altered by tuning these parameters, especially the solution pH, to modulate the pore size and thus the permeability of the shell membrane.While the effect of solution pH and ionic strength have been thoroughly explored to reversibly adjust the release profile, postload, and trap actives, the interdigitated structure of the resulting membrane limits the rapid response of the microcapsule to the local pH environment.Moreover, the net interaction among polyelectrolytes in electrostatic interactiondriven complexation depends not only on pH but also ionic strength of the media which make it difficult to precisely tune the pH condition at which the capsule releases the encapsulated actives.
These limitations can be mitigated to some extent by utilizing hydrogen bonding polymers in microcapsules.Unlike electrostatic interaction between oppositely charged polycation DOI: 10.1002/sstr.202300200One-step microfluidic approach in which interfacial complexation of hydrogen bonding polymers is utilized to prepare pH-responsive microcapsules is presented.Using water-in-oil-in-oil-in-water (W 1 /O 1 /O 2 /W 2 ) triple-emulsion droplets as templates, it is shown that polymeric microcapsules with an aqueous core and a shell that consists of two complementary hydrogen bonding polymers, poly(propylene oxide)(PPO) and poly(methacrylic acid)(PMAA), can be prepared in a robust manner.The presence of a buffer layer enables controlled interfacial complexation of PMAA and PPO at the water/oil interface, followed by spontaneous dewetting of the oil droplet to result in hydrogen-bonded microcapsules dispersed in aqueous media.In addition, it is demonstrated that the permeability of the PPO/PMAA membrane can be readily tuned by varying the molecular weight of PMAA.Furthermore, it is shown that the resulting PPO/PMAA microcapsules are stable at a high salt concentration (>1 M NaCl) unlike analogous capsules prepared through electrostatic complexation while they release the encapsulated protein above the critical pH of which the PMAA ionizes and results in disassembly of the shell membrane.and polyanion, hydrogen bonding polymer pairs comprise hydrogen acceptors and donors.For instance, poly(vinyl pyrrolidone) (PVPON) and poly(propylene oxide) (PPO) serve as hydrogen acceptors as they each have carbonyl and ether groups with lone electron pairs on electronegative atoms.On the other hand, weak polyacids such as poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) act as hydrogen acceptors at acidic pH where the acid groups are protonated.While the complementary interaction among these hydrogen bonding polymers at acidic pH leads to complex formation, the acid groups in hydrogen donor deprotonate and ionize at distinctive pH conditions above the pK a , resulting in rapid disassembly of the shell membrane.Moreover, the hydrogen-bonded complex is stable at high salt concentrations, [12] which makes this an ideal shell membrane that primarily depends on the solution pH.
These microcapsules based on hydrogen bonding polymers are conventionally prepared using layer-by-layer (LbL) assembly on colloidal substrates. [13]As LbL assembly allows precisely tuning the composition and the shell thickness by simply varying the type and number of cycles that the complementarily interacting polyelectrolytes are sequentially assembled, LbL assembly of hydrogen bonding polymers and subsequent dissolution and removal of the colloidal template yield hollow polyelectrolyte microcapsules comprising a shell with known composition and thickness. [14]However, LbL assembly requires a large number of repeated cycles with multiple rinsing steps in between, especially to acquire a thick and conformal shell.Moreover, the usage of sacrificial templates and removal implies the need for additional postloading of the desired actives after fabrication, limiting the broad applicability of such an approach.2b,15] In a separate work, a similar conceptual idea was extended to hydrogen bonding polymers but the demonstration was limited to microcapsules with an oil core, mandating additional oil core removal, resuspension in aqueous media, and postloading of actives for practical use. [16]Therefore, there is an unmet need for a new method that allows reliable and facile preparation of polyelectrolyte microcapsules with an aqueous core based on hydrogen bonding polymers.
