Semipermeable Microcapsules with a Block-Polymer-Templated

Subscriber access provided by READING UNIV

Article

Semipermeable Microcapsules with a Block Polymer-Templated Nanoporous Membrane Jaehoon Oh, Bomi Kim, Sangmin Lee, Shin-Hyun Kim, and Myungeun Seo Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b04340 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemistry of Materials is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials

Semipermeable Microcapsules with a Block Polymer-Templated Nanoporous Membrane Jaehoon Oh,,† Bomi Kim,,‡ Sangmin Lee,‡ Shin-Hyun Kim,*,‡ and Myungeun Seo*,† †

Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea ‡

Department of Chemical and Biological Engineering, KAIST, Daejeon 34141, Korea

ABSTRACT: Microcapsule with nanoporous membranes can regulate transmembrane transport in a size-dependent fashion while protecting active materials in the core from the surrounding, thereby being useful as artificial cell models, carriers for cells and catalyst, and microsensors. In this work, we report a pragmatic microfluidic approach to producing such semipermeable microcapsules with precise control of the cut-off threshold of permeation. Using a homogeneous polymerization mixture for polymerization-induced microphase separation (PIMS) process as the oil phase of water-in-oil-in-water (W/O/W) double emulsions, a densely crosslinked shell composed of a bicontinuous nanostructure that percolates through the entire thickness is prepared, which serves as a template for a monolithic nanoporous membrane of microcapsules with size-selective permeability. We demonstrate the nanopores with precisely controlled size by the block polymer self-assembly governs molecular diffusion through the membrane and renders manipulation of the cut-off threshold.

INTRODUCTION Microcapsules whose membranes possess uniform and well-defined pores can provide size-selective permeability.1,2 The membranes allow transport of materials smaller than the pores, while preventing the exchange of the larger. Such microcapsules can store large active materials without leakage and regulate transmembrane transport at the same time, which is beneficial for various chemical and biological applications, including catalytic reactions, microreactors, and immuno-isolation of cells.3–6 To produce semipermeable microcapsules, layer-by-layer deposition1,7 and interfacial colloidal assembly8 have been used on the particle or emulsion-drop templates. In addition, aerosol drops have also been used as templates to produce microcapsules with porous shells.9, 10 However, the conventional methods have limited control over cut-off threshold, capsule uniformity, and encapsulation efficiency. Droplet-based microfluidics has enabled the production of monodisperse semipermeable microcapsules with high encapsulation efficiency by employing double-emulsion drops as templates.11–13 The shell membranes have been designed to have uniform pores that interconnect the cores and surrounding by employing either colloidal self-assembly or phase separation of polymers.14–16 Colloidal assembly in the shell produces regular pores at the interstitial voids, of which size is determined by that of templating colloids;14 this enables the formation of nanopores with sub-100 nm colloids. However, the colloidal assemblies are fragile against mechanical stress, only providing limited use. Although phase separation between two distinct polymers15 or between a growing polymer and a liquid porogen16 yields mechanically stable microcapsules, precise control of pore

size is elusive, especially in sub-50 nm scale; this is because the characteristic length scale of separated domains are determined by interaction parameters and concentration or polymerization rate, both of which are very difficult to manipulate. Therefore, a robust microfluidic method to simultaneously achieve high mechanical stability of membrane and controllability over the cut-off threshold for permeation remains important yet unmet demand. Microphase separation of block polymers has provided highly ordered porous nanostructures with the pore size of typically 5–50 nm.17 The ordered lattice morphology is predominantly determined by the relative fraction of blocks, and the feature size is dictated by molar mass. Although the microphase separation in the shell of double-emulsion drops can produce regular nanopores in the membrane of microcapsules through selective removal of one block, it is challenging to create the nanopores that percolate the entire thickness of membranes.18,19 This is because bicontinuous phases, such as gyroid, can be prepared only with a highly limited window of stability. We have recently developed a facile strategy for production of three-dimensionally (3D) continuous nanopores by employing polymerization-induced microphase separation (PIMS).20,21,22 PIMS offers a synthetically feasible route to producing a disordered bicontinuous phase via in-situ synthesis and simultaneous crosslinking of block polymers that induces microphase separation during polymerization. Therefore, 3D network of continuous nanopores can be easily created in the crosslinked polymeric matrix through the removal of sacrificial domains. Nanoporous monoliths and free-standing films (ca. 100 μm) have been successfully prepared by the PIMS process by the bulk polymerization and the polymerization

