Three Compartment Liposome Fusion: Functional Protocells for Biocatalytic Cascades and Operation of Dynamic DNA Machineries

Nucleic acid‐functionalized liposomes modified at their boundaries with o‐nitrobenzyl phosphate‐caged hairpin units and pH‐responsive C‐G·C+ triplex forming strands are used for the concomitant light and pH‐triggered fusion of three types of loaded liposomes. The fusion processes are followed by light‐scattering size enlargement measurements, optical methods, and biocatalytic cascades activated upon the mixing of the liposomes loaded with enzymes and their substrates and their fusion into the cell‐like containments. The fused liposomes act as functional protocells for the integration of biocatalytic machineries. This is exemplified by the operation of an autonomous polymerization/nickase machinery synthesizing a Mg2+‐ion‐dependent DNAzyme and of a transcription machinery yielding the Malachite Green‐RNA aptamer product.

DOI: 10.1002/adfm.202302814 endocytosis, [2] signal transduction, [3] and viral infections. [4] For example, Nethylmaleimide-sensitive factor attachment receptors represent a broad class of proteins and protein subunits that induce membrane fusion. [5] Not surprisingly, research efforts are directed to emulate the cell fusion processes by biomimetic model systems. [6] In fact, the development of self-organized functional cell-like micro/nano containments, protocells, [7] is a major goal in the rapidly developing area of "Systems Chemistry". [8] Different protocell containments were suggested including liposomes, [9] polymersomes, [10] dendrosomes, [11] proteinsomes, [12] aqueous microdroplets [13] and hydrogel microcapsules. [14] By incorporation of loads into these assemblies catalytic, [15] photocatalytic, [16] and biocatalytic [17] transformations in the cell-like environments were demonstrated. In addition, by integrating stimuliresponsive units into the protocell boundaries triggered release of loads across cell-like interfaces were realized, [18] and switchable chemical transformations in the protocells were achieved. Triggers such as temperature, [19] light, [20] pH, [21] redox agents, [22] magnetic field [23] or ultrasound [24] were applied to induce the controlled release of loads from the artificial cells. Thus, the fusion of cell-like containments and the exchange of chemical agents between the interior of the protocell and the bulk exterior environment yielded functional micro/nano reservoirs revealing intracell-programmed catalytic functions or functional "smart" drug carriers for therapeutic applications. [25] Indeed, lipidated liposomes [26] and liposomes modified with molecular fusogenic systems [27] were applied to induce cell-like fusion processes. In addition, extracellular vesicles and their physiological functions in intercell communication and signaling by shuttling nucleic acid and proteins between cells attract growing biophysical interest. [28] Particularly, studies focusing on the development of synthetic analogs emulating native systems and efforts to identify clinical applications of synthetic extracellular vesicles were reported. [29] In addition, lipidated nucleic acids [30] or cholesterolmodified nucleic acids [31] (or peptides nucleic acids) [32] were incorporated into phospholipid vesicles or liposomes, and the formation of base-paired duplexes between the modified cell-like containments provided means to induce fusion. [33] Specifically, the fusion efficiency was affected by the geometries associated

Results and Discussion
o-Nitrobenzylphosphate ester-modified nucleic acids provide functional photoresponsive caged biopolymers. [43] Photochemical cleavage of the caged nucleic acid leads to tailored modified oligonucleotides for various uses. Diverse different applications of photoresponsive o-nitrobenzylphosphate-caged nucleic acids were reported including patterning of nucleic acid monolayer arrays on surfaces, [44] design of photoresponsive drug carriers, [45] light-simulated fusion of liposomes by deprotection of nucleic acid-functionalized liposomes, [46] fabrication of photoresponsive DNA hydrogels for spatiotemporal activation of cells responses, such as proliferation, [47] and cellular sensing [48] and imaging. [49] In addition, triplex nucleic acid structures play an important role in advancing the area of DNA nanotechnology. [50] pHresponsive and strand-responsive T-A•T or C-G·C + triplexes were used to reconfigure DNA nanostructers, [50] to design switchable DNA machines, [51] and to operate dynamic DNA networks. [52] Different applications of switchable triplex DNA structures were reported including their use for sensing, [53] their use as switching gates of drug carriers for controlled release [54] and their integration as functional constituents for switching the stiffness of hydrogels for controlled release, shape-memory and self-heating applications. [50] The principles for the stepwise fusion of three kinds of nucleic acid-functionalized liposomes using light and