In this work, we present a one-step microfluidic approach to prepare pH-responsive microcapsules.The interfacial complexation of hydrogen bonding polymers at water/oil interface of water-in-oil-in-oil-in-water (W 1 /O 1 /O 2 /W 2 ) triple-emulsion droplets prevents uncontrollable complexation and subsequent device clogging.The complementarily interacting hydrogen bonding polymer pair, PMAA and PPO, each dissolved in the aqueous core (W 1 ) and the outer oil phase (O 2 ), respectively, forms the shell membrane upon mixing of the two oil phases followed by interfacial complexation at the W 1 /O interface.Fluorescent dye permeability study on sets of PPO/PMAA microcapsules revealed that the permeability of the shell membrane can be readily tuned by varying the molecular weight of PMAA.Moreover, we show that the resulting microcapsules remain stable at high salt concentrations unlike the conventional polyelectrolyte microcapsules assembled through electrostatic interactions.
Furthermore, we show that the PPO/PMAA microcapsules rapidly disassemble above the critical pH of %5, thereby releasing the encapsulated protein.We envision that the strategy outlined in this work can be further extended to other hydrogen bonding polymer pairs to design smart microcapsules with tunable pH stability, offering new opportunities in microencapsulation technologies.

Synthesis of PPO/PMAA Microcapsules via Interfacial Complexation of Polymers
For reliable and facile preparation of hydrogen-bonded microcapsules, we utilized water-in-oil-in-oil-in-water(W 1 /O 1 /O 2 /W 2 ) triple-emulsion droplets as templates, as schematically illustrated in Figure 1a,b.To prepare these triple-emulsion droplets, we used a glass capillary microfluidic device, as described in our previous works, [17] and the details are in the Experimental Section.We injected pH 2-adjusted aqueous solution containing 1 wt% of PMAA (hydrogen donor) as the innermost aqueous phase (W 1 ) through the small tapered capillary in this device.Isopropyl myristate (IPM) containing 2 wt% Span 80 (surfactant) is supplied as the inner oil phase (O 1 ) through the hydrophobically modified injection capillary (left) to form a periodic stream of aqueous droplets dispersed in the oil phase due to preferential wetting of the oil on the injection capillary surface. [18]Next, IPM with the identical surfactant composition (2 wt% Span 80) but with an additional 1 wt% PPO (hydrogen acceptor) is supplied as the outer oil phase (O 2 ) through the interstices between the injection capillary and the square capillary.We note that due to the identical solvent used for both oil phases (O 1 and O 2 ), they mix immediately upon contact.Here, the inner oil phase (O 1 ) serves as a buffer layer, delaying the complexation between PMAA and PPO during emulsification, which often leads to clogging of the device.The triphasic coaxial flow is simultaneously emulsified at the entrance of the collection capillary (right) by shearing of the pH 2-adjusted outer aqueous phase (W 2 ) containing 10 wt% of poly(vinyl alcohol)(PVA, surfactant), yielding monodisperse triple-emulsion droplets that eventually become doubleemulsion droplets by mixing of the two oil phases.This leads to the diffusion of PPO to the W 1 /O interface followed by interfacial complexation of the two complementarily interacting hydrogen bonding polymers, PMAA and PPO.Due to the acidic aqueous environment, the carboxylic acid group of PMAA remains nonionized and hydrogen bonds with the ether group of PPO, leading to formation of the microcapsule shell membrane.Indeed, collection of the resulting emulsion droplets in a bath containing aqueous solution with a composition identical to the outer aqueous phase and monitoring the behavior of these droplets over time reveal that a polymeric membrane forms at the W 1 /O interface.This is followed by dewetting of the IPM droplet within a few minutes, resulting in hydrogen-bonded microcapsules, as shown in the scheme of Figure 1c.Interestingly, storage