ACS Paragon Plus Environment

Chemistry of Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of concentrated polymerization mixtures, respectively.20,21,22 We envisioned that if the polymerization mixture for the PIMS process can be segregated in confined space such as a thin middle layer (< 2 μm) of double-emulsion drops fabricated by the microfluidics and the polymerization proceeds akin to the bulk polymerization, it would be possible to construct microcapsules with a densely-crosslinked and also microphase-separated shell by the PIMS process, which could be readily converted into nanoporous microcapsules with a size-controlled 3D continuous nanopores. As the PIMS process starts with the polymerization mixture that is spontaneously transformed into the bicontinuous nanostructure, we anticipated that all the difficulties associated with controlling and aligning the microphase-separated morphology in the doubleemulsion drops could be avoided. Here we design microcapsules to have a spacious core and 3D nanoporous membrane by employing PIMS in the thin oil shell of water-in-oil-water (W/O/W) double-emulsion drops. With a capillary microfluidic device, the double-emulsion drops are prepared to have an ultra-thin oil shell that contains a solution for the PIMS process, including polylactide containing a chain transfer agent at the terminus (PLA-CTA) for reversible addition-fragmentation chain transfer (RAFT) polymerization, styrene as a monomer, divinylbenzene (DVB) as a crosslinker, and azobisisobutyronitrile (AIBN) as a thermal radical initiator to polymerize the styrenic monomers at 70C. During the polymerization, polylactide-b-poly(styrene-co-divinylbenzene) (PLA-b-P(S-co-DVB)) is formed and the polystyrenic block is simultaneously crosslinked in the shell due to incorporation of DVB. At the same time, the PLA and P(S-coDVB) blocks in the in situ-formed block copolymers are microphase-separated to form disordered bicontinuous microdomains. Selective removal of PLA domains leaves behind a monolithic membrane with a 3D network of uniform nanopores that percolate the crosslinked PS framework. The monolithic structures provide high mechanical stability of membranes thanks to the densely crosslinked PS framework. Also, the membranes provide size-selective permeability, of which cut-off threshold is precisely controlled by varying molar mass of PLA in the range of 5–30 nm. A systematic study on transmembrane transport behaviors of the semipermeable microcapsules through confocal microscope imaging with various sizes of dye molecules demonstrates such a unique ability of the membranes.

RESULTS AND DISCUSSION Microfluidic production of microcapsules with a nanoporous membrane. For the fabrication of the microcapsules with different pore sizes, PLA-CTAs with the number-average molar mass (Mn) of 13, 24, and 35 kg mol-1 were synthesized following the previously reported procedure21 and designated as PLA-CTA-13, PLA-CTA-24, PLACTA-35, respectively (see Figures S1, S2, and Table S1 in the Supplementary Information for synthetic details and characterization of PLA-CTAs). We confirmed that the synthe-

Page 2 of 9

sized PLA-CTAs can be effectively used in the PIMS process by conducting bulk copolymerizations of styrene and DVB in the presence of PLA-CTAs and AIBN at 70 C. The condition of WPLA-CTA = 30% and [styrene]:[DVB] = 4:1 was used for all three PLA-CTAs. The DVB content is known to affect onset of gelation that arrests the emergent microphase separated morphology, as well as crosslinking density which is important in maintaining the mesopore structure against pore collapse.22 At the 4:1 ratio, the produced porous monolith has been reported to exhibit an ultimate tensile strength of 6.4 MPa, an ultimate elongation of ~9%, and a Young’s modulus of ~170 MPa.20 [PLACTA]:[AIBN] was varied to 1:1, 1:1.8, and 1:2.7 for PLA-CTA13, PLA-CTA-24, and PLA-CTA-35, respectively. High AIBN concentration was used to conduct polymerization in air and achieve high crosslinking density by consuming more pendent double bonds of DVB repeating units in P(S-coDVB) matrix during the polymerization. The monolithic PLA-b-P(S-co-DVB) precursors were prepared, which were then treated with NaOH solution to remove PLA. The derived nanoporous monoliths were analyzed by Fourier transform infrared (FTIR) spectra, small angle X-ray scattering (SAXS), scanning electron microscope (SEM) images and nitrogen sorption experiments, which manifest the formation of 3D continuous nanopores based on the PIMS process20 as shown in Figures S3-S6 and Table S2. In the SAXS data shown in Figure S3, all the samples showed a broad principal scattering peak (at q*) accompanied by a broad shoulder at 2q* which was consistent with the PIMS process that produced a disordered bicontinuous morphology consisting of PLA and P(S-co-DVB) microdomains with some degree of local order,20 similar to those formed by spinodal decomposition at the late stage.23 The position of q* exhibited a shift to smaller q with the increasing molar mass of PLA-CTA, indicating increases in the domain spacing d (= 2π/q*) and also the size of PLA microdomain as the WPLA-CTA was fixed as 30%. Assuming densities of PLA and P(S-co-DVB) as 1.2524 and 1.0525 the average size of the PLA microdomains was roughly estimated as 5.8, 6.6, and 8.7 nm for PLA-CTA-13, PLA-CTA-24, and PLACTA-35, respectively. The shape of the SAXS pattern and the position of q* were virtually identical to those produced with low radical concentration,20 indicating that effect of P(S-co-DVB) homopolymer generated due to high AIBN loading26 in the early stage of polymerization would not significantly alter the microphase separated morphology27 and eventually join the crosslinked P(S-co-DVB) matrix during the polymerization. Complete removal of PLA was confirmed by FTIR spectra (Figure S4), and visualization by SEM corroborated formation of the desired nanopore structure (Figure S5). Huge scattering intensity with virtually no change in the shape of the SAXS pattern supports formation of the nanopore templated by the PLA microdomain (Figure S3). The H2type nitrogen sorption isotherms shown in Figure S6 were also consistent with the presence of 3D porous network28 with the increasing pore size as molar mass of PLA-CTA increased. The mean pore diameters were determined as 7.4, 8.8, and 10.9 nm for PLA-CTA-13, PLA-CTA-24, and


Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials PLA-CTA-35 respectively from Barrett-Joyner-Halenda (BJH) analysis29 of nitrogen sorption isotherms. Porosity of the nanoporous monoliths was in the range of 0.32 - 0.35 g mL-1, which matched well with the theoretical pore volume of 0.34 g mL-1 assuming the porous space was entirely templated by PLA.

