Effector delivery system

Abstract

The invention relates to system for delivering an effector molecule to biological cells or tissue, which system comprises: (a) a target region which comprises biological cells or tissue; and (b) a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets, wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet additionally contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, and at least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.. The invention also provides a synthetic droplet assembly and a process for delivering an effector molecule from a synthetic droplet assembly to biological cells or tissue.

Description

[0001] EFFECTOR DELIVERY SYSTEM

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a system for delivering an effector molecule to biological cells or tissue, and to a synthetic droplet assembly suitable for use in such a system. The invention also relates to a process for delivering an effector molecule from a synthetic droplet assembly to biological cells or tissue. The system, droplet assembly or process of the invention may be used to achieve a high-resolution, patterned delivery of the effector molecule.

[0004] BACKGROUND TO THE INVENTION

[0005] The patterning of cells is crucial in nature, where it is required for the stability of bacterial communities and their species diversity (C. D. Nadell et al., Nat Rev Microbiol 2016, 14, 589), tissue development (M. Cardoso-Moreira et al., Nature 2019, 571, 505), morphogenesis (A. M. Turing, Bltn Mathcal Biology 1990, 52, 153) and homeostasis (P. Rue et al., Molecular Systems Biology 2015, 11, 792) cell functions such as stress responses (E. de Nadal et al., Nat Rev Genet 2011, 12, 833) and the immune response (T. S. P. Heng et al., Nat Immunol 2008, 9, 1091).

[0006] Patterning is mediated by gene expression, which is tightly regulated by intercellular communication both in space and time, ensuring that cells precisely coordinate their roles within a group. Intercellular communication is mediated by chemical signals, including quorum sensing molecules (C. M. Waters et al., Annual Review of Cell and Developmental Biology 2005, 21, 319), growth factors and cytokines (S. Wemer, R. Grose, Physiological Reviews 2003, 83, 835), hormones (F. Dehkhoda et al, Front. Endocrinol. 2018, 9, DOI 10.3389 / fendo.2018.00035) and neurotransmitters (G. J. Augustine et al., Neuroscience, Oxford University Press, New York, 2024). Current ways to study intercellular communication include microfluidics (A. Burmeister, Current Opinion in Biotechnology 2020; A. Burmeister et al., Lab on a Chip 2019, 19, 98) and 3D printing (R. Krishna Kumar et al, Nat Commun 2021, 12, 857; R. Krishna Kumar et al, Microbial Biotechnology 2023, 16, 489; A. D. Graham et al., Sci Rep 2017, 7, 7004; Y. Jin et al., Nat Commun 2023, 14, 5986; L. Zhou et al, Advanced Materials 2020, 32, 2002183) with which multiple cell types can be positioned accurately with respect to each other. Furthermore, spatially-controlled gene expression has been achieved at the millimetre scale with chemical signals (S. Basu et al., Nature 2005, 434, 1130; T. Danino et al., Nature 2010, 463, 326; A. Tamsir et al., Nature 2011, 469, 212; T. Sohka et al., Proceedings of the National Academy of Sciences 2009, 106, 10135) and at the micrometre scale using light (J. Zhang, 2020; J. J. Tabor et al., Cell 2009, 137, 1272; R. Ohlendorf et al., Front Bioeng Biotechnol 2022, 10, 1029403; A. Levskaya et al., Nature 2005, 438, 441; E. Romano et al., Nat Chem Biol 2021, 17, 817). However, external control over gene expression via chemical signals, which is the basis of diffusion-mediated communication, hasnot been achieved at high spatial resolution. Therefore, a universal technology to both study and control patterned gene expression at micrometre resolution within a group of cells is desirable.

[0007] Meanwhile, a range of FDA approved platforms exist for altering cellular behaviour through the delivery of cargo (small molecules, drugs, peptide / proteins, DNA / RNA) via endocytic pathways such as cargo-conjugation, and the encapsulation of cargo within lipid / polymer nanoparticles (M. J. Mitchell et al, Nat Rev Drug Discov 2021, 20, 101; A. C. Anselmo et al., Bioeng Transl Med 2019, 4, el0143; O. S. Fenton et al., Advanced Materials 2018, 30, 1705328). Moreover, porous materials, such as metal-organic frameworks (S. He et al., Acta Pharmaceutica Sinica B 2021, 11, 2362), covalent organic frameworks (H. R. Abuzeid et al., Giant 2021, 6, 100054; C. W. Jones, JACS Au 2022, 2, 1504) and zeolites (M. Servatan et al., Drug Discovery Today 2020, 25, 642) are being developed as drug delivery platforms. Recently, synthetic cells (B. C. Buddingh’ et al., Acc. Chem. Res. 2017, 50, 769; M. Weiss et al., Nature Mater 2018, 17, 89) -micron-sized systems that mimic aspects of cell function - have been used to alter cellular behaviour in mycelial (O. Adir et al., Nat Commun 2022, 13, 2328), bacterial (P. M. Gardner et al., Nature Chem 2009, 1, 377; R. Lentini et al., Nat Commun 2014, 5, 4012; G. Rampioni et al., Chem. Commun. 2018, 54, 2090; X. Wang et al., Chem. Sci. 2019, 10, 9446; I. Gispert et al., Proceedings of the National Academy of Sciences 2022, 119, e2206563119; M. Walczak et al., Advanced Materials 2023, 35, 2301562; J. M. Smith et al., Nat Chem Biol 2023, 19, 1138) and eukaryotic cells (B. W. Drinkwater, Lab Chip 2016, 16, 2360; N. Krinsky et al., Advanced Healthcare Materials 2018, 7, 1701163; O. D. Toparlak et al., Science Advances 2020, 6, eabb4920; X. Wang et al., Small 2020, 16, 1906394; J. E. Hernandez Bucher et al., Biomaterials 2022, 285, 121522) through the triggered delivery of chemical signals that can change gene expression levels. However, the use of these systems to alter cellular behaviour is limited due to their inability to release chemical signals in a patterned manner. Moreover, synthetic cells show poor storage capacities (M. H. M. E. van Stevendaal et al., ChemSystemsChem 2021, 3, e2100009) of chemical signals due to their limited volume.