pH as trig-gers, and the physical, spectroscopic, and imaging methods to follow the fusion process are displayed in Scheme 1. Liposomes (a) were vacant, unloaded, containments, and functionalized at their boundaries with the cholesterol-functionalized onitrobenzylphosphate-modified DNA hairpin (1). Irradiation of the liposomes, = 365 nm, uncaged the hairpin structure to yield the duplex (1′)/(1″) modified liposomes. The resulting phototreated liposome (a) were reacted with liposomes (b) loaded with a high concentration of self-quenched sulforhodamine B, while their membranes were functionalized with a strand (2) complementary to the strand (1′) of liposomes (a). This resulted in the strand displacement of the strand (1″) from the duplexes (1′)/(1″) and the formation of interlinked (1′)/(2) duplexes that bridge the liposomes (a) and (b) leading to their fusion. The fusion of the liposomes generates larger liposomes where the dilution of the sulforhodamine B dye reduces the self-quenching effect, [35] resulting in the switched-on fluorescence of the dye. It should be noted that the (1′)/(2) bridged liposomes might lead to intimate-bridged interconnected liposomes or entirely fused liposome structures. The two interconnecting motives lead to largersized liposome structures, whilst the fused structures involve the exchange of the contents (loads) of the liposomes. While the size changes upon interactions of liposomes (a) and (b) cannot distinguish between the two mechanisms, the resulting enhanced fluorescence could point to, at least, partial real fusion and exchange of constituents (leading to dilution of the quenched dye). In the next step, the fused (a)/(b) liposomes were reacted with the nucleic acid (3)-modified liposome (c), at pH = 5.0. The sequence (3) associated with liposome (c) was engineered, however, to yield a C-G·C-stabilized triplex domain between (3) and the (1′)/(1″) duplex domain associated with (a), extended by a duplex region between (3) and (1″). The resulting inter-bridging of the fused (a)/(b) liposomes with (c) leads to the fusion of the three liposomes (a)/(b)/(c). This process is anticipated to enlarge the volume of the liposomes and further enhance the dilution of the sulforhodamine B fluorescent label. Figure 1A, curve (i), depicts the fluorescence changes of the liposomes system, upon the stepwise irradiation of liposomes (a) with (b), = 365 nm, followed by the treatment of the light-fused liposomes with the liposome (c), at pH = 5.0. The irradiation of liposomes (a) and (b), at = 365 nm, leads to a fluorescence enhancement that levels off after ≈1000 s, consistent with the fusion of the liposomes, and dilution of the fluorescent label. The further treatment of the fused liposomes with liposome (c) further intensifies the time-dependent fluorescence changes of the sulforhodamine B label that are, also, reaching a saturation level. The further increase in the fluorescence label entrapped in the liposome is consistent with the fusion-guided dilution of the fluorescent label in the three liposome fused containment. It should be noted that after the stepwise fusion of the liposomes, the liposome solution was separated chromatographically, and the presence of sulforhodamine B in the bulk surrounding solution was probed. No fluorescence signals of the diluted fluorescence dye could be detected in the eluted solution, thus possible leakage of the dye during the fusion process is eliminated, Figure S1 (Supporting Information). Moreover, it should be noted that the fluorescence changes of sulforhodamine B, in the second step of fusion are substantially lower than in the first step of fusion of liposomes (a) + (b). This is due to the non-linear fluorescence www.advancedsciencenews.com www.afm-journal.de Scheme 1. Schematic stepwise fusion of three kinds of liposomes (a), (b), and (c) using light and pH as triggers.
changes of the aggregated dye upon its dilution, Figure S2 (Supporting Information). Moreover, Figure S2 (Supporting Information) depicts the non-linear fluorescence changes of sulforhodamine B upon dilution. Using the stepwise size changes of the (a) + (b) and (a) + (b) + (c) fused liposomes, evaluated by dynamic light scattering, and realizing the quenched fluorophore label does not leak from the liposome containments, the dilution factors of the probe upon fusion may be estimated and the degrees of fluorescence changes in the two-step fusion, using Figure S2 (Supporting Information) may be evaluated. Using this process, the experimental fluorescence changes of the quenched fluorophore probe, depicted in Figure 1A, curve (i) are validated, suggesting a ≈80-85% fusion efficiency in each step. Knowing the initial concentration of the liposome and the overall fusion efficacy of the three-liposome ≈80-85%, we estimate the concentration of the fused three liposomes to be ≈2.7 × 10 12 liposome mL −1 , a value that is consistent with the concentration evaluated by the Nanocoulter counter device (see details supporting information).