Figure 1. Microfluidic fabrication of microcapsules with a membrane of bicontinuous nanostructure. (a) Optical microscope image showing the generation of double-emulsion drops in a glass capillary microfluidic device. (b) Schematic illustration of polymerization-induced microphase separation (PIMS) in the shell of double-emulsion drops to make bicontinuous domains of polystyrene (PS) and polylactic acid (PLA). The PIMS mixture in the shell is composed of PLA containing a chain transfer agent at the terminus (PLA-CTA), styrene, and divinylbenzene (DVB), as illustrated in left panel. (c) Optical microscope image of microcapsules obtained by polymerization of PIMS mixture. Inset is confocal microscope image of the microcapsule suspended in the solution of sulforhodamine B. The membrane is nonporous and impermeable for sulforhodamine B. (d) Scanning electron microscope (SEM) image of the dried microcapsules. Inset is a magnified view of the membrane surface which reveals no pores. Scale bars are 100 µm for a,c,d. Scale bar for inset of d indicates 200 nm.

The identical polymerization mixture was used in the microfluidic fabrication of double-emulsion drops as an oil phase to construct semipermeable microcapsules. To make the shells ultra-thin, we used a glass capillary device composed of two tapered cylindrical capillaries that are coaxially aligned in a square capillary.12 As the innermost and continuous phases, 10 w/w% aqueous solution of polyvinyl alcohol (PVA) was used; PVA adsorbs at the interfaces between the aqueous solution and middle oil phase and stabilizes them when double-emulsion drops are formed.30 The innermost water and middle oil phases spontaneously form a flow of water core and oil sheath. The continuous phase emulsifies the core-sheath flow at the capillary tip to form monodisperse double-emulsion drops with ultra-thin shell, as shown in Figure 1a and Supplementary Movie S1;

both double-emulsion drops and single oil drops were repeatedly generated due to the discontinuity of the coresheath flow.12 The drops passing through the hydrophilic collection capillary were collected in an aqueous solution of 9 w/w% PVA and 5 mM NaCl. The solution had the same osmolarity of 100 mOsm L-1 to the innermost phase but slightly lower density, leading to slow sedimentation of the double-emulsion drops with an ultra-thin shell while preventing water flux through oil shell. Floating single oil drops due to the lower density were readily separated from the double-emulsion drops, as shown in Figure S7. The double-emulsion drops were incubated at 70 °C for 2 days. During the incubation, styrene was copolymerized with DVB in the presence of PLA-CTA to form PLA-b-P(Sco-DVB) via the RAFT mechanism, and the emergent block polymer underwent microphase separation to yield solid membrane composed of two bicontinuous domains of crosslinked PS and PLA, as illustrated in Figure 1b. Under the identical condition, the polymerization mixtures consisting of PLA-CTA-24, styrene, and AIBN (1.8 eq to PLACTA-24), and PLA-CTA-24, styrene, DVB, and AIBN successfully produced PLA-b-PS and PLA-b-P(S-co-DVB) without degassing, supporting that the PIMS process proceeded via the RAFT mechanism in aerobic environments such as W/O/W double-emulsion drops by high AIBN loading (Figures S8 and S9, and Table S3). In case of PLAb-P(S-co-DVB), formation of high molar mass species due to incorporation of DVB was consistent with the literature.20,31 High transparency of shell was retained even after the polymerization as shown in Figure 1c; the colors are developed by thin-film interference in the membrane.32 The microcapsules were highly deflated and buckled upon drying of water (Figure 1d). Nevertheless, the membrane maintained its integrity without rupturing or fracturing, indicating high stability of the membrane originating from the densely crosslinked P(S-co-DVB) network. At this moment, the solid membrane was nonporous, and a smooth and featureless texture was observed from the membrane surface by SEM (inset of Figure 1d). Therefore, diffusion of small dye molecules such as sulforhodamine B into the microcapsules was not allowed (inset of Figure 1c). To remove the PLA block from the membrane, the microcapsules were immersed in the mixture of methanol and water with 4:6 volume ratio containing 0.5 M NaOH for 24 h at room temperature, then washed with distilled water. The etching leaves behinds network of nanopores in the crosslinked PS membrane, as illustrated in Figure 2a. The complete etching of PLA was confirmed by FTIR analysis (Figure S10). The microcapsules with nanoporous membrane were also highly deflated after drying, but showed no mechanical failure despite the large bending strain induced by folding (Figure S11a). The high mechanical integrity of the membrane against bending was again attributed to the monolithic P(S-co-DVB) network densely crosslinked at the molecular scale. Nanoporous texture was observed both from the membrane surface and the entire cross-section by SEM, consistent with removal of PLA by FTIR (Figure S11b and c). Slight differences in the porous texture between the nanoporous microcapsules and the



