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One-Step Fabrication of Supramolecular Microcapsules from Microfluidic Droplets

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Science  10 Feb 2012:
Vol. 335, Issue 6069, pp. 690-694
DOI: 10.1126/science.1215416

Abstract

Although many techniques exist for preparing microcapsules, it is still challenging to fabricate them in an efficient and scalable process without compromising functionality and encapsulation efficiency. We demonstrated a simple one-step approach that exploits a versatile host-guest system and uses microfluidic droplets to generate porous microcapsules with easily customizable functionality. The capsules comprise a polymer-gold nanoparticle composite held together by cucurbit[8]uril ternary complexes. The dynamic yet highly stable micrometer-sized structures can be loaded in one step during capsule formation and are amenable to on-demand encapsulant release. The internal chemical environment can be probed with surface enhanced Raman spectroscopy.

The encapsulation of materials for protection and phase separation has evolved into a major interdisciplinary research focus (1, 2). Synthetic microcapsules (3, 4), in which the composition of the shell structure and the core content can be controlled, have found importance in applications as diverse as cell encapsulation (5, 6), drug delivery (7), diagnostics (8), catalysis (9), food additives (10), and electronic displays (11). Preparation of conventional polymeric microcapsules via the layer-by-layer (L-b-L) technique (12, 13), although powerful, suffers from reduced encapsulation efficiencies as a result of postfabrication loading. Alternative self-assembly processes, either by forming polymersomes (14) or by colloidal emulsion-templating (15, 16), still lack monodispersity, stability, high loading efficiency, and material diversity in the resulting microcapsules, restricting their function and subsequent applications. As a subset of colloidal emulsions, microfluidic droplets (17) show great promise as a platform for many microcapsule fabrication techniques (1820) on account of their monodispersity, high fabrication throughput with economic use of reagents, and ease of scaleup.

We report a class of microcapsule prepared in a single step using a microdroplet platform, combining the advantages of microfluidic droplets and supramolecular host-guest chemistry with quantitative loading efficiency. Cucurbit[8]uril (CB[8]) (21) is used as the host molecule because it is capable of forming stable yet dynamic complexes with guest compounds in water with extremely high affinity. Moreover, this larger CB homolog is capable of simultaneously accommodating two guests to form a 1:1:1 ternary complex in water (21) [with an association constant (Ka) up to 1015 M−2] through multiple noncovalent interactions with an electron-deficient first guest such as methyl viologen (MV2+) and an electron-rich second guest, such as naphthol (Np) derivatives (Fig. 1A) (22). The ability of CB[8] to act as a supramolecular “handcuff” was further exploited by modifying gold nanoparticles (AuNPs) and water-soluble copolymers with complementary functionalities, thereby achieving a controlled dispersion of AuNPs in a polymer network held together by CB[8] (23).

Fig. 1

(A) The two-step, three-component formation of the CB[8] ternary complex in water with MV2+ (blue) and Np (red). (B) Schematic representation of the microdroplet generation process using a microfluidic T-junction device, consisting of an continuous oil phase perpendicular to a combination of three aqueous solutions of CB[8], 1a (AuNP functionalized with a mixture of neutral and viologen-containing ligands 3 and 4), and 2a (copolymer functionalized with Np) as the dispersed phase. (C) Microscopic image and the schematic of the T-junction and a wiggled channel for rapid mixing of reagents online. (D) The high monodispersity of microfluidic droplets is demonstrated by the narrow size distribution (diameter 59.6 ± 0.8 μm).

During microcapsule preparation, microdroplets were first generated in a microfluidic device, using a simple T-junction geometry (24) (Fig. 1B). The oil carrier phase was directed perpendicular to the aqueous dispersed phase, which consisted of three inlets for the aqueous solutions of CB[8], MV2+-AuNPs (1a), and Np-containing copolymer (2a). Droplets were generated as the oil phase sheared off the aqueous phase, before passing through a winding channel designed for thorough mixing of the three reagents (Fig. 1C). With an oil:water flow rate ratio of 2:1, droplets were generated at a frequency of 300 Hz and exhibited a high level of monodispersity when collected on a microscope slide, as indicated by the narrow size distribution with a mean diameter of 59.6 μm (Fig. 1D) and a low coefficient of variation of 1.3%.