For comparison, Figure 1A curve (ii), depicts the fluorescence changes of the system upon the stepwise mixing of the lipo-somes (a), (b), and (c) without irradiation at pH = 8.0. No fluorescence changes are observed, indicating that the coupled lightinduced/pH-acidification dynamic fusion of the liposomes is, indeed, needed to stimulate the fluorescence changes in the liposome mixture. Moreover, in a control experiment, Figure S3 (Supporting Information), liposome (a) and inactive liposomes (b) and (c) lacking the strand (2) and (3), yet loaded with the sulforhodamine B were irradiated at = 365 nm and treatment at pH = 5.0. No fusion or dilution of the dye was observed, implying that the functional liposomes (b) and (c) are, indeed, key constituents to induce the three-compartment fusion. Figure 1B depicts the temporal size changes of the liposome assemblies, followed by dynamic light-scattering, upon their fusion using the light/pH triggers. The individual liposomes (a), reveal an average size of 225 ± 5 nm, and irradiation of liposomes (a) and (b), = 365 nm, enlarges the liposomes to structures exhibiting an average size of 240 ± 5 nm. Treatment of the (a)/(b) fused liposomes with liposomes (c), at pH = 5.0, generates liposome structures revealing a size corresponding to 265 ± 5 nm curve (i). The size distribution changes upon the steps of fusion are displayed in Figure S4 (Supporting Information). Meanwhile, the sequential treatments of the liposomes (a) with liposomes (b) and then with liposomes (c), in the absence of the respective fusing triggers lead to unchanged sizes of the liposomes, curve (ii), 225 ± 5 nm, confirming that no fusion occurred. Figure  trimer liposomes or further enlarged globular liposomes exhibiting sizes of ≈250 nm, panel III. Whilst the SEM images and size changes demonstrate that subjecting the liposomes to light and acidic pH guides morphological inter-liposome structural interactions supporting the fusion of the liposomes and the existence of germinate contacts of liposomes through "kissing" interactions, that cannot be excluded. Nonetheless, the fluorescence studies, Figure 1A, and the further experiments confirming the exchange of loads between the interacting liposomes, together with the supporting size and shape changes of the liposomes upon sequential light/pH triggers will provide convincing evidence regarding the fusion of the three liposome containments.
Further support that the sequential light/pH-induced fusion of the liposome (a), (b), and (c), indeed, proceeded, and resulted in the exchange of loads in the fused containment, was obtained by the successful operation of a glucose oxidase (GOx)-horseradish peroxidase (HRP) cascade in the fused containment. Beyond demonstrating that the fusion process guides the operation of the two-enzyme cascade, the emergence of the biocatalytic cascade provides useful tools to support the dynamic fusion of the liposomes Figure 2. A mixture of three liposomes (a), (b), and (c), each liposome at a concentration of 3.34 × 10 12 liposomes mL −1 was employed to induce the fusion process and the activation of the biocatalytic cascade. Liposomes (a), functionalized with the o-nitrobenzylphosphate nucleic acid (1) were loaded with GOx (2 U mL −1 ), liposomes (b), were modified with nucleic acid (2) and loaded with glucose (0.25 m) and liposomes (c) modified with nucleic acid (3), were loaded with Amplex-Red (0.1 m) and HRP (0.2 U mL −1 ), Figure 2A Figure 2C curve (i) shows the time-dependent fluorescence changes of the system upon the triggered fusion of the three liposomes. Only the two-step light/pH triggered the The light/pH-triggered fusion of the liposomes was further confirmed by optical labeling of the respective liposomes with fluorophores and following the fusion process by confocal fluorescence microscopy. In this experiment, Figure 3, the liposomes, L 1 , functionalized with nucleic acid (2) were