Modulation of Higher‐order Behaviour in Model Protocell Communities by Artificial Phagocytosis

Abstract Collective behaviour in mixed populations of synthetic protocells is an unexplored area of bottom‐up synthetic biology. The dynamics of a model protocell community is exploited to modulate the function and higher‐order behaviour of mixed populations of bioinorganic protocells in response to a process of artificial phagocytosis. Enzyme‐loaded silica colloidosomes are spontaneously engulfed by magnetic Pickering emulsion (MPE) droplets containing complementary enzyme substrates to initiate a range of processes within the host/guest protocells. Specifically, catalase, lipase, or alkaline phosphatase‐filled colloidosomes are used to trigger phagocytosis‐induced buoyancy, membrane reconstruction, or hydrogelation, respectively, within the MPE droplets. The results highlight the potential for exploiting surface‐contact interactions between different membrane‐bounded droplets to transfer and co‐locate discrete chemical packages (artificial organelles) in communities of synthetic protocells.

Laura Rodríguez-Arco,B .V .V .S .P avan Kumar,Mei Li, Avinash J. Patil, and Stephen Mann* Abstract: Collective behaviour in mixed populations of synthetic protocells is an unexplored area of bottom-up synthetic biology.T he dynamics of am odel protocell community is exploited to modulate the function and higher-order behaviour of mixed populations of bioinorganic protocells in response to ap rocess of artificial phagocytosis.E nzymeloaded silica colloidosomes are spontaneously engulfed by magnetic Pickering emulsion (MPE) droplets containing complementary enzyme substrates to initiate ar ange of processes within the host/guest protocells.Specifically,catalase, lipase,oralkaline phosphatase-filled colloidosomes are used to trigger phagocytosis-induced buoyancy,membrane reconstruction, or hydrogelation, respectively,w ithin the MPE droplets. The results highlight the potential for exploiting surfacecontact interactions between different membrane-bounded droplets to transfer and co-locate discrete chemical packages (artificial organelles) in communities of synthetic protocells.
Artificial aqueous microcompartments capable of mimicking biological functions,s uch as encapsulation, selective exchange of chemicals with the environment, and minimal metabolism, are currently under investigation as model protocells in synthetic biology,o rigin-of-life studies,a nd biotechnology. [1][2][3][4][5] Thee xternal membrane of these synthetic microcapsules can be tailored using aw ide range of building blocks,s uch as lipids,p olymers,p rotein-polymer conjugates, and inorganic nanoparticles,t omeet specific criteria. [2] For example,aprotocell model based on the spontaneous assembly of partially hydrophobic silica nanoparticles at the interface of water and oil to form water-in-oil Pickering emulsion droplets with amechanically robust membrane has been recently developed. Thei norganic membrane was crosslinked to produce shell-like micro-compartments (colloidosomes) that could be transferred to water and endowed with biomimetic functions,s uch as in situ gene expression, [6] enzyme-mediated catalysis, [6,7] microcapsule growth and divi-sion, [8] membrane gating, [9] enzyme-directed secretion of an extracellular-like matrix, [10] and energy capture and conversion. [11] Thee mergence of collective behaviour in mixed populations of synthetic protocells is relatively unexplored, even though increasing levels of protocell interactivity could provide more complex and synergistic functions,s uch as resilience to environmental stress,c hemical signalling for artificial quorum sensing,a nd multiplex tasking via collaboration and specialization. Chemical communication and signalling have been demonstrated between populations of vesicles and bacteria, [12,13] lipid vesicles containing gene circuitry, [14] vesicles and proteinosomes, [15] colloidosomes, [16] modified [17] or immobilized [18] coacervate droplets,a nd in water-in-oil emulsion droplets. [19][20][21] Hierarchically structured compartments involving different types of protocells,such as liposome-encapsulated coacervate droplets, [22] multi-compartmentalized polymersomes, [23] nested vesicles, [24] and giant vesicles comprising light-harvesting organelles, [25] have also been reported. Contact-induced adhesive interactions between proteinosomes [26] or lipid-coated emulsion droplets [27] have been developed to produce aggregates of protocells with coordinated functions (prototissues), and sophisticated population dynamics used as mechanisms of artificial predation [28] and parasitism [29] in dispersed binary protocell populations.
