Article pubs.acs.org/cm
Perforated Microcapsules with Selective Permeability Created by Confined Phase Separation of Polymer Blends Bomi Kim,†,‡ Tae Yong Lee,†,‡ Alireza Abbaspourrad,§ and Shin-Hyun Kim*,† †
Department of Chemical and Biomolecular Engineering and KINC, Korean Advances Institute of Science and Technology (KAIST), Daejeon, South Korea § School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, United States S Supporting Information *
ABSTRACT: Semipermeable microcapsules have a great potential in controlled release of drugs, protection of catalysts, and immunoisolation of cells. However, a method to create such microcapsules with precisely controlled cutoff value and high mechanical stability remains an important challenge. Herein we report microfluidic approach to create microcapsules with size-selective permeability using phase separation of polymer blends in ultrathin middle layer of double-emulsion drops. The blend strongly confined in two-dimensional space exhibit local phase separation, instead of global separation. This enables the perforation of microcapsule membrane by selectively removing one of the phase-separated polymeric domains. The resultant monolithic membrane has uniform pores which connect the interior and the exterior of the microcapsules, thereby providing size-selective permeability. The pore size can be precisely tuned by regulating the extent of phase separation; this enables the control of cutoff value for permeation.
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INTRODUCTION Microencapsulation technologies have enabled the stable storage and controlled delivery of active ingredients as well as efficient compartmentalization of display pigments.1−3 Microcapsule’s core provides sufficient loading of active materials and functionalized shell makes the microcapsules compatible for specific applications. Emulsion drops have been utilized as template to produce such core−shell structures; by exploiting interfacial polymerization or particle adsorption at drop interface, inner active materials can be enclosed by membrane.4−8 Recent advances in microfluidic emulsification techniques have enabled the production of double-emulsion drops, or drops-in-drop, with a high degree of controllability over size and number of inner drops. Due to their intrinsic core−shell geometry, double emulsions produced using microfluidics serve as efficient and flexible templates to fabricate microcapsules with high encapsulation efficiency.9,10 A variety of microcapsules have been designed using double-emulsion drops as template. For example, polymeric microcapsules whose membrane is highly dense and inert are prepared to encapsulate and retain materials without premature leakage.11 In addition, stimuli-responsive membranes are also fabricated to induce triggered release of encapsulants.12−16 Microcapsules that are able to regulate flux of materials through their membrane have great potential in a wide range of applications such as selective release of encapsulants, construction of artificial cells and immunoisolation of cells.1,17 The simplest form of such microcapsules will have consistent pores in the membrane, thereby providing size-selective © XXXX American Chemical Society
permeability. There have been attempts to create such porous microcapsules using double-emulsion drops. For example, evaporation-induced consolidation of nanoparticles dispersed in middle layer results in the formation of nanopores in their interstices.18 In another approach, small emulsion droplets are used as templates to create pores in middle layer.19 However, lack of precise control over pore size and porosity and low mechanical stability severely limit the efficacy of previous approaches. Therefore, designing microcapsules with semipermeable membrane possessing controlled cutoff value and high stability still remains an important challenge. In this paper, we report a facile microfluidic approach to create monodisperse microcapsules whose membrane is uniformly perforated and monolithic, thereby providing selective permeability and high mechanical stability. We generate double-emulsion drops with ultrathin middle layer using a glass capillary device, of which shell phase is organic solution of a polymer blend. The double-emulsion drops containing an ultrathin middle layer exhibit much enhanced stability due to strong lubrication resistance in the middle layer by comparison to regular double-emulsion drops.20,21 During consolidation of the middle layer, the polymer blends exhibit local phase separation. The phase separation allows us to create membrane including interconnected pores upon selective removal of one polymer. The extent of phase separation and Received: October 17, 2014 Revised: November 20, 2014
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ultrathin middle layer, as shown in Figure 1b and Movie S1 in the Supporting Information. The middle layer thickness is approximately 500 nm, which is not observable with the optical microscope because of its limits of resolution. Although the toluene solution between the plug-like drops results in the formation of single-emulsion drops, they can be easily separated by exploiting their density difference. We carefully formulate the collection liquid, an aqueous solution of 3 wt % PVA and 30 mM NaCl; this solution has lower density than that of doubleemulsion drops and the same osmolarity to that of innermost drops. Therefore, the double-emulsion drops are concentrated in the bottom of the collection vial and negligible osmotic pressure difference results in minimal water flux across the middle layer. To evaporate the toluene and obtain solidified shell, we incubate the suspension of double-emulsion drops in a glass petridish at 40 °C for 1 h. The resultant microcapsules can encapsulate water-soluble actives within their core enclosed by the solidified polymeric membrane with thickness as small as approximately 100 nm; for example, red dye, sulforhodamine B (Mw 558.67), is encapsulated in the core without any leakage, as illustrated in Figure 1c, d. Selective