[0008] There is therefore an ongoing need for systems capable of releasing chemical signals in a patterned manner, with high spatial and temporal resolution, in order to achieve aims such as controlling patterned gene expression at high resolution within a groups of cells, and delivering cargo (e.g. small molecules, drugs, peptide / proteins, DNA / RNA) to cells and tissues in a patterned manner.

[0009] SUMMARY OF THE INVENTION

[0010] The invention provides synthetic droplet assemblies that are capable of releasing chemical signals (effector molecules) with high spatial and temporal resolution and in a patterned manner if desired. This can be achieved via the patterning of compartments and signalling between compartments both within the droplet assembly and with the immediate external environment. Thedroplet assemblies, which may be produced using 3D droplet printing, are thus a platform that allows for controlled release of molecules (effectors) from synthetic tissues to living cells. This system of effector release is widely applicable, offering diverse applications in biology and medicine. The platform may for instance be used to direct cellular activity by the patterned release of chemical signals. Thus, by using a synthetic droplet assembly of the invention, the inventors have achieved the patterned release of a chemical signal with temporal control, to direct gene expression in underlying bacterial populations. The inventors have also demonstrated ways to store chemical signals in the droplet assemblies and to activate release at controlled points in time. The synthetic droplet assemblies of the invention may thus be used in fundamental biology and in medicine, in applications that require the delivery of cargo to a target group of cells or tissue in a patterned manner and / or that require the controlled formation of chemical gradients within a target group of cells.

[0011] The technology described herein is suitable for a wide variety of applications, including for example in the local control of gene expression in target cells and tissue, or for targeted drug delivery in a patterned manner. The synthetic droplet assemblies described herein may be used to release therapeutics in a highly controlled manner, and the use of the systems and droplet assemblies of the invention in medical applications is therefore envisaged, such as in the treatment of chronic wounds, cancer, neurodegenerative diseases and spinal cord injuries.

[0012] The technology may also be used in fundamental research, such as the study of cell communication or developmental biology. Patterned effector release, using the systems of the invention, may for example be useful in creating 3D living tissues of high complexity as the differentiation of iPSC-derived cells may be controlled by gradients of effector molecules when released from the synthetic tissues.

[0013] The advantages of the invention are numerous. The technology of the invention advantageously provides the means to alter cellular behaviour in space and time by the release of effector molecules. Furthermore, whereas synthetic cells of the prior art are limited, as discussed above, because of effector storage capabilities and their inability to induce gene expression in a precise patterned manner, the droplet assemblies of the invention can overcome such limitations by achieving high spatial resolution of induced patterned gene expression (e.g. 50 pm as opposed to millimetres), increased effector storage capabilities (in reservoirs that are not limited in volume) and controlled effector release at any given time (by connecting reservoir modules with release modules) independent of external stimuli, such as light. The technology of the invention also allows for the integration of stimuli-responsive membrane pores, allowing for triggered effector release if desired, and it allows for storage of a range of effector molecules to - for example -selectively control the expression of different genes when using a range of promoters.

[0014] The inventors have achieved these advantages by developing a universal platform that allows for controlled release of molecules (effectors) from a 3D-printable synthetic dropletassembly to living cells (e.g. bacteria) or tissue. The synthetic droplet assembly can interface living tissue, or for instance a cell-laden hydrogel, through a layer of amphipathic molecules and membrane pores can incorporate into that layer to facilitate release of effector molecules. Effector molecules can be released from the 3D-printed droplet assembly into the target tissue or cells. Living cells take up the effector molecule, which can achieve an effect in the cells, e.g. drive gene expression. By controlling effector and pore concentration in the 3D-printed synthetic tissues, the release dynamics of effector molecules into the cell-laden hydrogel can be precisely controlled. In this way, the inventors were able to induce patterned gene expression at high spatial resolution (50 pm) in bacterial cells. The synthetic droplet networks can be composed of patterned mask layers and an effector-storing reservoir, allowing for efficient storage while maintaining patterned effector release. By connecting two types of synthetic tissues, the release module and the storage module, effector release could be initiated at a controlled point in time.

[0015] Accordingly, the invention provides a system for delivering an effector molecule to biological cells or tissue, which system comprises:

[0016] (a) a target region which comprises biological cells or tissue; and

[0017] (b) a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0018] wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet additionally contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, and

[0019] at least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.

[0020] The invention also provides a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0021] wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, andat least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.

