Spatial Positioning and Chemical Coupling in Coacervate‐in‐Proteinosome Protocells

Abstract The integration of molecularly crowded microenvironments into membrane‐enclosed protocell models represents a step towards more realistic representations of cellular structure and organization. Herein, the membrane diffusion‐mediated nucleation of either negatively or positively charged coacervate microdroplets within the aqueous lumen of individual proteinosomes is used to prepare nested hybrid protocells with spatially organized and chemically coupled enzyme activities. The location and reconfiguration of the entrapped droplets are regulated by tuning the electrostatic interactions between the encapsulated coacervate and surrounding negatively charged proteinosome membrane. As a consequence, alternative modes of a cascade reaction involving membrane‐ and coacervate‐segregated enzymes can be implemented within the coacervate‐in‐proteinosome protocells.

was then dialyzed against 65%, 40% and 20% ethanol/water for 2 h, then against Milli-Q water for 1 day to complete the transfer of the cross-linked proteinosomes into water.
Trace amounts of polyelectrolytes in the external water phase accompanying adventitious fragmentation of the proteinosomes during work-up were removed by graduated centrifugation. For this, 1 mL dispersions were centrifuged at 800 rpm for 1 min, followed by further centrifugation at 2000 rpm for 1 min, removal of the supernatant (400 μL) and replacement with 400 μL of Milli-Q water. This process was repeated three times to ensure removal of all extraneous components.
In situ preparation of a PDDA/PAA coacervate (monomer molar ratio = 1 : 1 or 2 : 3 at constant total polymer concentration) within proteinosomes containing PAA, PDDA and NaCl (see above methods) was achieved by dialysis of the crosslinked proteinosomes containing PDDA/PAA at the desired monomer molar ratio against 65, 40 and 20 vol% ethanol/water for 2 h, and then against Milli-Q water for 1 day (see above methods) to remove the NaCl to induce formation of an encapsulated PDDA/PAA coacervate phase and transfer the crosslinked proteinosomes into water.
Enzyme-mediated peroxidation in coacervate-in-proteinosome protocells Typically, 100 μL of an aqueous dispersion of BSA-NH2/PNIPAAm proteinosomes (pH 7.5, Tris-HCl (20 mM), NaCl (20 mM)) prepared with encapsulated HRP-FITC (0.8 mg mL -1 ) and PDDA (10 mM, MW = 100-200 kDa) was added to a 96-well well plate, followed by addition of ATP (60 μL, 50 mM) to produce a ATP/PDDA coacervate inside the proteinosomes. An aqueous solution of ABTS (100 μL, 1 mM) was then added and the mixture left for 60 s to ensure that sequestration of the substrate into the coacervate phase had reached equilibrium. A solution of H2O2 (40 μL, 1 mM) was then added to initiate the HRP-mediated peroxidation reaction. The increase in absorbance at 410 nm due to formation of the ABTS radical cation (ε410 = 36000 M -1 cm -1 ) was recorded as a function of time using a BMG labtech Clariostar plate reader. The enzyme activity in the absence of ATP/PDDA coacervate micro-droplets was carried out following the same procedure except for replacing the ATP solution with Milli-Q water. The kinetic rates were calculated based on the following equation: [V0]=∆A/∆t.ε.c, where ∆A/∆t was the change in absorbance at 410 nm over a given time period, ε molar attenuation coefficient of oxidised ABTS (ε410 = 36000 M -1 cm -1 ) and c the path length (1 cm).
Optical and confocal fluorescence microscopy. Optical microscopy experiments were carried out on a Leica DMI 3000B optical microscope. Fluorescence imaging was performed using a Leica DFC 310FX set up. Confocal fluorescence microscopy measurements were performed using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope equipped with a resonant scanner and an adaptive focus control to correct focus drift during time-courses with a 65 mW Ar laser (488 nm for FITC excitation, 15% power), 50 mW 405 nm diode laser (DyLight-405 excitation, 10% power). Detection bands were set at 500-560 nm with 20% gain (FITC) and 415-465 nm with 20% gain (DyLight-405). All experiments were carried out in an environmental chamber maintained at 25 °C.
Fluorescence-activated cell sorting (FACS). Freshly prepared dispersions (≈ 1 mL) of CMD-FITCcontaining proteinosomes, bulk CMD-FITC/CHXD coacervates (monomer molar ratio = 2 : 1) or coacervate-in-proteinosome protocells (CMD-FITC/CHXD, monomer molar ratio = 2 : 1) were investigated using a FACS Canto II flow cytometer operating at low pressure with a 100 µm sorting nozzle. 2D dot plots of the side-scattered light area (SSC-A) versus forward-scattered light area (FSC-A) were determined for a total of between 10,000-20,000 particles for each population. The samples were analysed immediately after preparation to minimize the effect of coalescence.
Diameter of entrapped CMD-FITC/CHXD coacervate droplets. The diameters of the proteinosomeentrapped CMD-FITC/CHXD (monomer molar ratio = 2 : 1) coacervate droplets were obtained by recording confocal microscopy images approximately in the middle section of individual proteinosomes. The diameters were measured using imageJ image analysis software; typically an average of 100 measurements in groups of approximately 5 individual proteinosomes, with each group representing a specific diameter.
Equilibrium partitioning constant for ABTS. The partition constant (K) was determined under equilibrium conditions from K = CCOA/CS where CCOA was the concentration of ABTS in a coacervate bulk phase and CS the concentration of ABTS in the supernatant phase. Typically, 1300 μL of the coacervate micro-droplet solution was prepared by mixing 600 μL of 50 mM PDDA solution with 600 μL of 50 mM ATP solution (both at pH 8) followed by the addition of 100 μL of 10 mM ABTS solution. This solution was left for 10 mins to come to equilibrium and then centrifuged for 15 mins at 800 rpm to separate the bulk and supernatant phases. The concentration of ABTS in each phase was determined by recording the absorbance of ABTS at 340 nm. The ATP/PDDA coacervate phase was disassociated by addition of 0.5 M NaCl prior to UV-Vis analysis to avoid scattering effects associated with the turbid coacervate phase.

