Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arrays

Abstract

Micro-arrays of discrete or hemifused giant unilamellar lipid vesicles (GUVs) with controllable spatial geometries, lattice dimensions, trapped occupancies and compositions are prepared by acoustic standing wave patterning, and employed as platforms to implement chemical signaling in GUV colonies and protocell/living cell consortia. The methodology offers an alternative approach to GUV micro-array fabrication and provides new opportunities in protocell research and bottom-up synthetic biology.

Fig. 1. (a) Schematic of sucrose-containing GUVs in isotonic glucose solution used for acoustic trapping. (b–d) Simulation of the acoustic pressure distribution in the acoustic trapping device for one pair of piezoelectric transducers (PZTs) (b), two pairs of PZTs with a square arrangement (c), and three pairs of PZTs with hexagonal arrangement (d); high pressure (blue, antinodes), low pressure (red, nodes). (e–h) Representative fluorescence microscopy images of micro-arrays of green GUVs (5% NBD-PE) produced in a 1D acoustic pressure field (6.71 MHz, 10 V) (e); 2D field (6.69/6.71 MHz, 10 V) (f); 2D field (5.06/9.13 MHz, 10 V) (g), and 2D field (6.70/6.71/6.72 MHz, 10 V) (h). Centre-to-centre line spacings: ca. 110 μm (e), 110 × 110 μm (inter-vesicle distance, ca. 50 μm) (f), 146 × 81 μm (g). All scale bars are 100 μm.

Fig. 2. (a–c) Representative fluorescence microscopy images of acoustically trapped rectangular micro-arrays consisting of single (a), double (b) and multiple (c) GUVs at each pressure node; samples prepared using GUV/node number ratios (R_G/N) of 1.2 (a), 2.2 (b) and 3.4 (c). All scale bars are 100 μm. (d) Plots of the percentage of unoccupied nodes (no GUVs, black) and occupied nodes containing single (red), double (blue) or multiple GUVs (≥3, cyan) against R_G/N. In total, three independent replicates were conducted at each GUV/node number ratio (R_G/N). In each replicate, 500 nodes were counted. Error bars represent the standard deviation.

Fig. 3. (a) Schematic of a hemifused heterogeneous pair of acoustically trapped GUVs containing either HRP or GOx, followed by addition of melittin, Amplex red and glucose. (b) Superimposition of bright field and fluorescence microscopy images of a rectangular micro-array consisting of colonies of co-trapped NBD-PE-labeled HRP-containing GUVs (green fluorescence) and unlabeled GOx-containing GUVs. (c) As for (b), but after Ca²⁺-induced hemifusion. (d) As for (c), but superimposition of red and green fluorescence image recorded 11 min after addition of melittin and Amplex red. All scale bars are 50 μm. (e) Time-dependent changes in mean fluorescence intensity for GOx/HRP-mediated formation of resorufin in acoustically trapped micro-arrays comprising melittin-functionalized hemifused multiple GUVs (red), melittin-functionalized non-fused multiple GUVs (black) and hemifused GUVs without melittin (blue). The increase of fluorescence intensity at the single GUV level was followed and the error bars represent variations between GUVs during a single experiment. Initial rates; 2.37 ± 0.48 and 0.67 ± 0.16 a.u for hemifused and non-fused GUVs, respectively.

Fig. 4. (a) Schematic showing H₂O₂-induced killing of HepG2 cells by co-trapped melittin-functionalized GOx-containing GUVs. (b) Superposition of optical and green fluorescence microscopy images of co-trapped clusters of melittin/GOx-GUVs and HepG2 cells (dark objects) recorded 4 h after addition of glucose (GUV/HepG2 number ratio, 5.7). (c) Red fluorescence microscopy image of (b) after staining with PI to determine the number of dead cancer cells. (d) Superposition of (b) and (c). (e) Superposition of optical and green fluorescence microscopy images of co-trapped clusters of melittin/GUVs and HepG2 cells (dark objects) without GOx encapsulated in the GUVs recorded 4 h after addition of glucose (GUV/HepG2 number ratio, 5.7). (f) Red fluorescence microscopy image of (e) after staining with PI to determine the number of dead cancer cells. (g) Superposition of (e) and (f). Scale bars in (b–g) are 50 μm. (h) Corresponding histogram of percentage of dead HepG2 cells after 4 h for GUV/HepG2 number ratios of 0.5 (1), 1.4 (2), 2.9 (3) and 5.7 (4). Approximately 56 000 cells were used in each experiment; (n = 3, **p < 0.01, ***p < 0.001). (i) Control experiments undertaken at a GUV/HepG2 number ratio of 5.7; histograms showing low percentage (<6%) of dead cells after 4 h for HepG2 cells alone (1), HepG2/melittin-GUVs (no GOx) (2) and HepG2/GOx-GUVs (no melittin) (3) compared with HepG2/melittin/GOx-GUVs (4); (n = 3, ***p < 0.001). Three independent replicates were conducted in each experimental group and control group. Error bars represent the standard deviation.

Fig. 5. (a) Schematic showing IPTG-induced GFP expression in E. coli by co-trapped melittin-functionalized IPTG-containing GUVs. (b–d) Representative fluorescence microscopy images of GFP expression in E. coli cells (green fluorescence) (b), co-trapped GUVs (red fluorescence, 0.5% TR-DHPE) (c) and green/red superimposition (d) of co-trapped clusters of IPTG (150 mM)-containing GUVs and E. coli recorded 210 min after addition of melittin. (e) Time-dependent changes in mean fluorescence intensity for IPTG-induced GFP expression in E. coli by co-trapped melittin-functionalized GUVs containing IPTG with different concentrations. (f–h) Representative bright field image (f) and fluorescence microscopy images recorded at 463 nm excitation (green fluorescence) (g) and 535 nm (red fluorescence, 0.5% TR-DHPE labelled GUVs) (h) of co-localized IPTG containing GUVs and E. coli recorded 6 h without melittin. All scale bars are 50 μm. The darker dots observed at the nodes (white arrows in the inset in (f)) are E. coli cells. Image g indicates no GFP expression in E. coli in the absence melittin. Scale bars are 50 μm.