[0022] The invention also provides a process for delivering an effector molecule from a synthetic droplet assembly to biological cells or tissue,

[0023] wherein the synthetic droplet assembly comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0024] wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, and at least one of the n droplets is an effector droplet, wherein each effector droplet further comprises the effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n,

[0025] and wherein the process comprises:

[0026] contacting each target interface droplet with a target region which comprises the biological cells or tissue, so that each target interface droplet contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 shows localized gene expression, and in particular: (a) a schematic of the droplet printing process, where droplet interface bilayers (DIBs) are formed between the droplets (left image). Through 3D printing, networks of droplets are formed (center image), producing droplet networks, which can be transferred with a pipette (right image), (b) A schematic of a 3D-printed droplet network interfacing a bacterium -laden hydrogel 18 h after transfer to the lipid-in-oil solution on top of the hydrogel. Magenta and yellow represent bacteria in the hydrogel expressing mCherry at high and low levels, respectively, (c) Zoomed-in schematic of (b) at the droplet network-hydrogel interface. A droplet hydrogel bilayer (DHB) forms between the droplet network and the hydrogel, -hemolysin (aHL) is incorporated into the bilayer, allowing arabinose to be released from the droplet network onto the immobilized bacteria, activating the expression of mCherry. (d) and (e) Epi-fluorescence microscopy images of a droplet network containing 33 RIM arabinose, 50 pg mb'1aHL and 250 pvi cascade blue dextran on top of a bacterium-laden hydrogel (composite bright-field (d) and fluorescence images (e) at t = 0 h (when the droplet network is placed on top of the hydrogel) and t = 18 h ( 18 hours after the droplet network is placedon top of the hydrogel)), (f) A z-stacked 3D confocal microscopy image of a droplet network on top of a bacterium -laden hydrogel at 18 h. In (d)-(f), cyan is cascade blue dextran fluorescence, yellow represents sfGFP fluorescence and magenta represents mCherry fluorescence, (g) A graph of the total mCherry expression, IT, overtime in bacterium-laden hydrogels with droplet networks on top containing 33 RIM arabinose and 50 pg ml / 1aHL (orange line), only 33 RIM arabinose (blue line), or no arabinose (green line). Solid lines and shaded regions are the mean and standard deviation values of n = 3 technical repeats.

[0029] Figure 2 shows patterned gene expression by controlled chemical signal release, and in particular: (a) a schematic of the image analysis workflow for calculating the pattern fidelity index (PF). First, raw epi-fluorescent microscopy images of both mCherry expression and the droplet network containing cascade blue dextran are binarized, before comparing the binarized pixel values of the two epi-fluorescent channels. Based on the area of intended gene expression (Ai) the area of unintended expression (Au) and the area of no gene expression (AN) within Ai were computed. Then, PF as a measure of controlled release of the chemical signal was calculated by subtracting Au from Ai before normalizing to Ai (see Equation (1) in text and Methods). Total gene expression, IT, and mean gene expression, IM, were determined based on raw pixel values, (b) and (c) Graphs of PF and total mCherry expression, IT (see Methods), against aHL concentration (b) and arabinose concentration (c) within the droplets of the droplet networks. Solid lines and shaded regions, respectively, are the mean and standard deviation values of n = 4 technical repeats for droplet networks on top of bacterium -laden hydrogels after 18 h. (d) Schematic of cross-sectional patterns incorporated into all layers of the 3D-printed droplet networks. White, droplets with no arabinose, no aHL, and no cascade blue dextran, and cyan, droplets containing 33 RIM arabinose, 25 pg mL'1aHL and 250 pvi cascade blue dextran, (e) Corresponding epi-fluorescent images of droplet networks constructed according to the patterns in (d). Cyan fluorescence is cascade blue dextran, (f), Binarized fluorescence images of the corresponding expression of mCherry (magenta) in bacteria underneath the droplet networks as shown in (e). (e) and (f) Images 18 h after placement of the droplet networks on top of bacteriumladen hydrogels, (g), PF, unintended area of gene expression, Au (normalized to the intended area of gene expression, Ai), area of no gene expression, AN (normalized to the intended area of gene expression, Ai), and mean mCherry expression, I , (see Methods) corresponding to the patterns in (e)-(f).

[0030] Figure 3 shows chemical signal storage and release in droplet networks, in particular: (a) a schematic of droplet networks of 4, 8 and 16 droplet layers on top of a bacterium -laden hydrogel, (b) Graph of the relationship between PF, total mCherry expression, IT, and normalized areas of no expression, AN, within Ai (normalized to Ai) with increasing droplet layer numbers. Solid lines and shaded regions, respectively, are the mean and standard deviation values of n = 3 technical repeats for droplet networks on top of bacterium -laden hydrogels after 18 h. (c) Epi-fluorescence images ofm Cherry at 18 h after transfer of droplet network networks composed of 4, 8 and 16 layers, (d) and (e) Schematics depicting a droplet network composed of a mask (cross-like pattern) with a reservoir on top (in cyan) from the side or bottom, respectively. Magnetic beads (grey spheres) are attached to the comers of the droplet network, (f) Schematic of a droplet network composed of a mask and reservoir on top of a bacterium -laden hydrogel after 18 h. A magnet (grey oval) directs controlled landing during the transfer of the droplet network to the bacterium -laden hydrogel, (g) Epi -fluorescence image of mCherry at 18 h after droplet network transfer. The red dashed line indicates an area where the bottom droplets did not form the intended contact with the hydrogel. In the schematics (a) and (d)-(f) and the corresponding experiments in (b) and (g) cyan droplets contain 33 RIM arabinose, 50 pg m '1aHL and 250 pvi cascade blue dextran (see Methods), whereas white droplets in the schematics do not contain these three components. Magenta and yellow are representations of bacteria in the hydrogel expressing mCherry at high and low levels, respectively.