Page 4 of 9

Table 1. Transmembrane transport of dye molecules for microcapsules made from PLA-CTA-13 FITC-dextran Dye molecules

Sulforhodamine B

Mw = 4 kg

Mw =

mol-1

10 kg

Mw = mol-1

20 kg

Mw = mol-1

70 kg mol-1

Hydrodynamic diameter, dH (m)

1.09×10-9

2.80×10-9

4.60×10-9

6.60×10-9

12.0×10-9

Permeability, P (m s-1)

3.27 ×10-7

7.90 ×10-8

2.54 ×10-8

4.34 ×10-9

0.00

(m2 s-1)a

×10-13

×10-14

×10-14

×10-15

0.00

Diffusivity, D

3.27

7.90

2.54

4.34

Free diffusivity, DO (m2 s-1)

4.50 ×10-10

1.75 ×10-10

1.07 ×10-10

7.43 ×10-11

4.09 ×10-11

D/DO

7.26 ×10-4

4.51 ×10-4

2.38 ×10-4

5.85 ×10-5

0.00

aEstimated

by product of permeability and membrane thickness (approximately 1 μm).

nanoporous monoliths was tentatively attributed to plastic deformation generated during the sample preparation. The thickness of the nanoporous membrane varied capsule-bycapsule but mostly on the order of 1 μm, as we confirmed with the cross-sectional SEM images; a representative cross-sectional image in Figure 11c shows a thickness of 1.5 μm.

Cut-off threshold and permeability of nanoporous membranes. Permeability of the membrane made from PLA-CTA-13 was studied by monitoring fluorescence intensity in the core of the microcapsules (I(t)) suspended in the aqueous solutions of dye molecules with different hydrodynamic diameters (dH) over time and shown in Figure 2b; sulforhodamine B as a molecular dye and fluorescein isothiocyanate (FITC)-tagged dextrans with average molar mass (Mw) of 4, 10, 20, and 70 kg mol-1 were used, which have dH of 1.1, 2.8, 4.6, 6.6, and 12.0 nm, respectively. A confocal microscope was used to clearly distinguish the fluorescence signal from the inner volume of the microcapsule from that of the surrounding and quantitatively determine concentration of the dye molecule diffused into the microcapsule. For all the dye molecules, I is linearly proportional to the concentration (c), as shown in Figure S12. Therefore, I(t)/Imax can be interpreted as c(t)/cmax, where Imax and cmax are the maximum intensity and concentration of dye, respectively. While I(t) increased over time for the other four dye molecules indicating diffusion through the nanoporous membrane from the surrounding, I(t) remained zero for nonpermeable FITC-dextran with Mw 70 kg mol-1. This indicates that the cut-off threshold of permeation is between 6.6 nm and 12.0 nm in the case of PLA-CTA-13. FITCdextran with Mw 20 kg mol-1 required a much longer time for I(t) to reach a plateau (Imax) compared with the other permeable molecules (Figure S13). We roughly estimated permeability of each molecule by fitting the data using a diffusion equation (simplified solution of Fick’s law): 𝐼(𝑡)

Figure 2. Microcapsules with a nanoporous membrane. (a) Schematic illustration of selective etching of PLA domains to produce nanoporous membrane made of crosslinked PS framework and molecular diffusion through the membrane. (b) Temporal dependence of normalized fluorescence intensity in the core of microcapsules for sulforhodamine B (squares), FITC-tagged dextrans with Mw 4 kg mol-1 (circles), 10 kg mol-1 (triangles), 20 kg mol-1 (inverted triangles), and 70 kg mol-1 (diamonds). Fits with equation (1) are shown by solid lines. The curve for Mw 20 kg mol-1 is obtained from a fit for a time lapse of 30000 s.

𝐼𝑚𝑎𝑥

3𝑃

= 1 − 𝑒 −( 𝑟 )𝑡

(1)

, where P is permeability, r is the radius of the microcapsule, and t is diffusion time.14 Although the equation (1) fitted well to the temporal variation of fluorescence intensity for relatively small molecules, it was inappropriate to describe those for large molecules whose diffusion is highly hindered by pores. Nevertheless, the fits with equation (1) provided rough estimation of the permeabilities. The permeabilities of sulforhodamine B and FITC-tagged dextran with Mw of 4, 10, and 20 kg mol-1 were measured as 3.27 ×10-7, 7.90 ×10-8, 2.54 ×10-8, and 4.34 ×10-9 m s-1, respectively. Then their diffusivity (D) was estimated as the product of P and membrane thickness and compared with the calculated


Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials free diffusion constants (DO) using Stokes-Einstein equation33 (Table 1). Apparently, the diffusion through the nanopores was more inhibited as the molecule size was closer to the cut-off threshold, as the molecule with comparable size to the pore would have less chance to progress through the pores. For example, the diffusivity of FITC-dextran with Mw 20 kg mol-1 was 75.2 times smaller than that of sulforhodamine B, although free diffusion constant is only 6.1 times smaller. These data support that 3D continuous nanopores templated by the PIMS process regulate transmembrane transport. The dye molecules that are much smaller than the cut-off threshold follow the solution of Fick’s law as confirmed in Figure 2b. Therefore, a dye molecule initially loaded in the inner volume of the microcapsule is expected to diffuse into the surrounding by following the Fick’s law. That is, the concentration in the inner volume exponentially decreases from initial value to zero with the same characteristic timescale of r/3P; the solution of the Fick’s law is different because initial condition is opposite in this case.