Individual stable microcapsules were observed immediately after dehydration of the microdroplet precursor. After 150 s, the spherical shape of the droplet was slightly distorted (Fig. 2A), until the capsules eventually collapsed on a glass surface. The individual microcapsules (Fig. 2B) can be easily isolated after the evaporation of the oil phase. The diameters of the initial droplets (ddroplet) and the corresponding microcapsules (dcapsule) decreased with the increasing ratio of the flow rate of the oil phase (QOIL) to the flow rate of the aqueous phase (QAQ) (Fig. 2C). By merely varying QOIL/QAQ, stable droplet precursors and microcapsules could be generated with a ddroplet of 42 to 67 μm and a dcapsule of 10 to 24 μm, with polydispersities of 0.9 ± 0.4%. Upon rehydration, rupture of some isolated microcapsules was observed, revealing their hollow nature (Fig. 2D). A scanning electron microscopy (SEM) image of a hollow capsule that has collapsed because of a lack of internal support is shown in Fig. 2E.

Fig. 2

(A) Brightfield images of the capsule formation process as the aqueous phase evaporates, resulting in a collapsed microcapsule-like structure. (B) Brightfield image of isolated microcapsules. (C) Variation in the mean diameters of initial droplets (ddroplet, blue) and corresponding stable microcapsules (dcapsule, red) as a function of the QOIL/QAQ ratio of aqueous solution for QAQ = 60 μl/hour (squares), 100 μl/hour (triangles), and 140 μl/hour (circles). (D) Brightfield image of burst capsules, showing the relics of the capsule shell. (E) SEM image of a dried microcapsule. (F) TEM image of the microcapsule shell, showing AuNPs approximately 5 nm in diameter dispersed in a mesh of polymer. (G) Schematic representation of the proposed microcapsule formation process from the initial droplet stage with a diameter ddroplet to the dehydrated stable capsules of a diameter of dcapsule.

A transmission electron microscopy (TEM) image (Fig. 2F) shows that the capsule shell consists of a supramolecular self-assembled network of AuNPs 1a and copolymer 2a, where individual AuNPs are interlinked via a mesh of the polymer. The formation of a supramolecular microcapsule is schematically represented in Fig. 2G, where the deposition of the supramolecular composite of CB[8], 1a, and 2a at the oil/water interface is assisted by interfacial tension stabilization through polymers and nanoparticles (25). The fabrication process showed that the microcapsules were resistant to heat (100°C) and reduced pressure (20 Pa), on account of the highly stable CB[8] 1:1:1 ternary complexes. Microcapsule formation was not observed when CB[8] was replaced with the smaller homolog CB[7], which is unable to form ternary complexes (23). Furthermore, when the AuNPs were not functionalized with the MV2+ ligand, no stable microcapsules were formed (fig. S1).

The microdroplet-assisted loading of water-soluble cargo was investigated by simultaneously incorporating a rhodamine-B functionality onto the polymer backbone (2b) (Fig. 3, A and B) and a fluorescein isothiocyanate–labeled dextran (FITC-dextran) into the capsule cavity. Aqueous solutions of 2b and FITC-dextran were injected into the microfluidic device with solutions of CB[8] and 1a. Droplets were collected in a reservoir, and fluorescence images were recorded with a laser scanning confocal microscope (LSCM) (26). The integrity of the capsule shell was not compromised by such loading, as shown by a clearly defined layer of rhodamine fluorescence confined to the water/oil interface of the droplets, whereas the interior of the capsule was filled evenly with FITC-dextran, as is apparent from the FITC fluorescence (Fig. 3C). The fluorescence intensity plot clearly shows that the rhodamine exterior is distributed in the “shell” forming outside the “cargo,” whereas the FITC fluorescence is only observed inside the microcapsules. Other types of cargo were also similarly encapsulated, including bacterial cells (Fig. 3D).

Fig. 3

(A) Chemical structure of rhodamine-B–containing copolymer 2b. (B) LSCM image of empty capsules containing 2b in the shell. (C) LSCM image of capsules containing aqueous solutions of CB[8], 1a, 2b, and FITC-dextran. (D) LSCM image of a capsule containing aqueous solutions of CB[8], 1a, 2b, and green fluorescent protein–expressing Esherichia coli cells. The inserts in (B) to (D) display the corresponding fluorescence intensity profiles (I, intensity; D, distance). (E) Fluorescence images of the process of disintegration of the microcapsule wall material in an aqueous solution of Na2S2O4 or in H2O over 12 hours (h) in a N2 environment at 25°C.