[0031] Figure 4 shows increased resolution of patterned gene expression. In particular (a)-(c) are schematic, composite and epi-fluorescence images, respectively, of a droplet network containing an aHL-mediated diffusion pathway comprising a single linear chain of droplets after 18 h on top of a bacterium -laden hydrogel, (d) Schematic of a droplet network during the heat-induced shrinking process, where a droplet network is printed in lipid-in-oil (left image), before placement on top of a heat plate (center image), which initiates the shrinking process through droplet annealing and volume decrease (right image), (e) Bright-field microscopy images overtime of a droplet network in lipid-in-oil solution on top of a heat plate set to 43 °C. (f) Time-dependence of the percentage changes in network side length (normalized to initial side length) during heating at 36 °C and 43 °C (see Methods), (g) Diameters overtime of single droplets, droplets forming a single droplet interface bilayer with another droplet, and central and peripheral droplets of one-layered droplet networks and eight-layered droplet networks heated at 36 °C. In (f) and (g) solid lines and shaded regions, respectively, are the mean values and standard deviations of n = 3 technical repeats, (h)-(j) Composite (bright-field and fluorescence) and epi-fluorescence images of an unheated droplet network (triangular pattern, left) and a shrunken network (rectangular pattern, right) at 18 h after transfer on top of a bacterial -laden hydrogel. The droplet network in (i) was shrunken in lipid-in-oil solution for 170 min at 43 °C before transfer on top of the bacterium-laden hydrogel. In (a)-(d) and (h)-(j) cyan droplets contain 33 RIM arabinose, 50 pg m '1aHL and cascade blue dextran, while white droplets do not contain these components. Magenta and yellow represent bacteria in the hydrogel expressing mCherry at high and low levels, respectively. In (j) the dashed lines represent the widths of the frame-like gene expression pattern.

[0032] Figure 5 shows droplet networks as switchable modules. In particular, (a) and (c) are schematics of two droplet networks comprising droplets that contain: cyan compartments: 33 RIM arabinose, 50 pg ml / 1aHE and 250 pM cascade blue dextran; yellow compartments: only50 pg mL1aHL and 250 pM cascade green dextran; white compartments: neither aHL nor arabinose. The constructs are shown before and after establishing contact between the two modules. In (a) and (c), the modules are connected so that an aHL-mediated diffusion pathway is either formed (c) or not formed (a). Orange circles represent arabinose, (b) and (d) show composite (bright-field and fluorescence) microscopy images and epi-fluorescence images of connected droplet networks showing the corresponding gene expression patterns after 18 h where there is a diffusive pathway (d) or no diffusive pathway (b). In (b) and (d), cyan, yellow and magenta fluorescence are cascade blue dextran, cascade green dextran, and mCherry fluorescence, respectively. Dashed lines represent cross-sectional outlines of droplets that contained aHL and arabinose (cyan) or only aHL (yellow). Yellow full lines represent cross-sectional outlines of droplets that interfaced with the bacterium -laden hydrogel and contained only aHL.

[0033] Figure 6 shows patterned competition between bacteria. In particular, (a) provides an overview of the competing strains: E7-inducible (BZB1011 Pmax:sfgfp::Tn7 pKCl-PBAD -ColE7-AMP, magenta) encoding the colicin E7 operon, and an mCherry reporter protein, inducible by arabinose; susceptible cells (BZB1011 p\JA66-PcolE2:sfgfp, orange) which are susceptible to colicin E7 and contain a plasmid encoding sfGFP downstream of the PCOIE2 promoter, which upregulates gene expression upon DNA damage; and E8 (BZB1011 Pmax:mrfpl : :Tn7 pColE8, green) which express colicin E8 at basal rates and regulate expression depending on DNA damage, (b) and (c) Schematics and epi-fluorescent images depicting local lysis of E7 -inducible and local DNA damage in susceptible cells upon release of chemical signal molecules from droplet networks, respectively, (d) and (e) Heat maps of mean sfGFP expression, IM, and pattern fidelity (PF), respectively, in S-GFP cells as a consequence of arabinose release from droplet networks into a mixed population of E7R-inducible (BZB1011 pW\-PBAD.-ColE7 -mCherry- AMP and S-GFP cells (1: 1) at a range of arabinose and aHL concentrations after 18 h. (f) Epi-fluorescence microscopy images of bacterial populations containing E7R-inducible and S-GFP cells (1:1) under droplet networks that released arabinose in a patterned manner using / -‘ / -'-maximizing arabinose (66 HIM) and aHL concentrations (30 pg mL1) from (d) and (e) heatmaps. Images are at t = 18 h, orange reflects sfGFP fluorescence, and white dashed lines represent patterns, (g) Schematic of the competition between E7-inducible cells (BZB1011 Pmax:sfgfp::Tn7 pKCl-PBAD:-ColE7-AMP, magenta) and E8 cells (BZB1011 pColE8), respectively, (h) Graph of the number of micro-colonies per mm2below a droplet network releasing arabinose into a homogenous population of E7-inducible and E8 cells (9:1 initial starting ratio) overtime, (i) Epi-fluorescence images of bacterial competition between E7-inducible (magenta) and E8 cells (green) representative for (h) at 12 h and 18 h. (j) Graph of the number of E8 micro-colonies in the center of the well with and without droplet networks (ST and CTR (control), respectively) at initial starting ratios of 1: 1 and 9: 1 between E7-inducible and E8 cells. The individual data points aredepicted for each condition with n = 4 technical replicates. It was tested whether the data was normally distributed using the Shapiro-Wilk test (p < 0.05). Given groups were normally distributed, significance between groups was tested performing a two-sample t-test. If data of at least one group was not normally distributed, significance between groups was tested by performing a Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0034] Figure 7 shows plasmid maps of plasmids used throughout the work in the Examples herein. In particular, (a)-(c) are schematics of plasmid maps of pJSl-PBAD:-mCherry-AMP, pKCl-PBAD:-ColE7-AMP and pYYl-PBAD:-ColE7-mCherry-AMP, respectively. Plasmid maps were created using Benchling (https: / / benchling.com, 2024).

