Triggerable Protocell Capture in Nanoparticle-Caged Coacervate Microdroplets

Abstract

Controlling the dynamics of mixed communities of cell-like entities (protocells) provides a step toward the development of higher-order cytomimetic behaviors in artificial cell consortia. In this paper, we develop a caged protocell model with a molecularly crowded coacervate interior surrounded by a non-cross-linked gold (Au)/poly(ethylene glycol) (PEG) nanoparticle-jammed stimuli-responsive membrane. The jammed membrane is unlocked by either exogenous light-mediated Au/PEG dissociation at the Au surface or endogenous enzyme-mediated cleavage of a ketal linkage on the PEG backbone. The membrane assembly/disassembly process is used for the controlled and selective uptake of guest protocells into the caged coacervate microdroplets as a path toward an all-water model of triggerable transmembrane uptake in synthetic protocell communities. Active capture of the guest protocells stems from the high sequestration potential of the coacervate interior such that tailoring the surface properties of the guest protocells provides a rudimentary system of protocell sorting. Our results highlight the potential for programming surface-contact interactions between artificial membrane-bounded compartments and could have implications for the development of protocell networks, storage and delivery microsystems, and microreactor technologies.

Figure 1

Construction of nanoparticle-caged coacervate protocells and membrane unlocking. (a) Scheme showing self-assembly and triggered membrane dynamics in caged PDDA/CM-dex coacervate droplets. Tannic-acid-coated Au nanoparticles are rendered amphiphilic at the water/coacervate droplet interface by asymmetric ligand exchange with bidentate ligands TA-PEG, MTA-PEG, or TA-AE-PEG6k or monodentate TA-PEG-ME to produce a membrane of jammed Au/PEG Janus-like nanoparticles that can be unlocked by exogenous light-mediated dissociation (I; Au/TA-PEG, Au/MTA-PEG, and Au/TA-PEG-ME) or endogenous enzyme-mediated molecular cleavage (II; TA-AE-PEG6k). (b) Graphics showing proposed models of the jammed (i) and unjammed (ii) membranes; light- or chemically induced ligand dissociation results in translocation of PEG-depleted Au nanoparticles into the coacervate matrix and formation of apertures in the membrane. (c) Mechanism of glucose oxidase (GOx)/glucose-mediated ligand dissociation from Au/TA-AE-PEG6k nanoparticles. Addition of glucose in the presence of dioxygen generates gluconic acid within the caged protocell, which cleaves TA-AE-PEG6k and unlocks the membrane.

Figure 2

Caging and unlocking of coacervate protocells. (a) Dark field microscopy image showing multilayer population of nanoparticle-caged coacervate microdroplets. (b) Confocal laser scanning microscopy (CLSM) image of caged coacervate droplets; red fluorescence membrane (Au/RITC-labeled TA-PEG nanoparticles); and green fluorescence interior (FITC-labeled CM-dex coacervate). (c) Zeta-potential measurements showing variation of coacervate droplet surface charge before and after Au/MTA-PEG membranization. Samples were prepared at a range of PDDA/CM-dex molar ratios. (d) Time series of CLSM images of Au-nanoparticle-caged coacervate droplets recorded before (1), 30 s after photobleaching (2), 5 min after recovery (3), and 10 min after recovery (4). The photobleaching and control areas are delineated by blue or green rectangles, respectively. Red fluorescence, Au/RITC-labeled TA-PEG nanoparticles. (e) Plots of changes in fluorescence intensity for delineated areas shown in (d). Minimal recovery of the fluorescence is observed over 10 min due to the solid-like membrane. (f,g) Time series of CLSM images (f) and corresponding changes in membrane and interior (core) red fluorescence [(g) gray value] for an Au/RITC-TA-PEG nanoparticle-caged coacervate droplet after light illumination. (h) CLSM images of Au/RITC-labeled TA-PEG nanoparticle-caged coacervate droplets before (1) and after (2) exposure to light showing membrane disassembly and translocation of the de-capped Au nanoparticles into the coacervate interior. Application of a shear force reassembles the membrane (3), which can be subsequently disassembled by further light exposure (4). (i) Plot showing time-dependent decreases in pH associated with glucose-triggered GOx activity in Au/TA-AE-PEG6k-caged coacervate droplets (+) or membrane-less coacervate droplets containing GOx (−). The pH decrease is lower in the presence of TA-AE-PEG6k due to the consumption of protons in the cleavage reaction. GOx, glucose, and TA-AE-PEG6k concentrations are 0.2 mg/mL, 10 mM, and 0.2 mg/mL, respectively. (j) Time series of CLSM images of a single GOx-containing Au/RITC-labeled-TA-AE-PEG6k-caged coacervate droplet before and after addition of glucose showing chemically mediated membrane disassembly. (k) Corresponding fluorescence intensity (gray values) profiles of the caged coacervate droplet shown in (j), before (top) and 40 min after (bottom) addition of glucose. Scale bars: 50 (a), 10 (b), and 5 μm (d,f,h,j). Error bars represent the standard deviation; (c,e,g,i), n = 3, 3, 20, and 3, respectively.