Control of the cut-off threshold and permeability. The cut-off threshold of permeation through the nanoporous membrane can be controlled by adjusting the molar mass of PLA-CTA, which is directly correlated with the pore size, as confirmed in bulk copolymerization. The cutoff thresholds of permeation for the microcapsules were characterized by the diffusion behavior of FITC-dextrans with Mw of 10, 20, 40, 70, 150, and 500 kg mol-1 (corresponding to dH of 4.6, 6.6, 9.0, 12.0, 17.0, and 30.0 nm) into the microcapsules. We counted the microcapsules whose fluorescence intensity in the core was smaller than 30% of that in the surrounding as dye-rejected after incubating in the solutions of dye molecules for 2 days, and estimated fractions of dye-rejected microcapsules by inspecting more than 20 microcapsules per each dextran. 30% of the fluorescence intensity compared with that of the surrounding was set to distinguish impermeable capsules in our experimental condition. In Figure 3a, the fraction for dye-rejected microcapsules is plotted as a function of dH of FITCdextran. It is clear that the fraction of rejection sharply increases with increasing dH of the solute, and the dH corresponding to 100% rejection can controlled by Mn of PLACTA which determines the pore size. This trend is qualitatively consistent with the reported permeation phenomena of polymer solutes through the nanoporous membrane produced via block polymer self-assembly, where the data was interpreted by a hindered transport theory accounting for altered free volume and drag of the solute within the pores.34 We also note that the size distribution of the pores created by PIMS (Figure S6) may affect the cut-off behavior. In case of PLA-CTA-35, we posit that the increased solution viscosity and the relatively low CTA concentration in the polymerization mixture confined in the oil phase may have produced a relatively wider size distribution of PLA microdomains formed by the PIMS process, resulting in a broader cut-off profile. We extracted the cut-off thresholds of the microcapsules from fits with cumulative normal distribution functions at 50% rejection as 9.5 nm, 15.5 nm, and

22.6 nm for PLA-CTA-13, PLA-CTA-24, and PLA-CTA-35, respectively. We further investigated the influence of nanopore size on the permeability of FITC-dextran with Mw of 4 kg mol-1 (dH = 2.8 nm and DO = 1.75×10-10 m2 s-1) using the three membranes with differently sized pores; the porosity of the membrane was assumed identical since all the membranes were prepared with WPLA-CTA = 30%. As shown in Figure 3b, permeabilities of 7.90 ×10-8, 2.34×10-7, and 4.42×10-7 m s-1 and diffusivities of 7.90×10-14, 2.34×10-13, and 4.42×10-13 m2 s1 were estimated for the microcapsules made from PLACTA-13, PLA-CTA-24, and PLA-CTA-35, respectively, by fitting I(t) with equation (1). Accordingly, D/DO was increased as 4.51×10-4, 1.33×10-3, and 2.53×10-3 with the increasing Mn of PLA-CTA, manifesting that larger pores less inhibit the diffusion of molecules.

Figure 3. Control of the permeability and cut-off threshold. (a) Fraction of dye-rejected microcapsules as a function of hydrodynamic diameter, dH, of fluorescein isothiocyanate (FITC)-tagged dextrans. Molecular weight, Mw, of the dextrans are denoted by dotted vertical lines. The fractions are fitted with cumulative normal distribution function as denoted by solid lines, from which cut-off thresholds are extracted at 50% rejection. (b) Temporal dependence of normalized fluorescence intensity in the cores for three distinct microcapsules made from PLA-CTA-13, PLACTA-24, and PLA-CTA-35, where FITC- dextran with Mw 4 kg mol-1 is used.



CONCLUSION We designed semipermeable microcapsules with a controlled cut-off threshold by combining block polymer selfassembly with microfluidic emulsification technology. By employing PIMS in the shell of monodisperse doubleemulsion drops, a dense membrane with a disordered bicontinuous morphology composed of crosslinked PS and PLA domains was formed, and converted into the nanoporous one through selective etching of PLA domain. A 3D network of uniform nanopores that percolate the whole thickness of the membrane serves as diffusion paths for the molecules smaller than the pores, while preventing the transport for the larger, thereby providing molecular sizeselective permeability. As a block polymer self-assembly process, PIMS renders control over the cut-off threshold of permeation in the range of 5-30 nm by adjusting the molar mass of PLA-CTA. Moreover, high mechanical integrity and chemical stability of membrane are expected from the monolithic matrix structure of 3D nanopores made of crosslinked PS, as no rupturing or fracturing of the membrane was observed upon drying even after the basic treatment. Together with advantages of the microfluidic-based approach including high uniformity in microcapsule size and high efficiency of encapsulation, which are very difficult to achieve with the conventional approach, we believe our microcapsules will create new opportunities in various applications that stems from controlled cut-off threshold of permeation. For example, catalytic nanoparticles or enzymes may be encapsulated with semipermeable membranes to protect them from large contaminants, thereby maintaining high catalytic activity;3 the cut-off threshold will be selected to be smaller than the catalysts and contaminants, while being larger than small substrates and reaction products. Stimuli-responsive photonic nanostructures encapsulated in semipermeable microcapsules can be also envisioned to serve as capsule-type sensors to detect small target molecules without interference of large adhesives; the cut-off threshold will be selected to exclude the adhesives while allowing the diffusion of the target molecules.35