[0035] Figure 8 shows patterned gene expression using masks and reservoirs incorporated into 3D-printed droplet networks. In particular, (a) is a schematic of a droplet network (that contains a mask and a reservoir) flipping during placement on top of a bacterium-laden hydrogel, causing the droplet network to predominantly land with the reservoir directly contacting the bacterium-laden hydrogel. Arrows around the sinking droplet network represent oil displacement, which we reasoned drives the flipping process as the reservoir is smaller in size compared to the mask, (b) Schematic of a droplet network that flips and lands reservoir side down activating expression of mCherry in the bacterial population. Bottom right microscopy images (bright-field and epifluorescence) is an example of a droplet network landing reservoir side down onto the bacteriumladen hydrogel. Images were taken 18 hours after placement of the droplet network (containing 33 RIM arabinose and 50 pg mb'1aHL monomer). Cyan fluorescence represents cascade blue dextran, while magenta fluorescence represents mCherry expression, (c) Schematic of a droplet network that does not flip and lands mask side down due to magnetic beads (droplets composed of 1.5% w / v ultra-low gelling agarose and nickel magnetic beads) attached to each comer of the mask of a droplet network and a magnet placed underneath the bacterium -laden hydrogel during tissue transfer. The release of arabinose from the mask of a droplet network containing 33 RIM arabinose and 50 pg mL'1aHL monomer activated patterned (cross-like shape) expression of mCherry in the bacterial population. Bottom right microscopy images (bright-field and epi-fluorescence) is an example of a droplet network landing mask side down onto the bacterium -laden hydrogel. Images were taken 18 hours after droplet network placement and cyan and magenta fluorescence represent cascade blue dextran and mCherry expression, respectively, (a)-(c) In droplet networks cyan droplets contained 33 RIM arabinose, 50 pg mL'1aHL monomer, and 250 pM cascade blue dextran, whereas grey droplets did not contain these components, (d) Epi-fluorescent microscopy images depicting mCherry expression (magenta) induced by the release of arabinose from droplet networks composed of 1, 2, 3 or 4 mask layers (stripe-like pattern) and 4 reservoir layers 18 hours after tissue placement on top of the bacterium-laden hydrogel. The droplets within the stripe-like pattern of the mask and all reservoir droplets contained 33 RIM arabinose, 50 pg mL'1aHL monomer, and 250 pMcascade blue dextran, whereas droplets of mask layers located outside the stripe-like pattern did not contain these components. Shown are n = 4 replicates.

[0036] Figure 9 shows single -droplet gene expression with and without reservoirs. In particular, (a) is a schematic illustrating droplet networks composed of 8 patterned layers (without reservoirs) or 4 patterned mask layers and 4 reservoir layers (with reservoirs). Both types of droplet networks were composed of droplets that contained 33 RIM arabinose and 50 pg ml1aHL monomer (cyan droplets) or did not contain these components (white droplets), (b) Graph of the PF and mean mCherry expression induced by arabinose release from droplet networks with and without reservoirs on top of masks comprising of a single-droplet diffusive pathway that contacts the hydrogel surface forming a DHB.

[0037] Figure 10 shows an overlay assay of E7-inducible against colicin E7, colicin E8-expressing and susceptible cells. In particular, (a) to (d) are photographs of serial dilutions from 10° to 104(of an overnight liquid culture) of E7-inducible (BZB1011 pBAD-E7) spotted on top of agar plates containing E7 cells (BZB1011 pColE7) (a), E8 cells (BZB1011 pColE8) (b), or susceptible cells (BZB1011) (c) and (d), after 12 hours at 37°C where the agar contained between 0 RIM and 333 RIM arabinose. The agar plates were composed of 1.5% (w / v) of agar in LB (a)-(c) or M9 (d).

