Concentrisome synthesis and characterizationIn our initial experiments we sought to generate and characterize the base nano-architecture. To achieve this, we formed multi-compartment systems in which the outer membranes were layered upon the inner ones. Unilamellar liposomes—which would later serve as nucleation points around which additional bilayers could assemble—were first fabricated using MHF, with a flow rate ratio (FRR, defined as the aqueous flow rate divided by the ethanol flow rate) of 20 and a total flow rate (TFR) of 210 μl min–1. The lipid composition used was dipalmitoylphosphatidylcholine (DPPC):cholesterol:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(dibenzocyclooctyl(polyethylene glycol)-2000) (ammonium salt) (DSPE-PEG2K-DBCO) (53:42:5 mol%; 6.8 mM in ethanol). Dibenzocyclooctyne (DBCO), here tethered to the lipid via a PEG chain, is capable of efficient covalent bond formation with azides via a widely used ‘click’ reaction called strain-promoted azide/alkyne cycloaddition (SPAAC). A PEG2K spacer in a brush conformation (5 mol%) was included for two reasons: (1) to distance the site of conjugation away from the lipid bilayer, and (2) to define the dimensions of dinter, noting the precedent for PEG-mediated bilayer spacing30,31. This nanoparticle suspension was then introduced into a second MHF chip such that it sheathed a central ethanolic stream, containing lipids of composition DPPC:cholesterol:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(azido(polyethylene glycol)-2000) (ammonium salt)(DSPE-PEG2K-N3) (53:42:5 mol%; 6.8 mM in ethanol). An FRR of 20 was chosen again with a TFR of 210 μl min–1.Dynamic light scattering (DLS) was used to assess the size of each population and track the formation of higher-order structures. A slight increase in the mean diameter was observed from approximately 100 nm to 120 nm (PDI = 0.16 and 0.26, respectively)—taken to indicate possible growth of a second bilayer (Fig. 2a). Each particle population was imaged using cryo-TEM where a clear difference in particle lamellarity could be observed (Fig. 2b,c). Estimates from particle counting over 50 micrographs indicated the presence of double-bilayer vesicles at 60% of the total particle population (see Supplementary Fig. 1 for additional micrographs). Importantly, no vesicles with more than two bilayers were observed. The co-existence of smaller structures (thought to indicate the presence of either micelles or PEGylated bicelles) could occasionally be seen by DLS (~1–30 nm). These structures were directly imaged using cryo-TEM (Supplementary Fig. 2) and are understood to be a common by-product of PEGylated-lipid/DPPC binary mixtures, taking the form of flat disks composed of a single lipid bilayer32,33. A tentative mechanism for concentrisome formation was proposed: that micellar and/or bicellar structures conjugate via click chemistry to pre-formed vesicles, eventually closing to form an additional bilayer. This was supported via cryo-TEM: some vesicles were observed to be surrounded by curved bicelle portions, thought to represent an intermediate state between unilamellar and concentrisome morphologies (Fig. 2d–f).Fig. 2: Characterization of concentrisomes via DLS and cryo-TEM.a, Dynamic light scattering data shows a slight shift of the hydrodynamic diameter of input liposomes, functionalized with DBCO, from approximately 100 nm (PDI = 0.16) to 120 nm (PDI = 0.26), after exposure to a new lipid composition via an MHF chip containing azide-functionalized lipids. Correlograms (correlation coefficient (g2−1) versus time) for both Gaussians are shown in the inset. b, Representative cryo-TEM micrograph for input liposomes (yellow), which are unilamellar. c, Representative cryo-TEM micrograph for concentrisomes (blue), showing inner and outer compartments. d–f, A graphic illustrating the potential stages of concentrisome growth with corresponding cryo-TEM examples. Left to right: pre-formed liposomes (d) act as nucleation points in the MHF chip, around which bicelle/micelles with azide functionality begin to assemble (see central micrograph in e). We propose that these covalently tethered assemblies eventually close over to generate an external spherical bilayer, generating the concentrisome morphology (f).Source dataMechanistic insights and process optimizationWe investigated the role of click chemistry in concentrisome formation. First, we ran the concentrisome synthesis procedure as before, but replaced terminally functionalized alkyne lipids with DSPE-PEG2K, such that the lipid composition used in both stages was DPPC:cholesterol:DSPE-PEG2K (53:42:5 mol%; 6.8 mM). Particle suspensions were analysed using DLS and cryo-TEM. Although some double-bilayer vesicles were observed, the majority of particles remained unilamellar, with under 10% of the total population exhibiting an additional bilayer (Supplementary Fig. 3). This was taken