We recently developed ap rimitive form of artificial phagocytosis in as ize-mismatched binary population of water-in-oil Pickering emulsion droplets in which crosslinked semi-permeable silica nanoparticle-stabilized colloidosomes were spontaneously ingested by larger magnetic iron oxide Pickering emulsion (MPE) droplets comprising particle-free fatty acid stabilized apertures. [30] Thep article-free patches were formed by addition of oleic acid to the oil (dodecane) phase (typically 2mgmL À1 ), which partially destabilized the iron oxide shell by changing the oil-water interfacial tension at the surface of the MPE droplets. Engulfment of the silica colloidosomes through the apertures was associated with the formation of as urface-adsorbed oleate bilayer,w hich was facilitated by maintaining ap H within the MPE droplets close to the fatty acid pK a (9.8). [30] Significantly,s mall water-soluble molecules initially entrapped within the colloidosomes were released into the MPE droplets after phagocytosis.A saconsequence,b yi nitially encapsulating an enzyme substrate and complementary enzyme in the colloidosomes and MPE droplets,respectively, ad ephosphorylation reaction could be triggered in the aqueous phase of the magnetic microcapsules.H erein, we extend this approach to the delivery of enzymes contained in the silica colloidosomes as aw ay to induce higher-order structural and functional changes inside the MPE droplet host via artificial phagocytosis.A st he macromolecular payloads are too large to diffuse through the pores of the silica nanoparticle membrane,t he enzymes are delivered in the form of discrete chemical packages (artificial "organelles") with minimal contamination of the internal solution of the MPE droplets.S pecifically,w ep repare catalase-filled colloidosomes capable of activating the buoyant motion of hydrogen peroxide containing MPE droplets,l ipase-entrapped colloidosomes that trigger their own phagocytosis by changing the interfacial tension of the MPE droplets via in situ triglyceride hydrolysis,a nd alkaline phosphatase-containing colloidosomes that initiate the nucleation and growth of as upramolecular hydrogel network inside amino acid-containing MPE droplets.T hese higher-level changes are relevant in the context of protocell community dynamics, enabling phenomena such as the spontaneous segregation of two populations,s elf-triggering of phagocytosis/viral-like behaviour,a nd arrestment of phagocytosis by ap rey population. Taken together, our results show that the dynamics of model protocell communities can be exploited to modulate the function and higher-order behaviour of mixed populations of micro-compartmentalized colloidal objects in response to internal triggers.
Phagocytosis-inspired behaviour was induced by addition of oleic acid to adodecane dispersion of MPE droplets (mean size = 500 AE 250 mm; pH 10.2) and silica colloidosomes (mean size = 52 AE 10 mm; pore size ca. 3-4 nm;Supporting Information, Figure S1). [30] By encapsulating catalase and hydrogen peroxide (H 2 O 2 )i nt he silica colloidosomes and MPE droplets,r espectively,i ngestion of the colloidosomes (Supporting Information, Figure S2) triggered the almost instantaneous enzyme-mediated production of micrometre-sized oxygen bubbles inside the MPE droplets (Figure 1a-c; Supporting Information, Movie S1) provided that the encapsulated H 2 O 2 and catalase concentrations were higher than 1% and 1mgmL À1 ,r espectively ( Figure 1d). Bubble generation in MPE droplets containing 1% H 2 O 2 increased from ca. 20 to 50 %when the entrapped catalase concentration was increased from 5to30mgmL À1 whilst at 5% H 2 O 2 essentially all the MPE droplets contained gas bubbles (Supporting Information, Figure S3a). Although the oxygen bubbles were exclusively generated inside the aqueous interior of the MPE droplets,t hey were often released to the external environment through the fatty-acid stabilized patches in the magnetic membrane (Supporting Information, Movie S2). Nevertheless,for concentrations of H 2 O 2 higher than 2% (Supporting Information, Figure S3b), nucleation and formation of bubbles occurred faster than their release to the external oil phase,leading to accumulation within the MPE droplets and buoyancy ( Figure 1e;S upporting Information, Figure S3b, Movie S3). Significantly,t he formation of gas bubbles and generation of buoyancyr equired an interaction between the two protocell populations via phagocytosis in contrast to previous results involving the buoyant motion of as ingle population of organoclay/DNAp rotocells. [31] Theb uoyant translational motion was characterized by well-defined vertical trajectories towards the oil/air interface with an average speed of around 1.5 cm s À1 .P hagocytosis of one single colloidosome was often enough to generate buoyant MPE droplets for levels of encapsulated catalase above 30 mg mL À1 ,w hile several capture events were required to float the MPE droplets at lower enzyme concentrations ( Figure 1f). As ac onsequence,d epending on the entrapped enzyme concentration, deposition of the MPE droplets onto ah ighly crowded field of sedimented colloidosomes resulted in almost instantaneous or delayed (ca. 20 sa t1mg mL À1 ) buoyancy-induced segregation of the two protocell populations (Supporting Information, Figure S4).