EXPERIMENTAL SECTION Materials. Unless otherwise noted, all the chemicals were used as received. d,l-Lactide was kindly provided by Corbion Purac (Amsterdam, Netherlands), and stored under nitrogen in a glovebox after recrystallization from toluene. 1,8-Diaza-bicyclo[5.4.0]undec-7-ene (DBU) was purchased from TCI (Tokyo, Japan) and also stored in a glovebox. Styrene (99%) and divinylbenzene (DVB, 80% (tech.)) were purchased from Sigma-Aldrich (St. Louis, MO) and purified by passing through a basic alumina column prior to polymerization. Octadecyltrimethoxysilane, poly(vinyl alcohol) (PVA, Mw 13-23 kg mol-1), sulforhodamine B, and fluorescein isothiocyanate (FITC)-tagged dextrans with seven different molar masses (4, 10, 20, 40, 70, 150, and 500 kg mol-1) were also purchased from Sigma-Aldrich. 2[Methoxy(polyethyleneoxy)propyl]trimethoxysilane was purchased from Gelest, Inc.. Azobisisobutyronitrile (AIBN,

98%) and methanol (≥99.8%) were purchased from Junsei (Tokyo, Japan). AIBN was purified by recrystallization in methanol and stored at -20C. 2-Hydroxyethyl 2-(((dodecylthio)carbonothioyl)-thio)-2-methylpropanoate (CTA-OH) was prepared following literature procedure, and used for the synthesis of polylactide macro-chain transfer agents (PLA-CTAs) by ring opening transesterification polymerization (ROTEP) of d,l-lactide in the presence of CTA-OH as an initiator and DBU as a catalyst.21 For the purpose of pore size characterization, crosslinked nanoporous PS monoliths were prepared from the mixtures of PLA-CTA, styrene, DVB, and AIBN following the slightly modified the literature procedure.20 Characterization details of PLA-CTAs and the corresponding crosslinked nanoporous PS monoliths are described in the Supporting Information.

Preparation and operation of microfluidic devices. Two cylindrical capillaries (1B100F-6, World Precision Instruments, Inc.) were tapered by micropipette puller (P97, Sutter Instrument) to have 40 μm aperture. One of them was carefully sanded to have a 122-μm-orifice and treated with octadecyltrimethoxysilane to render it hydrophobic, whereas the other was sanded to have a 288-μm-orifice and treated with 2-[methoxy(polyethyleneoxy)propyl] trimethoxysilane to render it hydrophilic. These two cylindrical capillaries were coaxially assembled within a square capillary (OD 1.5 mm, ID 1.05 mm, Atlantic International Technologies, Inc.) to have separation of 137 μm. A small tapered capillary was inserted into the untapered opening of the hydrophobic capillary to inject innermost fluids through the hydrophobic cylindrical capillary. Identical polymerization mixtures of PLA-CTA, styrene, DVB, and AIBN used for the synthesis of the crosslinked nanoporous PS monoliths were utilized as a middle oil phase. An aqueous solution of 10% PVA (w/w) was used as inner and continuous phases. The innermost water and middle oil phases injected through the hydrophobic injection capillary spontaneously formed a flow of water core and oil sheath due to hydrophobic nature of the wall. The continuous phase which was injected through the interstices between the hydrophobic cylindrical and outer square capillaries emulsified the core-sheath flow at the tip of the hydrophobic capillary to form monodisperse double-emulsion drops with ultra-thin shell. Volumetric flow rates of innermost, middle, and continuous phases were typically set to 400, 200, and 2500 μL h-1 respectively, which were controlled using syringe pumps (Legato 100, KD Scientific). The production of double-emulsion drops was observed with an inverted optical microscope (Eclipse TS100, Nikon) equipped with a high speed camera (Phantom v7.3, Vision Research Inc.). The emulsion drops were collected in a glass vial containing an aqueous solution of 9% PVA (w/w) and 5 mM NaCl. Double-emulsion drops sank on the collection liquid, whereas single oil drops floated as shown in Figure S7a. The oil drops were removed by pipetting (Figure S7b).