Supplementary Notes
Phase transformation-mediated assembly of coacervate-in-proteinosome protocells Host-guest nested protocells were also produced by co-encapsulation of high ionic strength (0.5 M NaCl) solutions of poly(acrylic acid) (PAA) and PDDA in the cross-linked proteinosomes followed by removal of the salt to induce coacervate phase transformation by lowering of the charge screening. By controlling the macromolecular molar ratios and hence the surface charge on the incipient coacervate primary droplets (Supplementary Note: Figure S01), a few or multiple coacervate droplets could be produced inside the proteinosomes depending on their stability with regard to coalescence Supplementary Note: Figure S02a). Note: Figure S02b,c), whilst a single large coacervate droplet or a few droplets were produced within the proteinosomes under charge neutral conditions (Supplementary Note: Figure S02d,e). In the former, the entrapped droplets were stable to coalescence over a period of at least 30 d (Supplementary Note: Supplementary Figure S03). In contrast, the single coacervate micro-droplets were essentially stationary and resided predominately in the centre of the sedimented proteinosomes, presumably due to their higher density compared with the surrounding aqueous solution. In both cases, co-encapsulation of the polyelectrolytes with DyLight 405-labelled glucose oxidase (GOx) indicated that enzymes in the aqueous interior of the proteinosomes could be efficiently partitioned into the PAA/PDDA coacervate droplets (Supplementary Note: Figure S02c,e).
Assembly of the droplets within the proteinosomes was reversible such that addition of aqueous HCl (pH 4.5) or NaCl (0.5 M) resulted in fast dissolution of the coacervate phase (Supplementary Note: Supplementary Figure S04).
Supplementary Note: Methods: Disassembly of entrapped PAA/PDDA coacervates. PAA/PDDA coacervate micro-droplets were prepared at a monomer molar ratio of 3 : 2 within BSA-NH2/PNIPAAm proteinosomes by dilution-mediated phase transformation. 40 µL of the aqueous dispersion of nested protocells was mounted on an optical microscopy slide and 2 µL of 5 M HCl added to decrease the pH to ca. 4.5 to protonate the acrylic acid groups of PAA. Alternatively, 10 µL of a 5 M solution of NaCl was added (final concentration = 0.5 M). Both procedures resulted in disassembly of the entrapped coacervate phase.