Artificial cells with viscoadaptive behavior based on hydrogel-loaded giant unilamellar vesicles

Viscoadaptation is an essential process in natural cells, where supramolecular interactions between cytosolic components drive adaptation of the cellular mechanical features to regulate metabolic function. This important relationship between mechanical properties and function has until now been underexplored in artificial cell research. Here, we have created an artificial cell platform that exploits internal supramolecular interactions to display viscoadaptive behavior. As supramolecular material to mimic the cytosolic component of these artificial cells, we employed a pH-switchable hydrogelator based on poly(ethylene glycol) coupled to ureido-pyrimidinone units. The hydrogelator was membranized in its sol state in giant unilamellar lipid vesicles to include a cell-membrane mimetic component. The resulting hydrogelator-loaded giant unilamellar vesicles (designated as HL-GUVs) displayed reversible pH-switchable sol–gel behavior through multiple cycles. Furthermore, incorporation of the regulatory enzyme urease enabled us to increase the cytosolic pH upon conversion of its substrate urea. The system was able to switch between a high viscosity (at neutral pH) and a low viscosity (at basic pH) state upon addition of substrate. Finally, viscoadaptation was achieved via the incorporation of a second enzyme of which the activity was governed by the viscosity of the artificial cell. This work represents a new approach to install functional self-regulation in artificial cells, and opens new possibilities for the creation of complex artificial cells that mimic the structural and functional interplay found in biological systems.

1x and 5x phosphate buffered saline (PBS) was prepared using PBS tablets (Sigma Aldrich), which was filtered before use (MF-Millipore Membrane filter, 0.45 µm pore size).MQuant® pH-indicator paper was obtained from Merck.

Instrumentation
Confocal fluorescence experiments were performed using a Leica TCS SP8 inverted confocal microscope (Leica microsystems) equipped with a 63x objective (water or glycerin immersion objective).For DOPE-RhB imaging (membrane marker), a 552 nm laser and HyD detector (565-610 nm) were employed.For Cy5 imaging (hydrogelator marker), a 638 nm laser and PMT detector (650-715 nm) were employed.18-well glassbottom chamber slides (ibidi GmbH) were used for vesicle visualization, which were treated with 1 mg/mL BSA in Milli-Q for >30 min, followed by washing with Milli-Q.
Plate reader experiments were performed using a Tecan MC-Spark plate reader and 96 flat transparent Nunc plates.A potentiometric pH meter (FiveEasy Plus FEP20) equipped with a micro electrode (Mettler Toledo) was employed to set the pH of the working solutions.Micrograph images were quantified and processed with Fiji, a program developed by the NIH and available as public domain software at https://imagej.net/Fiji.

Hydrogel precursor preparation
The hydrogel precursor was prepared by dissolving UPy-PEG (20 mM, 20 wt%) in basic Milli-Q (pH ~11 adjusted with 1M NaOH) at 70 °C for 1h.After dissolving, the pH was adjusted to ca. 10 with 1M HCl or 1M NaOH.For visualization purposes, monofunctional UPy-Cy5 was added from a stock (5 mg/mL in DMSO) to the dissolved hydrogelator at a concentration of 100 µM.Hydrogelator solutions were prepared fresh and used no longer than 5 hours before encapsulation in the HL-GUV.

Assembly of hydrogel-loaded giant unilamellar vesicles (HL-GUVs)
HL-GUVs were prepared by adapting our previously reported protocol for preparation of GUVs by the inverted emulsion method. 3All lipids were prepared in stock solutions in chloroform, from which they were added to the paraffin oil.The main lipid components DOPC/POPC/Chol were combined in a 35/35/30 molar ratio in 200 µL of paraffin (total lipid concentration 10 mM).In addition, 1% of DSPE-PEG and 0.06% DOPE-RhB were added for membrane functionalization.This mixture was heated to 80 °C for 30 min in a sand bath.Inner phase solutions (20 µL) for HL-GUVs were prepared containing 200 mM sucrose and 10 µL of hydrogel precursor solution (final hydrogelator concentration in the inner phase=10 wt%).When required, urease (3.5 mg/mL), 0.1 mM pyranine, esterase (3.5 mg/mL), and/or HRP (3.5 mg/mL) were added to the inner phase.20 μL of the inner phase were added to the paraffin oil suspension and vortexed for 25 seconds while turning the tube to prevent sedimentation.For enzyme activity experiments, PBS 1X was added in the inner and outer phases.The entire suspension was taken and layered on top of an outer phase solution in a tube (200 mM glucose, pH ~9).Subsequently, the tube was centrifuged at 3,300 g for 20 minutes at room temperature.The HL-GUVs were obtained by puncturing the tube at the bottom and obtaining the aqueous layer.The HL-GUVs were purified by centrifuging for 2 minutes at 1,500 g and carefully washing with outer phase, removing the supernatant.This washing step was performed twice.
Preparation of giant unilamellar vesicles (GUVs) containing no hydrogel were performed in a similar manner, with the hydrogel precursor volume being replaced by basic MilliQ water.

Fluorescence recovery after photobleaching (FRAP) experiments
Using the confocal microscope, a circular area of 3 μm at the center of the selected vesicle was photo-bleached at 100% laser power (excitation at 638 nm) for 10 frames (1 frame/s).
Images were processed using the Leica Las X software.The fluorescence intensity was normalized by the prebleach steady state fluorescence intensity, correcting for background fluorescence.The half time recovery and mobile fraction were determined by using the FrapBot software using a single exponential fitting. 4For experiments depicted in Fig. 4, the pH of the environment was switched between basic (pH≥8.5)and acidic (pH≤7) by addition of small aliquots of HCl (0.1M) and NaOH (0.1M).For experiments depicted in Fig. 5, the pH of the environment was acidified by addition of small aliquots of HCl (0.1M); urea was added at a final concentration of 25 mM followed by incubation for at least 1 hour.FRAP measurements were performed at least three times using different vesicles.The intensity of the ratiometric pH probe HPTS (co-encapsulated in the HL-GUV lumen) was examined upon excitation at 405 nm and 488 nm.

Reaction kinetics experiments
For reaction kinetics experiments, inner and outer phases were supplemented with PBS (1x).After preparation, the prepared HL-GUVs were placed in PBS solution (1x, 200 mM glucose) at the corresponding pH (7 or 9).For calcein-AM conversion experiments, calcein-AM (400 nM) was added to the outer phase in a microscope well, followed by addition of HL-GUVs and incubation for 30 min.Calcein intensity (excitation at 488 nm, emission at 500-535 nm) was then monitored every 5 minutes by taking images at several spots containing HL-GUVs.For Amplex Red conversion experiments, HL-GUVs were placed in PBS solution (1x, 200 mM glucose) at pH 7 for 10 min followed by addition of urea (25 mM final concentration).After 1 hour incubation, Amplex Red (10 µM) and H2O2 (0.001%) were added and the resorufin intensity (absorbance at 572 nm) was monitored using a plate reader spectrophotometer (with spectra being taken every 3 minutes).

Fig. S2 .
Fig. S2.Confocal image of HL-GUVs after reducing the pH to 7, shown as the overlay

Fig. S4 .
Fig. S4.Confocal image of HL-GUVs after reducing the pH to 3 (acquired using a 20x

Fig. S6 .
Fig. S6.HPTS fluorescence as a function of pH upon excitation at 480 nm (emission at

Fig. S8 .
Fig. S8.(a) Half time recovery through multiple cycles upon subsequent addition of acid

Table SI - 1 .
Summary of reported artificial cell systems with regulation of catalytic function.