Figure 3

Stimuli-responsive uptake and capture in nanoparticle-caged coacervate microdroplets. (a) Scheme showing light-mediated relaxation of mechanical strain and appearance of membrane apertures in Au/TA-PEG nanoparticle-caged coacervate droplets, leading to the uptake and capture of external microparticles (protein@ZIF8; mean size, 0.5 μm; red) or guest protocells (PCVs; mean diameter, 4 μm; blue). (b) Time series of optical images of an individual caged coacervate droplet surrounded by a dense population of PCVs recorded before (left) and after 163 s (middle) and 630 s (right) of continuous light exposure. The PCVs are progressively taken up and captured within the unjammed caged coacervate droplets. Samples were mounted onto a pegylated glass substrate. (c) Overlay of fluorescence and bright field images of an individual caged coacervate droplet and RITC-labeled PCVs (red fluorescence) recorded 10 min after continuous exposure to light at 290–390 nm. (d,e) Overlays of fluorescence and bright field images of an individual caged coacervate droplet and RITC-labeled colloidosomes (red fluorescence) recorded in ambient daylight (d) and 10 min after continuous exposure to light at 290–390 nm (e) showing transformation from non-interacting protocells to contact-dependent transmembrane transfer. Scale bars, 20 μm. (f,g) CLSM images of red/green fluorescence overlay images showing a single FITC-labeled GOx-containing Au/TA-AE-PEG6k-caged coacervate droplet surrounded by RITC-labeled PCV and recorded before (f) and 60 min after (g) addition of glucose. Unjamming of the membrane by enzyme-mediated cleavage of TA-AE-PEG6k results in PCV transfer across the membrane. The focal plane is aligned with the PCVs not the caged coacervate droplet. White dash circles delineate the boundary of the caged coacervate droplet. Scale bars, 20 μm. (h–k), FACS-derived 2D dot plots of side-scattered light (SSC) vs forward-scattered light (FSC) for single population of PCVs (h), single population of GOx-loaded caged droplets (i), mixture of PCVs and GOx-loaded caged droplets in the absence of glucose (j) and 1 h after the addition of glucose (k).

Figure 4

Protocell sorting by nanoparticle-caged coacervate microdroplets. (a) Scheme showing chemical-mediated protocell sorting. PCVs (red) and PEG-grafted colloidosomes (green) are mixed with GOx-loaded Au/TA-AE-PEG6k-caged coacervate droplets in the presence of glucose. Chemical-mediated membrane unlocking leads to the selective uptake and capture of only the PCVs. (b,c) CLSM image of red/green fluorescence overlay (b) and bright-field image (c) showing selective uptake of RITC-labeled PCVs (red fluorescence) into the unlocked caged coacervate droplets, while FITC-labeled PEG-tagged colloidosomes (green fluorescence) remain in the external water environment. White dash circles indicate the boundary of the unlocked caged droplets. Scale bars, 20 μm. (d) FACS-derived dot plots showing RITC (PE) fluorescence vs FITC fluorescence for a mixture of FITC-labeled PEG-tagged colloidosomes (green shading) and RITC-labeled PCVs (red shading) (volume ratio = 1:1). (e) Same as for (d) but after protocell sorting showing presence of only FITC-labeled PEG-tagged colloidosomes (green shading) in the resulting supernatant phase.