Page 6 of 9

Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials Production of microcapsules with nanoporous membrane. The double-emulsion drops were incubated in a 70 C oven for two days to fully polymerize the oil phase. The polymerized microcapsules were washed with distilled water four times. To selectively remove PLA, the microcapsules were transferred into a mixture of methanol and water (4:6 by volume) containing 0.5 M NaOH and incubated for 24 h. The microcapsules were then washed with distilled water four times. The complete removal of PLA was confirmed by disappearance of a vibrational frequency at 1747 cm-1 corresponding to C=O stretching of PLA in Fourier transform infrared (FTIR) spectra obtained on a Bruker Alpha FTIR spectrometer using a Platinum ATR (attenuation total reflection) single reflection module (Figure S10). Characterization of microcapsules with nanoporous membrane. A Hitachi S-4800 SEM (Schaumburg, IL) was used to observe the surface and the cross-section of the microcapsule membranes. An aqueous suspension of the microcapsules was cast on a carbon tape and allowed to evaporate the solvent, and then remaining microcapsules were randomly cleaved with a razor blade to observe the membrane cross-section (Figure S11c). Samples were coated with Os prior to imaging. To estimate the permeabilities of molecules with various sizes and the cut-off thresholds of permeation, the microcapsules were suspended in aqueous solutions of fluorescent molecules and observed with a laser scanning confocal microscope (LSM 5 PASCAL, Carl Zeiss) over time to monitor a fluorescent intensity change inside the capsule.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Detailed methods for preparation of nanoporous PS monoliths, 1H NMR and SEC data for PLA-CTAs, SAXS and FTIR data for PLA-b-P(S-co-DVB) precursors and nanoporous monoliths, SEM images of the nanoporous monoliths, nitrogen adsorption isotherms and BJH analysis data for the nanoporous monoliths, images of droplet collection and separation, styrene and styrene/DVB polymerization kinetics in the presence of PLA-CTA with high AIBN loading, FTIR data for nonporous and porous microcapsules, SEM images of cross-section of nanoporous membrane, temporal dependence of normalized fluorescence intensity in the microcapsules, and movie clip showing the generation of double-emulsion drops.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Author Contributions B.K.

and J.O. contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This research was supported by Basic Science Research Program (NRF-2014R1A1A1004941), Midcareer Researcher Program (2014R1A2A2A01005813), and X-Project (NRF2016R1E1A2913875) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. Experiments at Pohang Accelerator Laboratory (PAL) were supported in part by Ministry of Science, ICT and Future Planning of Korea and POSTECH.

REFERENCES 1. Antipov, A. A.; Sukhorukov, G. B. Polyelectrolyte multilayer capsules as vehicles with tunable permeability. Adv. Colloid Interface Sci. 2004, 111, 49–61. 2. Price, A. D.; Zelikin, A. N.; Wang, Y.; Caruso, F. Triggered enzymatic degradation of DNA within selectively permeable polymer capsule microreactors. Angew. Chem. Int. Ed. 2009, 48, 329–332. 3 Nam, J. O.; Kim, J.; Jin, S. H.; Chung, Y. M.; Lee, C. S. Microfluidic preparation of a highly active and stable catalyst by high performance of encapsulation of polyvinylpyrrolidone (PVP)-Pt nanoparticles in microcapsules. J. Colloid Interface Sci. 2016, 464, 246– 253. 4. Axthelm, F.; Casse, O.; Koppenol, W. H.; Nauser, T.; Meier, W.; Palivan, C. G. Antioxidant nanoreactor based on superoxide dismutase encapsulated in superoxide-permeable vesicles. J. Phys. Chem. B 2008, 112, 8211–8217. 5. Orive, G.; Gascón, A. R.; Hernández, R. M.; Igartua, M.; Pedraz, J. L. Cell microencapsulation technology for biomedical purposes: novel insights and challenges. Trends Pharmacol. Sci. 2003, 24, 207–210. 6. Wang, T.; Lacík, I.; Brissová, M.; V Anilkumar, A.; Prokop, A.; Hunkeler, D.; Green, R.; Shahrokhi, K.; Powers, A. C. An encapsulation system for the immunoisolation of pancreatic islets. Nat. Biotechnol. 1997, 15, 358–362. 7. Kurapati, R.; Raichur, A. M. Graphene oxide based multilayer capsules with unique permeability properties: facile encapsulation of multiple drugs. Chem. Commun. 2012, 48, 6013–6015. 8. Dinsmore, A. D.; Hsu, M. F.; Nikolaides, M. G.; Marquez, M.; Bausch, A. R.; Weitz, D. A. Colloidosomes: selectively permeable capsules composed of colloidal particles. Science 2002, 298, 1006– 1009. 9. Carne-Sanchez, A.; Imaz, I.; Cano-Sarabia, M.; Maspoch, D. A spray-drying strategy for synthesis of nanoscale metal-organic frameworks and their assembly into hollow suprastructures. Nat. Chem. 2013, 5, 203-211. 10. Bang, J. H.; Suslick, K. S. Applications of ultrasound to the synthesis of nanostructured materials. Adv. Mater. 2010, 22, 1039-1059. 11. Utada, A. S.; Lorenceau, E.; Link, D. R.; Kaplan, P. D.; Stone, H. A.; Weitz, D. A. Monodisperse double emulsions generated from a microcapillary device. Science 2005, 308, 537–541. 12. Kim, S.-H.; Kim, J. W.; Cho, J.-C.; Weitz, D. A. Double-emulsion drops with ultra-thin shells for capsule templates. Lab Chip 2011, 11, 3162–3166. 13. Datta, S. S.; Abbaspourrad, A.; Amstad, E.; Fan, J.; Kim, S.-H.; Romanowsky, M.; Shum, H. C.; Sun, B.; Utada, A. S.; Windbergs, M.; Zhou, S.; Weitz, D. A. Double emulsion templated solid microcapsules: mechanics and controlled release. Adv. Mater. 2014, 26, 2205–2218. 14. Lee, D.; Weitz, D. A. Double emulsion-templated nanoparticle colloidosomes with selective permeability. Adv. Mater. 2008, 20, 3498–3503.