of the resulting microcapsules in an isotonic collection bath containing 10 wt% PVA leads to shrinkage of the microcapsules due to consumption of the PMAA through complexation at the W 1 /O interface.This results in a decrease in concentration of PMAA, which also serves as an osmolyte, and the solution becomes hypotonic compared to the 10 wt% PVA solution outside which is present in bulk.The osmotic imbalance across the shell membrane caused by the consumption of PMAA induces the outflux of water and shrinkage of the capsule shell.More importantly, we also observe that these microcapsules reswell back to a spherical shape.We attribute this behavior to the inward diffusion of PVA through the shell membrane over time until it reaches osmotic equilibrium.Subsequent transfer of these reswollen microcapsules in pH 2-deionized (DI) water without any PVA leads to removal of the residual PVA by outward diffusion and dilution.To verify this hypothesis of PMAA consumption and PVA diffusion that leads to shrinkage and reswelling after interfacial complexation and dewetting, we prepare another set of triple-emulsion droplets with identical composition but prelabeling the PMAA with a green fluorescent dye (fluorescein) to track the mass transport during the PPO/PMAA microcapsule formation process, as shown in Figure 1d-k.Monitoring the fluorescence signal over time showed that the initially uniform signal throughout the innermost aqueous core reduces in intensity over time and instead localizes at the shell membrane.The loss of fluorescence signal in the capsule interior after complete reswelling of the microcapsule clearly shows that PMAA consumption is the major cause of capsule shrinkage.To further verify whether the reswelling is primarily due to inward diffusion of PVA, we perform a separate set of experiments in which we redisperse the microcapsules in PVA solution with different molecular weights from 13-23 to 89-98 kDa (same mass concentration) and monitor the extent and time scale of reswelling.We observe that the extent of microcapsules shrinkage is much less while the reswelling time scale is longer for the higher-molecular-weight PVA, as shown in Figure S1, Supporting Information.Overall, these results indicate that the shrinkage and reswelling of the microcapsule are indeed attributed to the osmotic imbalance caused by the consumption of PMAA followed by subsequent inward diffusion of PVA through the shell membrane.

Characterization of PPO/PMAA Microcapsules
To confirm the uniformity of the resulting PPO/PMAA microcapsules after reswelling, we monitor their size distribution using an optical microscope after transfer to pH 2 DI water.The microcapsules exhibit an average diameter of 169 μm and a coefficient of variation (CV) of 6% as shown in Figure 2a,b and remain stable for more than 30 days without noticeable signs of disintegration.We note that the PPO/PMAA microcapsules are less monodisperse than typical microcapsules prepared via microfluidics due to the additional shrinkage and reswelling step involved.Fourier-transform infrared spectroscopy (FTIR) analysis of the resulting microcapsule shows that the shell membrane primarily consists of PPO and PMAA, as evidenced by the broad hydroxyl peak (3000-3750 cm À1 ) and the ketone peak (1700-1750 cm À1 ) from PMAA as well as the C-H stretching (3200 cm À1 ), C-H bending, and C-O stretching (<1500 cm À1 ), originating from PPO (Figure 2c).We note that the PVA used as the surfactant may also serve as a hydrogen acceptor [19] and hydrogen bond with PMAA.However, we find that the PVA adsorbed on the PPO/PMAA membrane can be easily removed by washing in pH 2 DI water as the majority of PMAA is already consumed through complexation with PPO (Figure S2, Supporting Information).On the other hand, visual inspection of the resulting microcapsules using an electron microscope after lyophilization and blading shows that the microcapsules exhibit a spherical shape with a liquid core and a solid shell (Figure 2d).