[0038] Figure 11 are graphs showing co-culture of E7-inducible and susceptible cells. In particular, (a) is a graph of the mean GFP and RFP expression of E7-inducible (upper line) and S cells (lower line), respectively, mixed homogeneously at equal starting ratios in M9 ultra low gelling temperature agarose (ULGA) gels at a combined cell starting density of 3.6 x 109cells mb'1at varying arabinose concentrations after 18 hours at 37°C. (b) is a graph of the number of microcolonies formed of E7-inducible in M9 ULGA gels after 18 h at 37°C at varying arabinose concentrations at a cell starting ratio of 1.6 x 107cells mL1. (c)-(d) are graphs of the number of micro-colonies formed of E7-inducible (lower line on graph) and S cells (upper line on graph) mixed homogeneously at starting ratios of 1: 1 (equal number between E7-inducible and S cells), 1:9 or 1:99 (E7-inducible to S cells), respectively, in M9 ultra low gelling temperature agarose (ULGA) gels at a combined cell starting density of 1.6 x 107cells mL4at varying arabinose concentrations after 18 hours at 37°C. The solid line and shaded area represent the mean and standard deviation of n = 4 technical replicates.

[0039] Figure 12 shows time -dependent release of arabinose from droplet networks. In particular, (a) and (b) are box plots of the number of E7-inducible (BZB1011 pBAD-E7) (a) and susceptible cells (BZB1011) (b) arising from competition between them, when droplet networks containing either 0 RIM or 33 RIM arabinose and 50 pg mL4aHL were placed on top of the bacterium-laden hydrogel for 18 hours after placement immediately after gel formation (placed after 0 h) or placement after 24 hours of cell growth (placed after 24 h). The starting ratio was 1:9 (E7 -inducible to susceptible cells) while the total starting cell density was 1.6 x 107cells mL1. Individual data points on the box plots are technical replicates, (c) and (d) Box plots of the number of E7-inducible(BZB1011 pBAD-E7) (c) and susceptible cells (BZB1O11) (d) arising from competition between them, when droplet networks containing 333 RIM arabinose and 50 pg ml1aHL were placed on top of the bacterium-laden hydrogel for 18 or 42 hours, respectively. The starting ratio was 1:9 (E7-inducible to susceptible cells) while the total starting cell density was 1.6 x 107cells mL1.

[0040] Individual data points on the box plots are technical replicates.

[0041] Figure 13 shows the importance of culture medium for colicin competition. In particular, (a) and (b) are graphs of the mean sfGFP expression (a) and mean intensity of intracellular propidium iodide (b), in ULGA gels composed of M9 supplemented with 24 RIM glucose, M9 supplemented with 24 RIM glycerol or LB at a range of arabinose concentrations (0 RIM, 6 RIM, 33 RIM and 66 RIM) after 18 hours at 37°C. The gels contained E7-inducible cells at a starting cell density of 3.6x 109. The solid line and shaded area represent the mean and standard deviation of n = 4 technical replicates.

[0042] Figure 14 shows localized expression of colicin E7 underneath droplet networks. In particular, (a) is a composite microscope image (bright-field and cascade dextran blue, top image) of a droplet network on top of a bacterium-laden hydrogel and sfGFP fluorescence images of bacterium-laden hydrogel when a droplet network was placed (bottom image) and was not placed on top of the image (center image). The white dashed line represents the outline of the tissue, which was used as a reference area for when no droplet network was placed on top of the bacterium -laden hydrogel. The bacterium -laden hydrogels contained E7-inducible cells (BZB1011 pBAD-E7) at a starting cell density of 1.6 x 107cells mL1. (b) Boxplot of the micro-colonies per mm2within the peripheral and central areas of E7-inducible cells (BZB1011 pBAD-E7) (Figure 7(c) and Table SI) seeded at a starting cell density of 1.6 x 107cells mL'1in ULGA gels composed of LB medium at 37°C for 18 hours after droplet networks containing 333 RIM arabinose and 0 pg mL1aHL monomer were (+ST) and were not (-ST) placed on top of the bacterium -laden hydrogels. The individual data points are depicted for each condition with n = 5 technical replicates. It was tested whether the data was normally distributed using the Shapiro-Wilk test (p < 0.05). Given groups were normally distributed, significance between groups was tested performing a two-sample t-test. If data of at least one group was not normally distributed, significance between groups was tested by performing a Wilcoxon rank-sum test. **p < 0.01, ***p < 0.001 and ****p < 0.0001. (c) Boxplot of the relative abundance (RA) of E7-inducible microcolonies at a range of starting ratios (1:9 to 9: 1, ratio between E7-inducible cells and susceptible cells) when droplet networks containing 333 RIM arabinose and 50 pg mL'1of aHL monomer were placed (+ST) or not placed (-ST) on top of the bacterium -laden hydrogel after 18 hours at 37°C. The total starting cell density was 1.6 x 107cells mL1. Shown are individual data points of n = 4 technical replicates and boxplots are mean and interquartile range.