The capsules benefit from properties arising from the supramolecular CB[8] host-guest network. For example, the incorporation of the rhodamine-B–containing polymer demonstrates the ease of adding customizable functionality using the supramolecular approach (27). Stimuli-triggered degradation of the capsules (28) is also possible by a one-electron reduction of the MV2+ moiety, leading to on-demand release of encapsulated cargo (29). One-electron reduction of MV2+ generates the radical cation MV+●, which rapidly forms a stable 2:1 (MV+●)2:⊂CB[8] inclusion complex (30). When microcapsules containing the 500-kD FITC-dextran were isolated into an aqueous solution of sodium dithionite (Na2S2O4) free of oxygen, controlled dissipation of the fluorescence over time was observed (Fig. 3E). Conversely, the microcapsules failed to release the encapsulated 500-kD FITC-dextran when an N2 atmosphere was not maintained or in the absence of Na2S2O4. Without external stimuli, these microcapsules also allowed passive diffusion of the encapsulated cargo with appropriate molecular mass. The 500-kD FITC-dextran was retained by the microcapsules after rehydration, whereas the 10-kD counterpart was capable of diffusing freely into the external environment (fig. S2).

The incorporation of AuNPs further allows these microcapsules to be used as substrates for surface-enhanced Raman spectroscopy (SERS) (3133). Using the modular supramolecular approach (26), two microcapsules were prepared, one containing 5-nm-diameter (1a) and a second with 20-nm-diameter (1b) AuNPs. When the samples were excited with a 633-nm laser, characteristic SERS signals for CB[8] and MV2+ were observed (Fig. 4A) (34). The signal strength derived from the capsules containing 1b was much greater than those containing 1a, because SERS field enhancement is dependent on the distance between AuNPs and the size of the AuNPs (35). SERS mapping of microcapsules showed that the SERS signals were uniformly localized within the capsules (fig. S3). To investigate the feasibility of detecting encapsulated materials by SERS, FITC-dextran was loaded into the microcapsules. Significant Raman enhancement of the FITC signals was observed in addition to those from CB[8] and MV (Fig. 4B). A similar dependence on the size of the AuNPs for the degree of enhancement was observed, demonstrating the potential of the porous microcapsule shell as a SERS substrate for the detection of encapsulated materials.

Fig. 4

(A) SERS spectra of empty microcapsules consisting of 5-nm-diameter and 20-nm-diameter AuNPs 1a and 1b using a 633-nm laser, showing characteristic peaks for CB[8] (asterisks) and MV2+ (squares): 830 cm−1, 1630 cm−1, 1560 cm−1, and 1308 cm−1. (B) SERS spectra of FITC-dextran encapsulated microcapsules consisting of AuNPs 1a and 1b using a 633-nm laser, showing characteristic peaks for FITC (circles) in addition to those for capsule shell materials: 1186 cm–1, 1232 cm–1, and 1400 cm−1.

We have described the preparation, characterization, and application of a microcapsule held together by supramolecular host-guest 1:1:1 ternary complexes of CB[8], MV2+-AuNPs, and a Np-containing copolymer at the liquid/liquid interface of microfluidic droplets. These microcapsules are produced from microdroplets in one step with high frequency and monodispersity. Upon dehydration, stable microcapsules with a hollow interior can be isolated within minutes. When an additional aqueous stream is incorporated, a wide variety of materials can be quantitatively encapsulated during capsule formation. Stimulus-triggered on-demand release of the encapsulated cargo is achieved as a result of the supramolecular host-guest chemistry incorporated in the capsule shell. Additionally, these microcapsules exhibit strong plasmonic properties on account of the AuNPs present and can be used as a SERS substrate for the encapsulated materials.

Supporting Online Material

www.sciencemag.org/cgi/content/full/335/6069/690/DC1

Materials and Methods

Figs. S1 to S3

References (3640)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: This work was supported by Engineering and Physical Sciences Research Council Basic Technology Translational grant EP/H046593/1, a European Research Council Starting Investigator Grant (ASPiRe) ERC-2009-StG-240629, and a European Union NanoSci-E+ (CUBiHOLE) grant EP/H007024/1. SERS experiments were performed with the assistance of S. Mahajan. The DH5α Escherichia coli strain was a kind gift from M. Welch and S. Bowden from the Department of Biochemistry, University of Cambridge. The naphthol-containing polymer was a kind gift from E. Appel from the Department of Chemistry, University of Cambridge. The authors thank X. Liu, C. A. Smith, S. Mahajan, J. J. Baumberg, and W. T. S. Huck for the helpful discussions. J.Z., R.J.C., O.A.S., and C.A. have filed a provisional application for a GB patent on the fabrication process.
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