loaded with the TAMRA labeled nucleic acid (4), and liposomes L 2 modified at their boundaries with nucleic acid (3), were further functionalized with the FAM nucleic acid labeled nucleic acid (5), to yield liposome L 2 . The nanometer-sized liposome L 1 and L 2 have interacted with the o-nitrophenyl phosphate hairpin-modified nucleic acid (1)-functionalized micrometer-sized liposomes L. (Note that the relative sizes of the liposomes were engineered to allow the optical probing of the fusion process by confocal microscopy). The mixture of liposomes was illuminated, = 365 nm at pH = 5.0 to induce the fusion of the three liposomes, Figure 3A. The successful fusion of the three capsules yields a hybrid fused containment demonstrating the red fluorescence of the TAMRAlabeled load (4), confined in the core of the fused containment, and the green fluorescence of the FAM nucleic acid units (5) associated with the membrane of the fused containment. Figure 3B shows the confocal microscopy images of the fused liposome by following the fused containments through the fluorescence channels of the respective labels. Panel I shows the red fluorescence of the TAMRA-modified load in the inner core of the fused containment, and panel II depicts the green fluorescence of the FAM fluorophore associated with the tethers (5) confined to the membrane of the fused containment. Panel III shows the bright field image of the fused containment, and panel IV shows the overlay of the fluorescence image shown in panel I, panel II and panel III, demonstrating that the fusion of the three liposomes yields, as expected, an integrated synthetic cell consisting of the red fluorescence core of the TAMRA-labelled nucleic acid (4) surrounded by a green fluorescence rim corresponding to the FAM labeled nucleic acid (5) confined to the boundary of the three liposomes fused containment. Zoom-out, larger area, confocal microscopy images of the liposomes are displayed in Figure 3B and Figure S6 (Supporting Information). It should be noted that the latter system involved the fusion of two small liposomes L 1 and L 2 into a "giant" liposome L. This allows for the effective fusion of the small liposome to the giant carrier and yields a highly loaded carrier system for the subsequent protocell machinery applications.
The different experiments described, so far, demonstrated the light/pH-triggered fusion of the respective nucleic acidfunctionalized liposomes that resulted in the mixing of the constituent loads in the fused liposome containment. The mixed constituents were preserved in the protocells without leakage to the bulk surrounding solution. (In fact, the as-prepared separated liposomes retained the intact composition and functional activity for at least three days, and the optical properties of the fused containments were retained for at least 24 h, without leakage of the component to the bulk solution, Figure S7, Supporting Information). These features suggested that appropriate programmed biocatalytic transformations could be engineered in the fused protocell assemblies. These functional features of the fused protocell assemblies are utilized to demonstrate the fusionguided operation of a polymerization/nicking DNA machinery synthesizing a metal-ion-dependent DNAzyme in the protocell assembly and a fusion-guided transcription process driven in the protocell containment. Figure 4 depicts the fusion-guided operation of the polymerization/nicking DNA machinery in the fused protocell. The liposomes consist of the o-nitrophenyl phosphateprotected hairpin, (1)-functionalized liposome M 1 , loaded with the Kelnow Fragment polymerase (≈3 U mL −1 ), the Nt.BbvCl nicking enzyme (≈4 U mL −1 ) and the dNTPs mixture (≈1.6 mm), the liposomes M 2 , functionalized with nucleic acid (2) is loaded with the DNA template T, hybridized with the promoter, P (the concentration of T/P was ≈300 nm), and liposome M 3 was modified with a strand (3) and loaded with the fluorophore (TAMRA)/quencher (BHQ2) modified strand S (≈900 nm). The mixture of liposomes was subjected at pH = 5.0 to the photochemical uncaging of the o-nitrophenyl phosphate units (1), resulting in the fusion of the three liposomes and the generation of a fused protocell that contained the mixture of constituents (loads) that were in the parent liposomes, Figure 4A.The scaffold T/P was designed, however, to include in the template sequence T, the promoter P binding domain (c′), the sequence domain (ii) that upon polymerization yields a sequence-specific nicking site for the nicking enzyme, Nt.BbvCI and domain (iii) that is complementary to the Mg 2+ -ion-dependent DNAzyme sequence, Figure 4B. Thus, the fusion process of the three liposomes leads to a functional protocell containment loaded with a DNA polymerization machinery that guides the autonomous synthesis of the Mg 2+ -ion-dependent DNAzyme strand. In the presence of polymerase and the dNTPs mixture, the replication of the template proceeds, followed by the nicking of the replicated product and displacement of the Mg 2+ -ion-dependent DNAzyme, resulting in the cyclic replication of the DNAzyme. The generated DNAzyme cleaves the substrate fluorophore/quenchermodified S, yielding the fluorescence of the fluorophore-labeled cleaved product, acting as the reporter of the replication/nicking machinery. Figure 4B. That is, the fusion process in the fused containment activates the autonomous operation of the cyclic polymerization/nicking machinery that synthesizes the Mg 2+ion-dependent DNAzyme sequence. Figure 4C, curve (a), depicts the time-dependent fluorescence changes generated in the fused protocell containment. The concentration of liposomes in this experiment corresponded to 1.85 × 10 12 mL −1 . For comparison, Figure 4C, carve (b), depicts the temporal fluorescence changes of the mixture consisting of the liposomes M 1 , M 2 , and M 3 that are not subjected to the light/pH fusion triggers. No fluorescence signals are observed, confirming that the fusion process is, indeed essential to operate the polymerization/nicking machinery, synthesizing the DNAzyme cascade. Further control experiments confirmed that the polymerization nicking machinery indeed proceeds in the fused protocell assembly: (i) In one experiment, the three liposomes mixture, including M 1 in liposomes where the dNTPs mixture was omitted, was subjected to the light/pH fusion process. No activation of the polymerization/nicking machinery and the synthesis of the Mg 2+ -ion-dependent cascade was observed ( Figure S8, Supporting Information, Curve i). (ii) In a second control experiment, the promoter P was omitted from the liposome M 2 . The light/pH-triggered fusion of the liposome mixture did not lead to the synthesis of the Mg 2+ -ion-dependent DNAzyme in the fused protocell assembly ( Figure S8, Supporting Information, Curve ii). (iii) In a set of two additional control experiments the enzymes polymerase or the nicking enzyme Nt.BbvCI was omitted from the light/pHfused protocell assembly. Again, the polymerization/nicking machinery synthesizing the DNAzymes was not activated in the systems ( Figure S8, Supporting Information, Curve iii and Curve iv). Figure 4D exemplifies the confocal microscopy images of a fused micrometer-sized protocell containment in which the polymerization/nicking machinery proceeded. Panel I shows the fluorescence generated in the fused protocell upon operating the polymerization/nicking machinery for 3 h and the concomitant cleavage of the TAMRA/BHQ2-functionalized substrate S. Panel II and III present the bright-field image and overlay image of the fused liposomes. For zoom-out, larger area, confocal microscopy images of the liposomes are shown in Figure 4D, see Figure S9 (Supporting Information). The results demonstrate the successful operation of DNA machinery in the fused-liposome protocell assembly.