The Effect of PMAA Molecular Weight and PPO Concentration on Dewetting Phenomena
As the shell membrane results from interfacial complexation of PPO and PMAA, dewetting time, the time at which the IPM droplet containing PPO dewets from the PPO/PMAA membrane, dictates the shell thickness.Measuring the dewetting time with variation in the molecular weight of PMAA from 4 to 9.5 to 100 kDa and PPO concentration from 1 to 5 to 10 wt% reveals that the dewetting time tends to be shorter as the molecular weight of PMAA is lower and when the concentration of PPO is higher, as shown in Figure 2e.This suggests that the dewetting time scale is related to the diffusion of the PMAA to the interface, which decreases with the molecular weight [20] as well as the diffusion flux of PPO that is proportional to the concentration gradient. [21]To also verify the origin of the dewetting phenomena, we conduct a separate experiment in which we prepare analogous PPO/PMAA microcapsules dispersed in IPM utilizing water-in-oil-in-oil (W 1 /O 1 /O 2 ) double-emulsion droplets as templates.Collection of the resulting emulsion droplets in a vial containing 10 wt% PVA aqueous solution shows that the microcapsules formed within the IPM phase spontaneously partition to the underlying aqueous phase after interfacial complexation due to the preferential interaction of the PPO/PMAA shell membrane with the aqueous phase than IPM (Figure S3, Supporting Information).Overall, these results indicate that a higher concentration of PPO and a lower molecular weight of PMAA may induce rapid formation of the shell at the interface that prefers to interact with the aqueous media, leading to faster dewetting.We note that the high concentration of PPO can also promote dewetting by depletion force. [22]Interestingly, as the dewetting of the IPM droplet halts the interfacial complexation of PPO and PMAA, this yields PPO/PMAA microcapsules with somewhat similar shell thickness regardless of PPO concentration and PMAA molecular weight.Indeed, we observe no significant difference in shell thickness when the PPO concentration was increased by 5-and 10-fold, as shown in the electron micrographs of Figure 2f-h.While a similar tendency in thickness is anticipated for microcapsules prepared with different molecular weight PMAA at constant PPO concentration (1 wt%), microcapsules consisting of high-molecular-weight PMAA (100 kDa) exhibit a crumpled morphology upon electron microscope imaging, as shown in Figure S4, Supporting Information.We attribute this behavior to the formation of a loosely complexed shell membrane with an increase in molecular weight of the constituent polymer that results in a decrease in the mechanical stiffness, as similarly observed by others. [23]

PPO/PMAA Microcapsules with Tunable Molecular Permeability
The ability to precisely control the molecular architecture of the shell membrane by simply altering the molecular weight of the PMAA has great potential for tuning the permeability of the resulting PPO/PMAA microcapsules.To demonstrate this, we prepare sets of microcapsules with different-molecularweight PMAA and separately monitor the molecular transport of green, fluorescent dye labeled dextran (FITC-dextran) with varying molecular weights across the microcapsule membrane over time using a confocal microscope as shown in Figure 3a-f.For simplicity, we will omit the PPO concentration from the microcapsule description hereafter, and thus all microcapsules are prepared with 1 wt% PPO solution unless specified.Close examination of these sets of confocal micrographs shows that both 4 kDa FITC-dextran (denoted as, FITC-dextran 4k ) and 2000 kDa FITC-dextran (FITC-dextran 2000k ) diffuse through the membrane of PPO/PMAA 100k microcapsules consisting of 100 kDa PMAA.Due to the larger hydrodynamic radius of the FITC-dextran 2000k compared to FITC-dextran 4k , we observe slower diffusion into the microcapsule, as shown in Figure 3a,b.Meanwhile, decreasing the molecular weight of PMAA from 100 to 9.5 kDa substantially reduces the permeability these FITC-dextrans with different molecular weights, as evidenced by the confocal micrographs of Figure 3c,d, where we observe a dramatic decrease in fluorescence intensity in the core of the PPO/PMAA 9.5 k microcapsules even after 1 h after exposure to FITC-dextran 2000k (Figure 3d).Performing analogous experiments on PPO/PMAA 5k microcapsules with further decrease in PMAA molecular weight confirms that utilizing lower-molecular-weight PMAA indeed yields microcapsules with more tightly structured shell membranes with lower permeability.