Removal of PLA Domains from Microcapsule Membrane Composed of a Blend of PS and PLA. We perforate the microcapsule membrane by selective removal of the phase-separated PLA segments embedded within the PS matrix, as schematically illustrated in Figure 2a. We dissolve the biodegradable PLA segments by incubating the microcapsules with a mixture of methanol and water containing NaOH; this mixture accelerates hydrolysis of ester groups exist in the PLA backbone.23 Morphology of the resultant perforated membrane is characterized by scanning electron microscope (SEM), as shown in Figure 2b, c and Figure S1 in the Supporting Information. Although PS is a brittle polymer, the ultrathin membrane is folded and the microcapsules are fully deflated upon drying, while maintaining membrane integrity, as shown in Figure 2b. The membrane is monolithic and shows regular pores on the surface; these pores connect the interior and the exterior of the microcapsule as shown in Figure 2c. Average pore diameter is 400 nm and diameter coefficient of variation (CV) is 30%, where largest pore has a diameter of 710 nm; we obtain these data from SEM analysis of 150 pores. The polymeric blend comprised of PS and PLA has large interaction parameter of approximately 0.2 at incubation temperature of 40 °C;23 usually blends with such a large interaction parameter must undergo macrophase separation.24 However, we observe a local phase separation in the microcapsule membrane; we attribute this phenomenon to the extreme confinement of the polymeric blend in an ultrathin membrane of approximately 100 nm in thickness. In this case, phase separation is mainly limited in lateral direction of the ultrathin membrane and results in formation of submicron-sized domains. Although PLA weight fraction in the blend is 10%, total pore volume, templated by PLA domains, is much smaller than 10%. We hypothesis that within the membrane, PS layer is sandwiched by two PLA layers due to higher affinity of PLA to the aqueous phase than that of PS; therefore, this unique geometry results in formation of fewer pores. Membrane comprised of threelayered PLA−PS−PLA is schematically illustrated in the middle part of Figure 2a. We confirm the higher affinity of PLA to the aqueous phase by fabricating microcapsules templated by double-emulsion drops whose middle phase is separately injected from two separate channels:25 We inject a solution of PS in toluene from one channel and use the second channel
thus the average pore size can be essentially controlled by adjusting interaction parameter between two polymers; therefore, polymer blend with an appropriate interaction parameter enables us to control cutoff value of permeation. We characterize the selective permeability of the microcapsule membrane using binary mixtures of fluorescent probes with different sizes.
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RESULTS AND DISCUSSION Preparation of Double-Emulsion Drops with Ultrathin Middle Layer. Double emulsions with ultrathin middle layer exhibit enhanced longevity; the emulsions retain their core− shell integrity without rupture during solvent evaporation from the middle layer.22 More importantly, extreme confinement of the polymer blend in such a thin middle layer and subsequently fast consolidation prevent global phase separation of the polymer blend, and instead lead to a local phase separation. To make thin-shelled double-emulsion drops, we employ emulsification technique of a biphasic flow in a glass capillary microfluidic device, as schematically shown in Figure 1a.22 We
Figure 1. (a) Schematic illustration of the microfluidic capillary device for production of water-in-oil-in-water (W/O/W) double-emulsion drops with ultrathin middle layer. (b) Optical microscope image showing generation of double-emulsion drops. (c, d) Confocal microscope images of mnodisperse polymeric microcapsules whose membrane is made of PS and PLA blends; red dye molecules, sulforhodamine B, are encapsulated.
simultaneously introduce two immiscible fluids, aqueous solution of 10 wt % poly(vinyl alcohol) (PVA) and toluene solution containing 9 wt % polystyrene (PS, Mw 785 400) and 1 wt % poly(lactic acid) (PLA, Mw 15 000), through a single hydrophobic injection capillary to form a core−sheath biphasic flow; because of the hydrophobic nature of the capillary surface, the toluene solution flows along the wall of the capillary, whereas the aqueous phase forms a train of plug-like drops without contacting the wall. This core−sheath flow is emulsified in a dripping mode into a continuous phase, 10 wt % aqueous solution of PVA, at the tip of the injection capillary, thus forming monodisperse double-emulsion drops with B
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great difference between the hydrophilicity of PS and PLA. When a mixture of PS and PLA is used to form the middle layer, such lateral separation is highly restricted. Instead, the blend forms a three layered structure, comprised of innermost PLA layer, middle PS layer perforated with PLA disks, and outermost PLA layer; this geometry prevents direct exposure of PS to water. To study the effect of the polymer ratio on the pore formation, we remarkably vary the membrane composition with PS to PLA ratio of 9:1, 7:3, and 5:5. The size and number density of the pores remain within small range of variations for all compositions, as shown in Figure 2d and Figure S1b−d in the Supporting Information. This experiment reveals that change in the polymeric composition only influences thickness of each layer within the membrane and does not affect the total area of localized PLA domains embedded in PS layer. The microcapsules with perforated membranes exhibit selective permeability depending on the size of the tracer particles used as schematically illustrated in Figure 3a. To
Figure 3. (a) Cartoon showing size-selective permeation through perforated membrane of microcapsule. (b−d) Confocal microscope images of a microcapsule dispersed in aqueous binary mixture of 1 μm PS particles labeled with red dye and 100 nm PS particles labeled with green dye.