[0043] Figure 15 shows that E7-inducible causes DNA damage in susceptible cells. In particular, (a) is a schematic depicting the workflow to characterize the correlation between GFP expressionand the relative colony forming units (CFU), whereby E7R-inducible or E7R-inducible and S-GFP cells are grown for 18 h at 37 °C in M9 ULGA gels, before re-suspension and plating on LB agar plates containing 50 pg mL1of kanamycin to select for colony-forming units arising from S-GFP cells, (b) Graph of the mean mCherry expression (mean fluorescence intensity of activated pixels, indicating colicin E7 expression), mean sfGFP expression (indicating DNA damage in susceptible cells), and the relative number of colony forming units (CFU) from susceptible cells over a range of arabinose concentrations (0 RIM to 8 mM) added to bacterium -laden hydrogels composed of M9 ULGA supplemented with 24 mM glycerol. The starting ratio between E7R-inducible and susceptible cells (BZB 1011, S-GFP cells) was 1 : 1 at a total starting cell density of

[0044] 1.6 x 107cells mL1. Mean mCherry expression and mean GFP expression of E7 -inducible and susceptible cells, respectively, were measured after 18 hours of co-culture, while the number of colony forming units from susceptible cells was measured 12 hours after solubilization of the M9 ULGA gels and plating on selective medium. ‘Single’ indicates M9 ULGA gels containing only susceptible cells seeded at 0.8 x 107cells mL'1without the addition of arabinose (0 mM), while ‘both’ indicates the co-culture of E7-inducible and susceptible cells at an equal starting ratio and a total starting cell density of 1.6 x 107cells mL'1without the addition of arabinose (0 mM). The solid line and shaded area represent the mean and standard deviation of n = 4 technical replicates.

[0045] (c) Boxplots of the number of micro-colonies per mm2of activated susceptible cells (expressing GFP as a consequence of DNA-damage) in areas underneath the droplet networks containing a range of arabinose concentrations (0 mM - 33 mM) and 50 pg mL'1of aHL monomer, and peripheral areas (outside of where the droplet networks were placed) at 18 hours after placement of droplet networks at 37°C. E7R-inducible (BZB1011, E7R-inducible) and susceptible cells (BZB 1011, S-GFP cells) were seeded at a 1:1 starting ratio and a total starting cell density of 1.6 x 107cells mL1. The ULGA gels contained M9 medium supplemented with 24 mM glycerol. Shown are individual data points of n = 4 technical replicates and boxplots display the mean and interquartile range.

[0046] Figure 16 shows localized DNA damage in susceptible cells. In particular, (a)-(d) are heatmaps of micro-colonies per mm2in areas underneath droplet networks (a), the total number of micro-colonies in peripheral areas outside of where droplet networks were placed (b), the mean GFP expression (c) and mean micro-colony size (d) of activated susceptible cells (S-GFP cells experiencing DNA-damage caused by colicin E7 expressed from E7R-inducible) after 18 hours at 37°C. E7R-inducible cells (BZB1011, E7R-inducible) and susceptible cells (BZB1011, S-GFP) were seeded at equal starting ratio and atotal cell density of 1.6 x 107cells mL1. The ULGA gels were composed of M9 supplemented with 24 mM glycerol. Droplet networks contained a range of arabinose concentrations (8 mM, 12 mM and 16 mM) and a range of aHL monomer concentrations (0 pg mL1, 10 pg mL1, 25 pg mL'1and 50 pg mL'1).Figure 17 shows a single aqueous droplet containing aHL and hydrochloric acid (HC1, denoted “small molecule” in Fig. 17(a)) deposited onto a layer of mammalian cells (astrocytes). Cellular responses were monitored using a Fluo-4 calcium assay. Transient fluctuations in intracellular calcium levels indicated that local pH changes induced by the droplet triggered a localized signaling response in the cells. Panel (a) presents a schematic representation of the droplet-cell interface (not to scale), while panel (b) shows the droplet positioned over the cells (left) and the corresponding calcium response (right).

[0047] Figure 18 shows a network of aqueous droplets, each containing aHL and hydrochloric acid (HC1), deposited onto a layer of OVCAR cells (an ovarian cancer cell line). Cellular responses were monitored using a Fluo-4 calcium assay. Transient fluctuations in intracellular calcium levels indicated that local pH changes induced by the droplets triggered a localized signaling response in the cancer cells. Panel (a) presents a schematic representation of the dropletcell interface (not to scale), while panel (b) is a graph showing the corresponding calcium response of the cells.

[0048] Figure 19 shows a single aqueous droplet containing aHL and a small molecule drug (either isoprenaline or doxorubicin) deposited onto a layer of mammalian cells (cardiomyocytes). Cellular responses were monitored by measuring the frequency of cardiomyocyte contractions in response to the drug released from the droplet via aHL. Panel (a) presents a schematic representation of the droplet-cell interface (not to scale). Panel (b) shows the percentage increase in beat frequency 5 minutes after drug release. Panel (bl) demonstrates that isoprenaline increases the frequency of contraction over a concentration range of 10 nM to lOOnM, with beat frequency rising proportionally across this range. Panel (b2) shows that doxorubicin decreases the frequency of contraction over a concentration range of 500 pM to 5 mM, with beat frequency decreasing as concentration increases.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The invention provides a system for delivering an effector molecule to biological cells or tissue, which system comprises:

[0051] (a) a target region which comprises biological cells or tissue; and

[0052] (b) a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0053] wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet additionally contacts the target region to form a layer of the amphipathicmolecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, and

[0054] at least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.