15. Kim, B.; Lee, T. Y.; Abbaspourrad, A.; Kim, S.-H. Perforated microcapsules with selective permeability created by confined phase separation of polymer blends. Chem. Mater. 2014, 26, 7166–7171. 16. Kim, B.; Jeon, T. Y.; Oh, Y.-K.; Kim, S.-H. Microfluidic production of semipermeable microcapsules by polymerization-induced phase separation. Langmuir 2015, 31, 6027–6034. 17. Olson, D. A.; Chen, L.; Hillmyer, M. A. Templating nanoporous polymers with ordered block copolymers. Chem. Mater. 2008, 20, 869–890. 18. Shim, J. W.; Kim, S.-H.; Jeon, S.-J.; Yang, S.-M.; Yi, G.-R. Microcapsules with tailored nanostructures by microphase separation of block copolymers. Chem. Mater. 2010, 22, 5593–5600. 19. Xu, J.; Li, J.; Yang, Y.; Wang, K.; Xu, N.; Li, J.; Liang, R.; Shen, L.; Xie, X.; Tao, J.; Zhu, J. Block copolymer capsules with structuredependent release behavior. Angew. Chem. Int. Ed. 2016, 55, 14633–14637. 20. Seo, M.; Hillmyer, M. A. Reticulated nanoporous polymers by controlled polymerization-induced microphase separation. Science 2012, 336, 1422–1425. 21. Oh, J.; Seo, M. Photoinitiated polymerization-induced microphase separation for the preparation of nanoporous polymer films. ACS Macro Lett. 2015, 4, 1244–1248. 22. Schulze, M. W.; Hillmyer, M. A. Tuning mesoporosity in crosslinked nanostructured thermosets via polymerization-induced microphase separation. Macromolecules 2017, 50, 997-1007. 23. Bates, F. S.; Wiltzius, P. Spinodal decomposition of a symmetric critical mixture of deuterated and protonated polymer, J. Chem. Phys. 1989, 91, 3258-3274. 24. Henton, D. E.; Gruber, P.; Lunt, J.; Randall, J. In Natural Fibers, Biopolymers, and Biocomposites; Mohanty, A. K.; Misra, M.; Drzal, L. T., Eds.; CRC Press: Boca Raton, FL, 2005; Chapter 16. 25. Quach, A.; Simha, R. Pressure-volume-temperature properties and transitions of amorphous polymers; polystyrene and poly(orthomethylstyrene). J. Appl. Phys. 1971, 42, 4592-4606.

26. Keddie, D. J. A guide to the synthesis of block copolymers using reversible-addition fragmentation chain transfer (RAFT) polymerization. Chem. Soc. Rev. 2014, 43, 496-505. 27. Park, J.; Saba, S. A.; Hillmyer, M. A.; Kang, D.-C.; Seo, M. Effect of homopolymer in polymerization-induced microphase separation process. Polymer 2017, 126, 338-351. 28. Lowell, S.; Shields, J. E.; Thomas, M. A.; Thommes, M. Characaterization of Porous Solids and Powders: Surface Area, Pore Size and Density; Springer: New York, 2004. 29. Barrett, E. P.; Joyner, L. G.; Halenda, P. P. The determination of pore volume and area distributions in porous substances. I. computations from nitrogen isotherms. J. Am. Chem. Soc. 1951, 73, 373–380. 30. Lankveld, J. M. G.; Lyklema, J. Adsorption of polyvinyl alcohol on the paraffin-water interface I. Interfacial tension as a function of time and concentration. J. Colloid Interf. Sci. 1972, 41, 454-465. 31. Liu, B.; Kazlauciunas, A.; Guthrie, J. T.; Perrier, S. One-pot hyperbranched polymer synthesis mediated by reversible addition fragmentation chain transfer (RAFT) polymerization. Macromolecules 2005, 38, 2131-2136. 32. Ikkai, F. Structural colored balloons consisting of polystyrene microcapsules in water. Langmuir 2008, 24, 3412–3416. 33. Edward, J. T. Molecular volumes and the Stokes-Einstein equation. J. Chem. Educ. 1970, 47, 261–270. 34. Phillip, W. A.; Amendt, M.; O’Neill, B.; Chen, L.; Hillmyer, M. A.; Cussler, E. L. Diffusion and flow across nanoporous polydicyclopentadiene-based membranes. ACS Appl. Mater. Interfaces 2009, 1, 472-480. 35. Chinnayelka, S.; McShane, M. J. Microcapsule biosensors using competitive binding resonance energy transfer assays based on apoenzymes. Anal. Chem. 2005, 77, 5501–5511.


Page 8 of 9

Page 9 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials Semipermeable microcapsules with nanoporous membranes are designed by using a polymerization-induced microphase separation (PIMS) in shells of double emulsions. The nanopores percolate through the entire thickness of the microcapsule membrane, which provides size-selective permeability. The cut-off threshold of permeation can be precisely controlled in the range of 5-30 nm.


9

Semipermeable Microcapsules with a Block-Polymer-Templated

Recommend Documents