(Figure 3e,f ) To further determine the change in permeability of the microcapsule with respect to variation in PMAA molecular weight, we monitor the relative fluorescence intensity ratio inside and outside the microcapsules over time using FITC-dextrans with an even broader range of molecular weights from 4 to 40 to 500 to 2000 kDa, as shown in Figure 3g-i.Comparing the relative fluorescence intensity among PPO/PMAA microcapsules reveals that for PPO/PMAA 100k microcapsules, all dextran molecules diffuse into the microcapsule, and dextrans with molecular weight higher than 500 kDa exhibit slower transport.In contrast, only FITC-dextran 4k readily permeates through the PPO/PMAA 9.5k and PPO/PMAA 5k microcapsules while dextrans with molecular weight higher than kDa do not diffuse into the capsules.As the hydrodynamic radius of dextrans with molecular weight of 4, 40, and 2000 kDa each corresponds to less than 1.86, 4.78, and 26.89 nm, respectively, [24] this indicates that the PPO/PMAA microcapsules synthesized using either 5 or 9.5 kDa PMAA are nonpermeable to molecules with a hydrodynamic radius of 4.78 nm or more while molecules with a hydrodynamic radius of 1.86 nm or less can diffuse out from the microcapsules.Likewise, much larger molecules with hydrodynamic radius of 26.89 nm or less will diffuse out of PPO/PMAA 100k microcapsules.We attribute this behavior to the effective pore size that depends on the molecular weight of the polymer constituting the shell membrane.As a result, PMAA with smaller molecular weight forms a denser network, which leads to smaller pore size and thus lower permeability, as observed similarly by others. [21]f note is that the permeability of the PPO/PMAA microcapsule also affects the reswelling behavior of the capsule in PVA solution after shrinkage.As reswelling is due to diffusion of PVA, the time scale of capsule reswelling not only depends on the molecular weight of the PVA but also on the pore size of the microcapsule shell, which is a function of PMAA molecular weight, as evidenced by the longer reswelling time for PPO/ PMAA microcapsules consisting of lower-molecular-weight PMAA (Figure S5, Supporting Information).

pH-Responsive Properties of PPO/PMAA Microcapsules
The use of weak polyacid as the hydrogen donor in microcapsules offers new opportunities in achieving pH-responsive microcapsules as the PMAA incorporated in the shell becomes ionized when exposed to pH conditions higher than the complexation pH of 2. When these hydrogen-bonded microcapsules are exposed above this critical pH, the hydrogen bonding interaction between PPO and PMAA is disrupted, and the shell disintegrates. [16]To first confirm whether the solution pH indeed has any effect on the integrity of the PPO/PMAA 9.5k microcapwe monitor the behavior of the microcapsules upon dispersion in pH 2-and pH 6-adjusted DI water.While the PPO/PMAA 9.5k microcapsules remain intact for more than 30 days in pH 2 solution, the microcapsule gradually failed to maintain its shape and suddenly disappeared within an hour of exposure to pH 6-adjusted DI water.To demonstrate the utility of this pH-responsive property of PPO/PMAA microcapsules in encapsulation and on-demand release of proteins, we select FITC-labeled pepsin as the model protein and encapsulate them in the innermost aqueous core of the PPO/PMAA 9.5k microcapsules.The microcapsules retained more than 90% of the pepsin within the microcapsule even after 3 days of storage in pH 2adjusted DI water.However, upon dispersion in pH 6-adjusted DI water, the microcapsules rapidly disintegrated.Timelapse confocal micrographs show that the encapsulated pepsin starts to release through the microcapsule pore 3 min 30 sec after exposure to pH 6-adjusted DI water due to membrane disintegration.After 5 min, the morphology of the capsule becomes significantly distorted, differing completely from its initial shape.This is followed by complete decomposition of the capsule and release of the remaining pepsin into the media (6 min 30 sec), as shown in Figure 4a.We note that due to the higher permeability of the PPO/PMAA 100k microcapsules, these microcapsules cannot effectively retain pepsin (35 kDa) within the microcapsule even in pH 2-adjusted DI water (Figure S6, Supporting Information).