to infer a positive covalent click interaction between terminally functionalized PEG spacers promoted the formation of concentrisomes.We then used the fluorogenic and membrane-permeable dye 3-azido-7-hydroxycoumarin to probe the reactivity of DBCO (ring-strained alkyne) functional groups before and after concentrisome formation. The dye is capable of SPAAC chemistry and, after generating a triazole with ring-strained alkynes, produces a fluorescent signal34. It was proposed that the number of available alkynes would reduce following the assembly of an additional bilayer via SPAAC around the pre-formed liposomes, leading to a lower intensity fluorescent signal. The concentration of DSPE-PEG2K-N3 in the outer bilayer was varied from 0 to 5 mol%, off-set by DSPE-PEG2K, such that the total PEGylated-lipid concentration was equal throughout. The particle concentration of the inner-alkyne-functionalized liposomes, and the flow rates used, were kept constant (FRR = 20; TFR = 210 µl min–1). We added 3-azido-7-hydroxycoumarin (with a final concentration of 6.15 μM) to each sample and analysed the emergence of a fluorescent signal over time. Results are shown in Supplementary Fig. 4b where an exponential decay in the fluorescence intensity for the triazole was observed as a function of azide-lipid present in the second bilayer. From this it was concluded that terminally functionalized DSPE-PEG2K alkyne lipids were interacting via SPAAC chemistry.Our assay was extended to explore the role of the (1) FRR and (2) TFR on the formation of concentrisomes via SPAAC. A lower fluorescence intensity for the triazole–coumarin adduct was taken as a proxy for the efficiency of concentrisome formation. The FRR values were varied from 10 to 60 and azidocoumarin was added to each sample as before. A second population at each FRR value was generated—this time using DSPE-PEG2K instead of DSPE-PEG2KN3—to serve as an internal reference from which the fluorescence intensity of the ‘click’ population could be compared (producing a change in fluorescence intensity ΔI). Under these assumptions, a larger ΔI corresponded to a more efficient SPAAC reaction, and, by extension, concentrisome formation. The TFR was varied from 200 to 450 µl min–1, and the assay (with internal references) was conducted as before (Supplementary Fig. 4c). We found that an FRR value of 20, as previously used, yielded the largest ΔI value. There was no clear dependence between concentrisome formation efficiency and TFR, and 210 µl min–1 remained the TFR of choice for future experimentation.Using PEGylated lipid to modulate d
inter
Cryo-TEM was used to provide a more detailed structural analysis of the particles we generated, in particular, the dimensions of dinter. It was proposed that this dimension could be modulated using PEG spacers with varying persistence lengths. To explore this, concentrisomes containing PEGylated lipids of different PEG lengths were generated under the same conditions as before; dinter was measured for each concentrisome. A point at the approximate centre of each concentrisome was chosen and the annular space calculated in four directions (Supplementary Fig. 5). When non-PEGylated liposomes without azide/alkyne functionality were generated, evidence of a higher percentage of multi-lamellar structures was observed, although the number of bilayers per particle was varied (average dinter ≈ 10 nm). On the other hand, clear two-compartment liposomes were observed when using PEG-tethered ‘clickable’ groups, with dinter increasing with the length of the PEGylated lipid of the inner and outer bilayers (histogram and cryo-TEM snapshots are shown in Fig. 3a,b). For a PEG2K DBCO/PEG2K azide combination, dinter averaged at 23 nm. For a PEG2K DBCO/PEG5K azide combination, the average was 34 nm, and a PEG5K DBCO/PEG5K azide combination averaged at 44 nm. This notable increase was taken to infer that PEG tethers were present in a largely uncoiled brush conformation perpendicular to each lipid bilayer, acting as steric scaffolds at 5 mol% (ref. 35).Fig. 3: Relationship between PEGylated-lipid length and intermembrane space.a, The variation of dinter between successive bilayers, using PEG chains of different molecular weights, and in various combinations, is shown. Micrographs correspond to combinations, with average dinter values of 10, 23, 34 and 44 nm, for no-linker, PEG2KDBCO:PEG2KN3, PEG2KDBCO:PEG5KN3 and PEG5KDBCO:PEG5KN3, respectively. Scale bars, 25 nm. b, A histogram with measured dinter values for each linker combination. Gaussian plots are added for clarity. A gradual increase in dinter is seen with increasing combined PEG length; >25 concentrisomes were analysed to generate each distribution.Source dataControlling bilayer compositionA fluorescence assay was developed to demonstrate the potential for concentrisomes to act as compartmentalized, multifunctional nano-capsules, and that