We sought to increase the complexity of the above artificial phagocytosis process by developing as trategy that would enable in situ triggering of protocell ingestion by auxiliary enzyme activity in the guest colloidosomes rather than relying on the external addition of oleic acid. Forthis,we prepared lipase-containing silica colloidosomes,a dded them to apopulation of MPEs dispersed in adodecane solution of at riglyceride (triolein), and investigated whether enzymemediated hydrolysis of triolein within the colloidosomes and concomitant release of surface-active agents (mono/diacyl glycerols,oleic acid) could induce the opening of apertures in the shell of initially intact MPE droplets (Figure 2a). Control experiments showed that the shell of the MPE droplets remained immobilized and closed when dispersed in a2 0mgmL À1 triolein solution in the presence or absence of lipase-free colloidosomes (Supporting Information, Figure S5). In contrast, when lipase-containing silica colloidosomes were added to the dodecane phase,t he fluidity of the iron oxide membrane progressively increased such that particle-free domains were produced within the MPE shell ( Figure 2b;Supporting Information, Movie S4). Formation of the particle-free apertures was dependent on the triolein and lipase concentrations (Figure 2c;S upporting Information, Figure S6a), consistent with asubsequent decrease of the oilwater interfacial tension at the surface of the MPE droplets owing to the production of mono/diacylglycerols and oleic acid (Figure 2d;S upporting Information, Figures S6b and  S7). Significantly,s egregation of the iron oxide membrane resulted in slow penetration of the lipase-containing colloidosomes into the aqueous phase of the MPE droplets through the particle-free domains exposed at the water-droplet-oil interface (Figure 2b;S upporting Information, Movie S4). Tr ansfer into the host interior occurred more slowly compared with when the colloidosomes were initially dispersed in a2mg mL À1 solution of oleic acid in dodecane (ca. 2min vs. 3s ,r espectively) even though the decrease in oil/water interfacial tension with surfactant concentration was similar for oleic acid and am onoacylglycerol such as monoolein (1oleoyl-rac-glycerol;S upporting Information, Figure S8). [32] Control experiments indicated that both surfactants produced particle-free apertures when added individually to dodecane dispersions of the MPE droplets,b ut that the rates of colloidosome penetration were negligible for monoolein. As transfer into the water phase of the MPE droplets is dependent on the formation of asurfactant bilayer/multilayer at the surface of the colloidosomes, [30] we attributed the difference in phagocytosis efficiencyt ot he facile interfacial assembly of oleic acid compared with mono-and diacylglycerols.
Given the above observations,w ec o-encapsulated catalase and lipase within the silica colloidosomes to produce ah ybrid protocell capable of triggering phagocytosis and inducing buoyancy( or disintegration) of the host MPE droplet. Optical microscope images showed that upon addition of the enzyme-containing silica colloidosomes to as uspension of 5% H 2 O 2 -containing MPE droplets (carbonate buffer, 1m,p H10.2) dispersed in a4 0mgmL À1 dodecane solution of triolein, the magnetic membrane was rapidly opened and bubbles were progressively formed in the aqueous phase lumen to produce buoyant MPE droplets (Figure 2e;Supporting Information, Movie S5).
Finally,w eu sed the process of artificial phagocytosis to trigger in situ structural reconfiguration of the MPE/colloidosome host-guest microcompartments.F or this,w em ixed apopulation of alkaline phosphatase (ALP)-containing silica Figure 2. a) The use of lipase-containing colloidosomes for triggering artificial phagocytosis. Lipase-mediated hydrolysis of triolein releases surface-active molecules that produce particle-free apertures in the MPE droplet membrane followed by engulfmentoft he colloidosomes. b) Time sequenceofoptical microscopyimages showing the formation of aparticle-free aperture in the initially intact shell of aMPE droplet (pH 10.2) dispersed in adodecane solution of triolein (10 mg mL À1 ) after addition of multiple silica colloidosomes containing encapsulated lipase (100 UmL À1 ). The silica colloidosomes in contact with the particle-free patches of the magnetic droplet are spontaneously transferred into the aqueous phase. See the SupportingInformation, Movie S4 for complete sequence. Scale bar = 100 mm. c) Plot showing time-dependent percentage changes in particle-free surface area for MPE droplets against triolein concentration after addition of silica colloidosomes containing encapsulated lipase (100 UmL À1 ). Increased levels of triolein hydrolysis are associated with larger apertures in the MPE droplets. d) Plots of the concentration of free oleic acid (column plot, left Yaxis) and oil/water interfacial tension (scatter plot, right Y axis) against the initial concentration of triolein in dodecanea fter the addition of lipase-containing colloidosomes (10 000 UmL À1 ). The concentration of released oleic acid increasesw ith the initial substrate concentration resulting in adecrease of the interfacial tension responsible for the formation of particle-free domains in the MPE droplet membrane.E rror bars correspond to standard deviations. e) Time sequence of optical microscopyi mages showing the formation of am embranea perture and bubble formation in an initially intact MPE droplet (pH 10.2) after addition of multiple silica colloidosomes containing both lipase (10 kU mL À1 )and catalase( 30 mg mL À1 ). Scale bar = 150 mm. White balance correction and increase of brightness have been applied to (b) and (e).