To determine the critical pH above which the PPO/PMAA 9.5 k microcapsules disintegrate, microcapsules are separately immersed into sets of pH-adjusted DI water ranging from 2 to 7, respectively, and the ratio of capsules remaining over time was recorded.While we observe that increasing the pH slightly above 2 does not deteriorate the integrity of the microcapsule, complete disintegration throughout the overall population is observed above pH 4.5 as shown in Figure 4b.At pH 4.5, more than 91 AE 6% of the capsules maintained their shape even after 2 h, but at pH 5 or higher, the extent of disintegration increased dramatically and almost no capsules remained after 2 h.This indicates that the PPO/PMAA microcapsules allow on-demand burst release of the internal substances at pH 5 or higher.More importantly, while the molecular weight of PMAA has a significant effect on the permeability, we observe negligible effect on the pH-responsive property (Figure 4c) Furthermore, unlike the electrostatic interaction-driven polyelectrolyte microcapsules which generally undergo disintegration at high salt concentrations by charge screening effect, [15] we find that the PPO/ PMAA microcapsules remain intact even in 1M NaCl solution due to the hydrogen bonding-driven complexation of polymers (Figure 4d).Overall, this clearly demonstrates that the one-step microfluidic synthesis of pH-responsive microcapsules outlined in this work through interfacial complexation of hydrogen bonding polymers provides the means to prepare smart microcapsules with tunable permeability and pH-responsive release properties.

Conclusions
In summary, pH-responsive microcapsules were microfluidically synthesized through interfacial complexation of hydrogen bonding polymers for encapsulation and on-demand release of proteins.Using triple-emulsion droplets as templates, hydrogenbonded microcapsules with an aqueous core were synthesized in one step and in a continuous and a robust manner without device clogging.In contrast to traditionally manufactured capsules using the LbL method, we demonstrated that hydrogenbonded microcapsules can be synthesized without the need of repeated cycles with multiple rinsing steps in between using centrifugation.More importantly, we showed that cargoes such as proteins can be encapsulated without the need for postloading, a process which can substantially limit the broader applicability of these microcapsules.Systematic investigation on the effect of molecular weight of PMAA revealed that a more densely structured membrane with lower molecular permeability can be achieved by utilizing PMAA with lower molecular weight.More importantly, it was shown that while the PMAA molecular weight has significant effect on the molecular permeability, the pH-responsive behavior does not depend on the molecular weight but only on the type of weak polyacid used.As there exists a library of weak polyacids with distinctive pK a values, we envision that the hydrogen-bonded microcapsule strategy outlined in this work would offer new perspectives in the design of smart microcapsules with the ability to selectively release the encapsulated actives above the predetermined pH condition.
Synthesis of PPO/PMAA Microcapsules: We used a custom-made glasscapillary microfluidic device for the preparation of water-in-oil-in-oil-inwater (W 1 /O 1 /O 2 /W 2 ) triple emulsion droplets.Briefly, glass capillary was processed using a micropipette puller (Sutter instrument model P-97) and polished with a sandpaper for the injection and collection capillaries to have ID of 100 and 300 μm, respectively.Injection and collection capillary were each treated with ODTS and 3-[methoxy (polyethyleneoxy)9-12]propyltrimethoxysilane, respectively, for 2 h or more and subsequently washed with DI water.After surface treatment of these capillaries, they were aligned within the square tubing.An additional thin capillary was prepared by pulling above a torch and was used as the innermost capillary.The flow rates of the W 1 , O 1 , O 2 , W 2 phases were set to 1000, 1000, 2500, 8000 μL hr À1 , respectively, using syringe pumps (KDS Legato 100).The production of triple-emulsion droplets was monitored with an inverted microscope (Eclipse Ts2, Nikon) and recorded using a camera (FASTCAM Mini UX50, Photron).After the microcapsules were formed, the dewetted IPM droplets on top of the collection bath were removed and the microcapsules were washed three times with pH 2-adjusted DI water to remove the remaining PVA.For the experiment where PPO/PMAA microcapsules dispersed in IPM were prepared by using water-in-oil-in-oil (W 1 /O 1 /O 2 ) double-emulsion droplets as templates, a similar glass-capillary microfluidic device was used except for the absence of innermost capillary and the treatment of the collection capillary with ODTS instead of 3-[methoxy(polyethyleneoxy)9-12]propyltrimethoxysilane.The flow rates of W 1 , O 1 , O 2 phases were set to 1000, 2000, 6000 μL hr À1 , respectively, during operation.