the composition of each bilayer can be user-defined. The assay used the self-quenching dye calcein and a thermoresponsive lipid composition capable of cargo release following heating to a gel/fluid phase transition temperature (Tm). We were able to develop a system where one bilayer was thermoresponsive and the other was non-thermoresponsive (graphically illustrated in Fig. 4a; a full list of each composition used is given in a table as part of Supplementary Fig. 7).Fig. 4: Validation of layering and compositional control.a, A graphic illustrating the main events of the assay, showing the ability to control the composition of each bilayer (in this case making them thermoresponsive and non-thermoresponsive). Initially the dye is quenched (non-fluorescent). For concentrisomes with a non-thermoresponsive outer bilayer, calcein was not expected to undergo sufficient dilution via efflux to unquench and produce a fluorescent signal. b, Percent calcein release profiles at 42 °C for each population. When both membranes were thermoresponsive, the fluorescence increase was much higher than when only the inner one was, because, in the latter, calcein remained encapsulated in a small enough volume to prevent unquenching. The release profiles for the additional controls mentioned in the main text can be found in Supplementary Fig. 6, whereas compositional details can be found in Supplementary Fig. 7. Values were calculated from fluorescence intensities after addition of surfactant (Triton X-100, 5 wt%, 2.5 µl min–1). Error bars indicate the s.d. of the average intensities for n = 3. The <20% calcein release for ThermoInner:Non-thermoOuter concentrisomes was thought to originate from unilamellar vesicles that had not undergone click chemistry.Source dataThe dye was loaded at self-quenching concentration (40 mM) into thermoresponsive liposomes (DPPC:cholesterol:DSPE-PEG2K-DBCO; 6.8 mM; 90:5:5 mol%), represented in Fig. 4a as a blue circle. For clarity, this composition was termed ThermoInner. A suspension of these liposomes was exposed to a second lipid composition via MHF, a non-thermoresponsive composition denoted Non-thermoOuter (DPPC:cholesterol:DSPE-PEG2K-N3; 6.8 mM; 53:42:5 mol%), represented as a yellow bilayer in Fig. 4a, with no dye present in the intermembrane space. The resulting concentrisome composition was termed ThermoInner:Non-thermoOuter. Upon heating to 42 °C a gel-to-fluid phase transition was initiated for ThermoInner, coupled with the release of calcein from the inner compartment to the lumen of the surrounding non-thermoresponsive liposome. The outer bilayer was expected to retain any released calcein in a volume such that the dye remained at a self-quenched concentration.A number of control populations were prepared. The first was a scenario in which both inner and outer bilayers were thermoresponsive, leading to full cargo release at elevated temperatures. These concentrisomes were formed using the same flow conditions and sheathing stream liposomes (ThermoInner) as before. The first had an outer bilayer consisting of ThermoOuter (DPPC:cholesterol:DSPE-PEG-N3; 6.8 mM; 90:5:5 mol%) where both bilayers of the concentrisome were rendered thermoresponsive (denoted ThermoInnerThermoOuter). The release profiles, shown in Fig. 4b, revealed a considerable decrease in percent calcein release for concentrisomes with a non-thermoresponsive outer bilayer, indicating that a new bilayer of a different composition had successfully formed around pre-existing liposomes.Incorporating cholesterol into membranes modulates their thermoresponsive properties. To address the possibility that enough cholesterol was unintentionally inserted into the inner bilayer during recirculation, enough to alter the Tm of ThermoInner to render it non-thermoresponsive and producing a false positive, we ran an experiment in which only cholesterol was present in the recirculating ethanol stream, at a concentration equivalent to that used in our previous experiment (2.86 mM). No change in the calcein release profile was noted for these liposomes (see Supplementary Fig. 6b). We also generated a concentrisome population with a fluid phase outer bilayer (that is, non-thermoresponsive) without cholesterol, consisting of DOPC:DSPE-PEG2K-N3 (6.8 mM; 95:5 mol%, DOPCOuter). The calcein release profile of this final control (termed ThermoInner:DOPCOuter) resembled that of ThermoInner:Non-thermoOuter (Supplementary Fig. 6d), once again suggesting that a change in calcein release could not be attributed to cholesterol migration between bilayers.Finally, to further examine whether formation of concentrisomes was aided by covalent linkage between bilayers (building on previous experiments) we ran another control using our Non-thermoOuter composition without azide-functionalized lipids (DPSE-PEG2K). Calcein release for this concentrisome system (termed ThermoInner:PEGOuter) resembled that