A further fused-liposome-guided DNA machinery is presented in Figure 5, demonstrating the transcription of an mRNA in the fused liposomes protocell. Specifically, the transcription of the Malachite Green (MG) RNA aptamer is addressed. Three liposomes N 1 , N 2 , and N 3 are used to assemble the functional protocell. Liposomes N 1 are functionalized with the photoprotected o-nitrophenyl phosphate caged nucleic acid hairpin units (1), and loaded with the T 7 RNA polymerase, RNAp (≈3 U mL −1 ), and the base mixture NTPs (≈4 mm). Liposomes N 2 are functionalized with the nucleic acid (2) and loaded with the duplex AA′ (≈600 nm), acting as the transcription template. Liposomes N 3 , are modified at their boundaries with the strand (3) and loaded with Malachite Green, MG, ligand (≈1.5 μm), Figure 5A. Subjecting the mixture of liposomes to light, = 365 nm, at pH = 5.0, results in the fusion of the three liposomes and the mixing of the loads into the integrated fused containment. This activates the transcription machinery displayed in Figure 5B. The strand A of the template includes the domain x that binds the promoter, and the domain z that is complementary to the MG RNA aptamer sequence. The strand A′ hybridized with the template strand act as the promoter strand that activates the transcription machinery. Accordingly, the fusion process of the three liposomes leads to the activation of the MG transcription process in the protocell containment. The transcription of the MG aptamer is followed by the fluorescence of the resulting aptamer-MG-complex ( ex = 618 nm, em = 658 nm). Figure 5C curve (a) depicts the time-dependent fluorescence changes upon transcription of the MG aptamer. Figure 5C, curve (b) depicts the temporal fluorescence changes of a control system consisting of the non-fused mixture of the three liposomes N 1 , N 2 , and N 3 . No fluorescence changes are observed indicating that the light/pH fusion of the liposome is essential to activate the transcription process. Also, in a set of control experiments where the promoter strand A′ was omitted from liposome N 2 or the enzyme RNAp or NTPs constituents were deleted from the liposomes N 1 did not yield functional transcription machineries in the light/pH-fused microcapsules and no fluorescence changes of the MG-aptamer were observed. Thus, the operation of the transcription machinery in the fused liposome requires the presence of all constituents outlined in Figure 5A. Confocal microscopy measurements of the fused liposomes allowed the imaging of the transcription machinery in the protocell. Figure 5D, panel I exemplifies the confocal fluorescence image of the fused liposomes upon operating the transcription machinery for a time interval of 3 hours. The red fluorescence of the liposome demonstrates the presence of the MG-aptamer complex generated by the transcription machinery. Panel II and III show the bright-field image and overlay image of the fused protocell in which the transcription machinery proceeds. For zoom-out, larger area, confocal microscopy images of the liposomes shown in Figure 5D, see Figure S10 (Supporting Information). Figure 5D, panel IV-VI depicts a control experiment. In this experiment, the NTPs mixture was omitted from the N 1 liposomes, and the light/pH-fused liposomes N 1 /N 2 /N 3 were imaged after a time interval of 3 hours. No red fluorescence of the transcribed MG-aptamer was observed, indicating that the presence of all constituents in the fused liposome assembly is essential to activate the transcription machinery in the fused liposome.

Conclusion
The present study has introduced a versatile method to induce, by two concomitant light and pH triggers, the fusion between three liposomes. By employing nucleic acid-functionalized liposomes modified at their boundaries with o-nitrobenzyl phosphate-caged hairpin units and pH-responsive C-G·C + triplex forming strands, the guided fusion of three types of liposomes was achieved, and the exchange of their loads into integrated protocell assemblies was demonstrated. The fusion processes were followed by lightscattering and size enlargement of the liposome, optical methods, and by activation of a biocatalytic cascade of mixed enzyme loads in the fused liposome containment. In contrast to previous reports demonstrating the stepwise fusion of three liposomes by duplex-guided fusion motives, [35] the advantages of the present method rest on the ability to retain the "dormant" mixture of the three liposomes and to activate the fusion process in a single spatiotemporal step that comings light and pH triggers. The present study has emphasized the structural and stimuliresponsive properties of the nucleic acid constituents embedded in the liposome membranes on the dynamics of liposome fusion. Nonetheless, the contents of the nucleic acid ingredients in the liposome membranes and their spatial distribution could be a further means to control the fusion dynamics. At present, by evaluating the contents of the non-incorporated strands (1), (2), and (3) within the synthesis of liposomes (a), (b), and (c), Scheme 1, we estimate a loading efficiency of 85%, 90%, and 85%, respectively. Indeed, a systematic study probing the effects of nucleic acid loading of the liposomes on the fusion dynamics would be a further challenge to follow. The fusion of three liposomes enabled the loading of the fused liposomes with diverse catalytic and chemical constituents. This enabled the assembly of functional protocell assemblies that consists of complex biocatalytic machineries. This was exemplified by the integration of DNA machinery consisting of polymerase/nickase that led to the autonomous synthesis of the Mg 2+ -ion-dependent DNAzyme, and by the assembly of the transcription machinery synthesizing the MG-RNA aptamer in the cell-like containment. The ability to integrate through the fusion of three loaded liposomes a complex mixture of intercommunicating biocatalysts and chemical agents paves the way to design programmed chemical transformations in the protocell. The engineering of the transcription machinery in the protocells is particularly interesting, as it provides the primary step for programmed transcription and translation of proteins in such cell-like containments.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.