Synthesis of PPO/PMAA Complex for PVA Adsorption Study: To prepare the PPO/PMAA complex for PVA adsorption study, 1 wt% of PPO dissolved in IPM was added to pH 2 adjusted, 1 wt% PMAA solution.The mixture was vortexed for 5 min and then centrifuged at 8000 rpm for 1 h to obtain the PPO/PMAA complex.To remove the residual IPM, PPO/PMAA complex was washed three times using hexane.For comparison, the half of the PPO/PMAA complex was left for control, while the other half was dispersed in pH 2 adjusted, 10 wt% of PVA solution for 3 h and was washed using pH 2-adjusted DI water.The resulting PPO/ PMAA complex was dried at room temperature for subsequent FTIR spectra acquisition.
Synthesis of Fluorescein-Labeled PMAA: PMAA was first dissolved in 20 mL of DI water and adjusted to pH 5. The final molar concentration of methacrylic acid groups was set at 0.138 M and 1 mol% of methacrylic acid groups were activated using 21.16 mg of 1-ethyl-3-(3-(dimethylamino) propyl)carbodiimide (EDC) and 12.71 mg of N-hydroxysuccinimide (NHS).This corresponded to molar ratio of EDC, NHS, and methacrylic acid groups to 4:4:1 and were stirred for 1 h at room temperature.Then, 9.59 mg of 6-aminofluorescein was added and the mixture was stirred for additional 12 h at room temperature without exposure to light.The reacted solution was dialyzed for 48 h at 4 °C to remove the unreacted 6-aminofluorescein and the final solution was freeze dried at 4 °C.
Synthesis of Fluorescein Isothiocyanate (FITC)-Labeled Pepsin: 1 g of pepsin was dissolved in 10 mL of DI water.10 mg of FITC dissolved in DMSO was added to the pepsin solution.The mixture was stirred for 2 h at room temperature without exposure to light.The reacted solution was dialyzed for 24 h at 4 °C to remove the unreacted FITC and freeze dried to acquire the FITC-pepsin.
pH Responsivity Measurement of PPO/PMAA Microcapsule: To monitor the pH responsivity of the PPO/PMAA microcapsules, 20 μL of microcapsule dispersion (pH 2) was separately added to each glass well filled with 4 mL of pH-adjusted DI water.The DI water in each well was pH adjusted a priori so that the final mixture yielded pH ranging from 2 to 7. Ten or more microcapsules were monitored in triplicate for each measurement to acquire the remaining capsule ratio over time.FITC-labeled pepsin was added to PMAA solution at a concentration of 0.5 mg mL À1 in preparation of pepsin-encapsulated microcapsules.Release of pepsin over time was observed using a confocal laser scanning microscope.
Other Characterizations: PPO/PMAA microcapsules dispersed in pH 2 DI water were deposited on a silicon wafer using a micropipette and dried at room temperature.The dried microcapsules were frozen using liquid nitrogen and subsequently cut with a razor blade for cross-sectional imaging using scanning electron microscope.For confocal laser scanning microscope imaging in the permeability test, FITC-labeled dextran was first dissolved in pH 2-adjusted DI water at a concentration of 0.5 mg mL À1 .After filling the glass well with 2 mL of FITC-dextran solution, 20 μL of microcapsule dispersion was added for imaging.Ten or more microcapsules were monitored over time using confocal laser scanning microscope and the fluorescence intensity inside and outside the microcapsule were analyzed using the ImageJ software.FTIR spectra were collected using FTIR spectroscope (PerkinElmer) in transmittance mode.