of ThermoInner:ThermoOuter, further supporting our theory that concentrisome formation was promoted by covalent linkages between successive bilayers (see Supplementary Fig. 6c).These results allowed us to reach a number of key conclusions about the concentrisome system: (1) the outer bilayer composition could be tuned to have different physical and stimuli-responsive properties to the inner bilayer; (2) click chemistry and a covalent linkage between opposing bilayers is necessary for concentrisome synthesis; and (3) cholesterol, under these conditions, does not passively insert into pre-formed vesicles (at least enough to alter the Tm of the inner bilayer).Multi-stage releaseAn additional fluorescent assay was developed to show that concentrisomes were capable of encapsulating multiple different payloads in different compartments, each released sequentially via a defined external stimulus (see Fig. 5a for a graphical illustration). In these experiments, two different dyes were encapsulated in the outer (methylene blue) and inner (calcein) compartments. Using a combination of multi-angle DLS measurements and fluorescence calibration, approximate encapsulation efficiencies were determined for (1) calcein in the inner core of the concentrisomes, and (2) methylene blue present in the intermembrane space (see Supplementary Fig. 9). By employing different lipids, the outer and inner membranes were designed to release at lower and higher temperatures, respectively. Sequentially exposing our sample to a low temperature followed by a high temperature would lead to the release of methylene blue first, then calcein.Fig. 5: Multi-stage payload release.a, A graphic illustrating multi-stage sequential release of two different cargos from two concentrisomes compartments. The two cargos are self-quenching dyes and thus release leads to an increase in fluorescence. b, Percent release of methylene blue over time from the concentrisome system with outer and inner bilayers composed of DPPC and DSPC, respectively. As methylene blue was isolated to the intermembrane space, the dye was released at 42 °C (above the transition temperature of the outer membrane DPPC composition). Data are represented as mean values ± 1 s.d. for n = 3. c, Percent calcein release over time for the same concentrisome system. In this case calcein was isolated to the inner liposome, thus release was observed only at 52 °C (which is above the transition temperature of this DPSC composition, the main component of the inner bilayer). Data are represented as mean values ± 1 s.d. for n = 3. d, A graphic illustrating the events of a multi-stage sequential release of the same cargo (calcein dye) from the two compartments of a concentrisome. Calcein was encapsulated in both the inner liposome and intermembrane space of a concentrisome system, with the inner bilayer composed of our DSPC composition (Tm ≈ 52 °C), and the outer bilayer of our DPPC composition (Tm ≈ 42 °C). e, Percent release of calcein dye over time, showing two discrete bursts when the samples are heated to the phase transition temperatures of the inner and outer membranes. Data are represented as mean values ± 1 s.d. for n = 3. Further controls can be found in Supplementary Fig. 8.Source dataTo test this, thermoresponsive liposomes with composition DSPC:cholesterol:DSPE-PEG2K-DBCO (1 mM; 80:15:5 mol%), termed ThermoInner 2 (Tm ≈ 50 °C), were generated using MHF, with encapsulated calcein (40 mM). Once purified, these liposomes were subjected to the MHF layering method and introduced to their azide counterpart in the form of ThermoOuter (Tm ≈ 42 °C) with methylene blue dissolved at 40 mM in the sheathing stream. This was done to encapsulate the dye within the intermembrane volume. A second purification step was required to remove excess methylene blue. The concentrisome population was denoted ThermoInner 2:ThermoOuter. A number of control populations were generated. Control 1 was produced in the same way (with both calcein and methylene blue), this time using DPPC-based thermosresponsive compositions for both the inner and outer bilayers, that is, ThermoInner:ThermoOuter. Control 2 reversed the order of the DSPC and DPPC compositions giving ThermoInner:ThermoOuter 2. Finally, control 3 had the composition ThermoInner 2:ThermoOuter, this time replacing methylene blue with additional calcein (40 mM).The release profile for methylene blue for the concentrisomes in our test system, ThermoInner 2:ThermoOuter, was analysed at 42 °C and at 52 °C. As expected, a ~40% release of methylene blue was observed at the lower temperature, which corresponded to release of the dye from the outer compartment through the outer DPPC-based membrane (Fig. 5b). The release profile for encapsulated calcein was then analysed (using a fresh concentrisome sample) at 42 °C and 52 °C; ~50% calcein release was observed only at the higher temperature (see Fig. 5c), in