colloidosomes with ad ispersion of MPE droplets prepared from an aqueous solution of the phosphorylated amino acid precursor, N-fluorenylmethyloxycarbonyl-tyrosine-(O)-phosphate (Fmoc-TyrP) as am eans of generating the phagocytosis-mediated self-assembly of as upramolecular hydrogel network within the confined interior of the protocell construct. Engulfment of the silica colloidosomes resulted in ALP-mediated dephosphorylation of Fmoc-TyrP and selfassembly of N-fluorenylmethyloxycarbonyl-tyrosine (Fmoc-Ty rOH) into afilamentous hydrogel (Figure 3a). Optical and fluorescence microscopy images recorded in the presence of the hydrogel binding dye Hoechst 33 258 showed blue fluorescence associated with the engulfed colloidosomes (Figure 3b-e). Thef luorescence intensity increased rapidly within 1h of engulfment, after which there was as low increase over 24 h ( Figure 3f). Ten-fold changes in the concentration of either the substrate or enzyme had minimal effect on the final outcome of the hydrogelation reaction after 24 h( Supporting Information, Figure S9). We attributed this to the spatial restrictions placed on hydrogel growth owing to confinement within the limited aqueous volumes of the MPE/ colloidosome host-guest microcompartments.Hoechst 33258 fluorescence was mainly associated with the colloidosome interior (Figure 3d), consistent with diffusion of the Fmoc-Ty rP molecules through the cross-linked silica nanoparticle membrane and hydrogelation in close proximity to the entrapped ALP.B lue fluorescence was also observed in the associated MPE droplet phase and in partially engulfed or surface-connected silica colloidosomes (Supporting Information, Figure S10), indicating ac ounter-flow of Fmoc-TyrOH monomers from the colloidosomes into the surrounding aqueous microenvironments.When left to dry overnight, the hydrogelled MPE droplets remained intact, while in the absence of phagocytosis the MPE droplets irreversibly collapsed (Supporting Information, Figure S11). Scanning electron microscopy (SEM) images of dried MPE droplets recorded 7days after the onset of hydrogelation showed spheroidal porous microcompartments (Figure 3g)w ith ashell comprising anetwork of iron oxide particles,hydrogel filaments,a nd embedded silica colloidosomes (Figure 3g; Supporting Information, Figure S12). Immobilization of the iron oxide particles in the hydrogel prevented any further membrane restructuring of the MPE droplets by addition of adodecane solution of oleic acid (15 mg mL À1 ).
In conclusion, we have demonstrated that higher-order structural and functional changes can be enzymatically activated in ab inary population of bioinorganic protocells consisting of MPE droplets engineered to artificially phagocytose enzyme-containing silica colloidosomes.Phagocytosis of catalase-containing colloidosomes catalyses the decomposition of hydrogen peroxide encapsulated inside the MPE droplets to produce oxygen bubbles that give rise to buoyancy-induced vertical motion and removal of the MPE droplet population from the mixed protocell community. Alternatively,e ncapsulation of lipase in the silica colloidosomes triggers the phagocytosis response by generating particle-free domains in the MPE droplet membrane via colloidosome-mediated hydrolysis of triolein to produce free oleic acid in the continuous oil phase.C ombining these two strategies,encapsulation of both catalase and lipase inside the silica microcontainers transforms them into "viral" protocells capable of self-triggered phagocytosis and removal of the MPE proto-phagocyte population. Finally,w eh ave demonstrated that the artificial phagocytosis of silica colloidosomes containing encapsulated ALP generates an internal hydrogel network by enzyme-mediated dephosphorylation of an amino acid gel precursor trapped inside the MPE droplets.T aken together our results highlight the potential of exploiting surface-contact interactions between different membranebounded droplets to trigger encapsulated biochemical reactions that give rise to higher-order behaviours in protocell consortia. Although our observations are exploratory,collective interactivity between compartmentalized microscale objects could provide as tep towards new applications in Bars on data points represent standard deviations. g) Scanning electron micrograph of adried single Fmoc-TyrP-containing MPE droplet after phagocytosis of multiple ALP-encapsulating colloidosomes (intact small spheres)s howing ahydrogelled network of iron oxide particles and embedded silica colloidosomes. Scale bar = 100 mm. Increase of brightness has been applied to (g). biomimetic storage and delivery,a nd microreactor technology.