Statistical Analysis: Microcapsule diameter was measured using NIS-Elements Basic Research software.Among the observed microcapsules, capsules that were excessively crushed and failed to maintain their spherical shape were excluded from the measurement.FTIR spectra were normalized using OriginPro software.Relative fluorescence intensity was measured using the ImageJ software.For each fluorescence image, the relative fluorescence intensity was obtained by dividing the fluorescence intensity inside the capsule excluding the capsule shell by the intensity outside the capsule.The microcapsule dewetting, shrinkage, and swelling time were measured after recording the screen.The dewetting time was set by the time when the microcapsule was completely separated from the oil droplet, while the shrinkage and reswelling time were each set by the time when the microcapsule was most deformed and completely returned to its original spherical shape, respectively.

Figure 1 .
Figure 1.Microfluidic synthesis of hydrogen-bonded microcapsules.a) Schematic illustration and b) optical micrograph of the glass capillary microfluidic device used to generate triple-emulsion droplet templates for preparation of microcapsules.c) Schematic illustration describing the interfacial complexation and subsequent dewetting, shrinkage, and reswelling process.d-g) Series of optical micrographs and h-k) fluorescence micrographs depicting the PPO/PMAA microcapsule formation which includes (d,h) interfacial complexation, (e,i) dewetting, (f,j) shrinkage, and (g,k) reswelling.All scale bars represent 100 μm.

Figure 2 .
Figure 2. Characterization of PPO/PMAA microcapsules.a) micrograph of PPO 1wt% /PMAA 9.5 k microcapsules.The subscript in PPO and PMAA each denotes the concentration and molecular weight, respectively, and the scale bar represents 100 μm.b) Plot showing the resulting microcapsule size distribution.c) FTIR spectra of PPO 1wt% /PMAA 9.5 k microcapsule, PPO, and PMAA 9.5 k , respectively.d) Top-view scanning electron micrograph of PPO 1wt% /PMAA 9.5 k microcapsules.Scale bar represents 10 μm.e) Plot showing the variation in dewetting time with respect to PMAA molecular weight and PPO concentration.Molecular weight of PPO was set at 4 kDa while PMAA molecular weight was varied from 4, 9.5, 100 kDa, respectively.The bar graphs represent the mean and standard deviation (n = 10).f-h) Sets of cross-sectional electron micrographs showing the microcapsule shell formed at different PPO concentrations.f ) 1 wt%, g) 5 wt%, and h) 10 wt% PPO, respectively.

Figure 3 .
Figure 3. Molecular permeability of PPO/PMAA microcapsules.a-f ) Sets of timelapse confocal micrographs showing the diffusion of FITC-dextran with varying molecular weight into PPO/PMAA microcapsules consisting of PMAA with different molecular weight.The concentration of the FITC-dextran solutions was set at 0.5 mg mL À1 and adjusted to pH 2. All scale bars represent 100 μm.a,b) PPO/PMAA 100k microcapsules.c,d) PPO/PMAA 9.5 k microcapsules.e,f ) PPO/PMAA 5k microcapsules.g-i) Plots showing the change of relative fluorescence intensity ratio inside and outside the microcapsules over time.The data points in the plots represent the mean and standard deviation (n = 10).g) PPO/PMAA 100k microcapsules.h) PPO/PMAA 9.5 k microcapsules.i) PPO/PMAA 5k microcapsules.

Figure 4 .
Figure 4. pH responsivity of PPO/PMAA microcapsules.a) Timelapse confocal laser scanning micrographs and optical micrographs showing the disintegration of PPO/PMAA 9.5 k microcapsules containing FITC-pepsin in pH 6-adjusted DI water.Scale bar represents 100 μm.b) Plot showing the effect of pH on disintegration of PPO/PMAA 9.5 k microcapsules.c) Plot showing the effect of PMAA molecular weight on disintegration of PPO/PMAA microcapsules in pH 5-adjusted DI water.d) Plot showing the effect of salt concentration (NaCl) on the disintegration of PPO/PMAA 9.5 k microcapsules.All data points in the plots represent the mean and standard deviation of three measurements of each sample containing 10 or more capsules.