accordance with the Tm value of our DSPC composition.Using the same concentrisome composition, we also demonstrated that it is possible to achieve multi-stage, sequential release of the same payloads in two discrete bursts by loading calcein in both the inner and outer compartments (see Fig. 5d,e). Here we observed two sharp increases in fluorescent signals, corresponding to release events: the first upon reaching the phase transition temperature of the outer membrane (42 °C) and the second upon reaching that of the inner membrane (52 °C).All control populations followed the expected order of release (see Supplementary Fig. 8), demonstrating it was possible to simultaneously control both successive bilayer composition and location of multiple encapsulants within a concentrisome. From these results we concluded that discrete cargos can be encapsulated in distinct compartments within the same concentrisome, and that two defined stimuli can be used to release those cargos.Compartmentalized biochemical synthesisHaving demonstrated the ability to sequentially release multiple encapsulants, we went on to develop a compartmentalized system capable of the controlled mixing of an enzyme and substrate within a single particle, by defining the physical properties of each successive bilayer as before. The system chosen was that of the β-galactosidase (β-Gal)-mediated hydrolysis of non-fluorescent fluorescein di-β-d-galactopyranoside (FDG) to produce fluorescein (fluorescent)2. In these experiments, the enzyme and substrate would initially be housed in different compartments, with the particle being dormant. Only upon permeabilization of the inner compartment would the substrate meet the enzyme, leading to a chemical reaction confined in the lumen of the outer compartment.The substrate FDG (0.15 mM) was first encapsulated in thermoresponsive liposomes (ThermoInner) formed using extrusion, and then purified by size exclusion chromatography. As before, these liposomes where then fed through the MHF chip via the aqueous sheathing stream. This was performed in the presence of β-Gal (0.5 U ml−1) in the aqueous stream and ThermoOuter lipids dissolved in the ethanol stream, trapping the enzyme between the two bilayers (Fig. 6a). Trypsin (0.25 wt%) was added to collected samples to catalyse the proteolysis of unencapsulated β-Gal and prevent a false positive (that is, bulk hydrolase activity). The effect of trypsin on the bulk enzymatic reaction is shown in Supplementary Fig. 10. A sample of single bilayer liposomes of ThermoInner composition containing FDG was used as a control (trypsin was added to the bulk medium).Fig. 6: Triggered biochemical synthesis within the nanoparticle.a, Graphic illustrating in situ enzymatic synthesis in attolitre concentrisome reactors. Thermoresponsive liposomes containing the FDG substrate were recirculated with non-thermoresponsive lipids and β-Gal using an MHF flow regime identical to the flow conditions as before. In the control, liposomes were recirculated with ethanol and β-Gal only. b, Fluorometric data tracking the hydrolysis of FDG to fluorescein (λex = 498 nm; λem = 517 nm) at room temperature. No fluorescence increase was observed indicating enzyme and substrate remained compartmentalized. c, The same populations at 42 °C, showing an increase in fluorescence in the concentrisome sample. This indicates the thermoresponsive inner compartment permeabilizes, leading to the content mixing and subsequent enzymatic reaction occurring within the concentrisome. Error bars indicate the s.d. of the mean between three separate experiments, for both control (blue) and test (yellow) results. Further control experiments can be found in Supplementary Figs. 10–12.Source dataWe monitored the production of fluorescein first at room temperature, and then at 42 °C. We observed negligible enzyme activity at room temperature for both the concentrisome system and single bilayer liposome control (Fig. 6b). However, raising the temperature and crossing the gel/fluid transition of the inner bilayer (containing the FDG substrate) led to an increase in fluorescence intensity for the concentrisome population (Fig. 6c). The fluorescent product was largely isolated to the lumen of the concentrisome system and could be collected using centrifugal filtration (see Supplementary Fig. 11). The single bilayer liposome control showed a negligible increase with all unencapsulated β-Gal degraded by trypsin. From this we inferred that enzyme activity was isolated solely to the double-bilayer system. This was supported by an additional control, detailed in Supplementary Fig. 12, where the enzyme and substrate were encapsulated in two separate unilamellar vesicle populations and then mixed. Even at an elevated temperature, no fluorescein product was detected.
Engineering a nanoscale liposome-in-liposome for in situ biochemical synthesis and multi-stage release