confirm this, we disperse the perforated microcapsules made of 9:1 PS:PLA blend into a binary mixture of 1 μm PS particles labeled with red dye and 100 nm PS particles labeled with green dye. Because the average pore size is 400 nm and the largest is 710 nm, 100 nm PS particles easily diffuse through the membrane, whereas 1 μm PS particles remain in the exterior, as exemplified in Figure 3b−d. We acquire these images after incubating microcapsules within the dispersion of the binary PS particles for 24 h. The confocal microscope images taken in low magnification is shown in Figure S3 in the Supporting Information. Formation of Nanoporous Membrane Using a Blend of PMMA and PLA. To obtain membrane with smaller pore size, we can carefully decrease the degree of phase separation by employing a polymer blend with smaller interaction parameter. In this case the extent of phase separation can be significantly reduced which results in formation of membrane with smaller pore size. To exploit the effect of interaction parameter and realize selective permeability at a molecular level, we use a blend of poly(methyl methacrylate) (PMMA, Mw 606 000) and PLA (Mw 15 000); these two polymers have high molecular affinity and therefore low interaction parameter as small as
Figure 2. (a) Schematic illustration for preparation of perforated microcapsules using local phase separation of PS and PLA blend confined in ultrathin membrane. (b, c) Scanning electron microscope (SEM) images of dried microcapsules whose membrane is perforated. Inset of c shows pores in the membrane in a higher magnification. (d) Diameter (filled circles) and number density (filled squares) of pores on the membrane as a function of weight fraction of PLA in blends. Error bars denote standard deviation.
to inject solution of PLA in toluene, as schematically shown in Figure S2a in the Supporting Information. Although the produced double-emulsions exhibit homogeneous middle layer immediately after drop generation, upon toluene evaporation, a boundary emerges between PS and PLA domains and results in two different macro domains, as exemplified in Figure S2b in the Supporting Information. The PLA domain spreads to cover the innermost water drop, whereas the PS domain shrinks to form a small dark patch as shown in Figure S2c in the Supporting Information; this behavior indicates that there is a C
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0.001.26 We fabricate microcapsules using a 9:1 blend of PMMA and PLA dissolved in toluene as middle layer, as schematically shown in Figure 4a. After selective removal of
PLA segments, we examine morphology of dried microcapsules using SEM as shown by images in Figure 4b, c; highmagnification image does not show any macropore exist on the surface as illustrated in the inset of Figure 4c. With such a small interaction parameter of 0.001 and low degree of polymerization of PLA (∼200), phase separation is insignificant, thus leading to a homogeneous blend throughout the membrane even after complete evaporation of toluene.27 Therefore, removal of PLA from the PMMA matrix results in formation of nanopores in the membrane. To approximate the membrane pore size, we disperse the microcapsules within a mixture of fluorescent probes with two different molecular sizes, fluorescein isothiocyanate (FITC)-tagged dextran (M w 20 000) and sulforhodamine B (Mw 558.67). The red dye molecules with smaller size can diffuse through the PMMA membranes, whereas the dextran molecules remain in the exterior of the microcapsules, as shown in Figure 4d−f; lowmagnification images are shown in Figure S4 in the Supporting Information. Using these data, we can deduce that pores are larger than the effective diameter of red dye molecules (∼2 nm) and smaller than that of the dextran molecules (∼6.6 nm). Estimation and Control of Membrane Permeability. We estimate permeability of fluorescein (Mw 332.31) through the microcapsule membrane using three different polymeric compositions, 9:1 blend of PS and PLA, 9:1 and 5:1 blends of PMMA and PLA; the pores of membrane made of PS and PLA blend is a couple of orders of magnitude larger than fluorescein, while the pores of membrane made of PMMA and PLA blend is just several times larger than fluorescein. To do this, we disperse these microcapsules into 1 × 10−5 M aqueous solution of fluorescein and observe them with confocal microscope. The fluorescein intensity in the interior of the microcapsules
Figure 4. (a) Schematic illustration for preparation of microcapsule whose membrane is made from PMMA and PLA blend. (b,c) SEM images of dried microcapsules without any macropore on the membrane. (d−f) Confocal microscope images of a microcapsule dispersed in aqueous binary mixture of fluorescein isothiocyanate (FITC)-tagged dextran (Mw 20 000) and sulforhodamine B (Mw 558.67).
Figure 5. (a) Schematic illustration of molecular diffusion through perforated membrane of microcapsule. (b) Time dependence of normalized fluorescence intensity in the interior of microcapsules which are dispersed in aqueous solution of fluorescein at t = 0; circles correspond to microcapsules made from 9:1 PS and PLA blend, and triangles and squares correspond to microcapsules made from 5:1 and 9:1 PMMA and PLA blends, respectively. (c, d) Series of confocal microscope images of (c) a macroporous microcapsule made from 9:1 PS and PLA blend, taken in 15 s interval and (d) a nanoporous microcapsule made from 9:1 PMMA and PLA blend, taken in 120 s interval; both are dispersed in aqueous solution of fluorescein. D
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increases over time as the fluorescein molecules diffuse through the membrane; this process is schematically illustrated in Figure 5a. We plot the time-dependent change of fluorescence intensities normalized by their maximum values, I/Imax as a function of time, as shown in Figure 5b. These data points can be fitted with a diffusion equation to obtain permeability values, P 3P I (t ) = 1 − e−( r )t Imax
permeability. This class of semipermeable microcapsules with controlled cutoff value has great potential as advanced microcarriers. For example, two distinct encapsulated materials with different molecular size can be sequentially released when these microcapsules are used as delivery vehicles for drugs, cosmetics, and nutrients. In addition, selective permeability enables the potential use of the microcapsules as immuneisolators of live cells; pores permit diffusion of nutrients and wastes for metabolism, while protecting the cell from immune system.1,17
(1)
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where r is radius of microcapsules and t is diffusion time:18,28 PS microcapsules with macropores show P of 0.3954 μm/s, whereas PMMA microcapsules with nanopores made from 9:1 and 5:1 blends show P of 0.0262 and 0.0401 μm/s, respectively. The relatively high permeability of PS membrane is attributed to large pores which enable the free diffusion of fluorescein molecules; a series of confocal microscope images of PS microcapsules, taken with 15 s intervals, are shown in Figure 5c. By contrast, pore size of PMMA membrane is comparable to the size of fluorescein, thereby severely restricting the diffusion of molecules through the pores; a series of confocal microscope images of PMMA microcapsules made of 9:1 blend, taken in 120 s intervals, are shown in Figure 5d. Nevertheless, the PMMA membrane shows only 10-fold smaller permeability than that of PS membrane, although pore size of PMMA membrane is approximately 100-fold smaller than that of PS membrane. We attribute this to much higher pore density in PMMA membranes than that of PS membrane. In the case of PMMA and PLA blend, solvent evaporation leads to formation of a nearly homogeneous single-layered structure, in which PLA entirely contributes to formation of nanopores; by contrast, consolidation of PS and PLA blend results in formation of a three-layered structure, in which only part of the PLA contributes to pore formation. Therefore, we can tune the pore density of PMMA membrane, while roughly maintaining pore size by adjusting composition of PLA in the polymeric blend. For an increase in the PLA fraction from 0.1 to 0.167, the permeability also increases by a factor of 1.53. This rough consistence between pore fraction and permeability enables us to control the membrane permeability, while maintaining the cutoff value of permeation.
ASSOCIATED CONTENT
S Supporting Information *
Experimental details, optical and confocal microscope images of microcapsules, SEM images of capsule membranes, and movie clip showing generation of double-emulsion drops. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions ‡
B.K. and T.Y.L. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by KAIST HRHRP (Project N10130008), the Midcareer Researcher Program (2014R1A2A2A01005813) through the National Research Foundation (NRF) grant funded by the MSIP, and the Industrial strategic technology development program (10045068) of the Korea Evaluation Institute of Industrial Technology (KEIT) funded by the MOTIE.
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REFERENCES
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CONCLUSION In this work, we report a facile microfluidic approach combined with phase separation phenomena to create microcapsules with porous membrane, providing size-selective permeability. Double-emulsion drops with ultrathin shell serve as highly stable templates to fabricate microcapsules. Polymer blends, confined in the ultrathin membrane, exhibit local phase separation due to limited lateral diffusion; thick-shelled double-emulsion drops exhibit global phase separation, which are therefore not applicable for creation of regular pores. Upon selective removal of one polymeric constituent from the blend, we perforate the membrane to create microcapsule with regular pores, providing the selective permeability. The pore size can be controlled by adjusting interaction parameter between two polymers, which regulates the extent of phase separation; a blend of PS and PLA with large interaction parameter leads to macroporous membrane, while a blend of PMMA and PLA with low interaction parameter results in nanoporous membrane. These monolithic microcapsules with uniform pores offer high mechanical stability as well as selective E
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