[0055] The system of the invention comprises a synthetic droplet assembly as defined above. A synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, and wherein each of said n droplets contacts at least one other of said n droplets to form a layer of the amphipathic molecules as an interface between the contacting droplets, may be produced by a process for producing droplet assemblies by 3D-printing of droplets, for instance as described in any of WO 2014 / 087175 Al, WO 2021 / 234394 Al, G. Villar et al. Science 2013, 340, 48, and I. Cazimoglu et al, ACS Nano 2021, 15, 20214.

[0056] The number of droplets, n, in the synthetic droplet assembly of the system of the invention is equal to or greater than 2. Thus, n is an integer equal to or greater than 2. Often, however, the droplet assembly comprises more than 2 droplets. For instance, typically, the number of droplets, n, in the synthetic droplet assembly is equal to or greater than 3, or equal to or greater than 5. Usually, the number of droplets, n, in the synthetic droplet assembly is equal to or greater than 10, or, for instance, equal to or greater than 20, or equal to or greater than 30. Often, the number of droplets, n, is equal to or greater than 50, or, for instance, equal to or greater than 100. For instance, n may be equal to or greater than 200, equal to or greater than 500, or equal to or greater than 1,000. Indeed, the number of droplets, n, may be equal to or greater than 20,000, equal to or greater than 50,000, or, for instance, equal to or greater than 100,000.

[0057] There is in principle no upper limit to the number of droplets, n in the synthetic droplet assembly because very large droplet assemblies of this kind may be produced. Indeed, the number of droplets, n, in the synthetic droplet assembly of the system of the invention may be very large, for instance, at least 1,000,000 or even larger. The number of droplets, n, may for instance be at least 10,000,000, or, for instance, at least 1,000,000,000. In some embodiments, the number of droplets, n, is at least 10,000,000,000, for instance at least 50,000,000,000. If, for instance the droplet assembly is a cube comprising at least 4000 layers of droplets, the number of droplets, n, in the droplet assembly may be at least 64 billion droplets.

[0058] Thus, for instance, the number of droplets, n, in the synthetic droplet assembly may be from 2 to 100,000,000,000, for instance from 2 to 50,000,000,000, from 2 to 10,000,000,000, from 2 to 1,000,000,000, from 2 to 100,000,000, from 2 to 10,000,000, or from 2 to 1,000,000. The number of droplets, n, may for instance be from 2 to 100,000, for instance from 2 to 10,000, or, for example, from 2 to 5,000, or from 2 to 2,000, for instance from 2 to 1,000. The number ofdroplets, n, in the synthetic droplet assembly may alternatively for instance be from 10 to 100,000,000,000, for instance from 10 to 50,000,000,000, from 10 to 10,000,000,000, from 10 to 1,000,000,000, from 10 to 100,000,000, from 10 to 10,000,000, or from 10 to 1,000,000. The number of droplets, n, may for instance be from 10 to 100,000, for instance from 10 to 10,000, or, for example, from 10 to 5,000, or from 10 to 2,000, for instance from 10 to 1,000. Alternatively, for instance, the number of droplets, n, in the synthetic droplet assembly may be from 100 to 100,000,000,000, for instance from 100 to 50,000,000,000, from 100 to 10,000,000,000, from 100 to 1,000,000,000, from 100 to 100,000,000, from 100 to 10,000,000, or from 100 to 1,000,000. The number of droplets, n, may for instance be from 100 to 100,000, for instance from 100 to 10,000, or, for example, from 100 to 5,000, or from 100 to 2,000, for instance from 100 to 1,000. The number of droplets, n, in the synthetic droplet assembly may alternatively for instance be from 200 to 100,000,000,000, for instance from 200 to 50,000,000,000, from 200 to 10,000,000,000, from 200 to 1,000,000,000, from 200 to 100,000,000, from 200 to 10,000,000, or from 200 to 1,000,000. The number of droplets, n, may for instance be from 200 to 100,000, for instance from 200 to 10,000, or, for example, from 200 to 5,000, or from 200 to 2,000, for instance from 200 to 1,000. The number of droplets, n, in the synthetic droplet assembly may alternatively for instance be from 500 to 100,000,000,000, for instance from 500 to 50,000,000,000, from 500 to 10,000,000,000, from 500 to 1,000,000,000, from 500 to 100,000,000, from 500 to 10,000,000, or from 500 to 1,000,000. The number of droplets, n, may for instance be from 500 to 100,000, for instance from 500 to 10,000, or, for example, from 500 to 5,000, or from 500 to 2,000, for instance from 500 to 1,000. Alternatively, the number of droplets, n, in the synthetic droplet assembly may be from 1,000 to 100,000,000,000, for instance from 1,000 to 50,000,000,000, from 1,000 to 10,000,000,000, from 1,000 to 1,000,000,000, from 1,000 to 100,000,000, from 1,000 to 10,000,000, or from 1,000 to 1,000,000.

[0059] At least some, if not all, of the n droplets in the synthetic droplet assembly may be arranged in a hexagonal close-packed structure.

[0060] As mentioned above, each of the n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium.

[0061] The aqueous droplet medium may be any suitable aqueous medium. For instance, the aqueous medium may be pure water, or an aqueous buffer solution, or an aqueous solution of one or more salts. Alternatively, the aqueous medium may comprise a hydrogel.

[0062] Often, the aqueous droplet medium is an aqueous solution.

[0063] The aqueous droplet medium may for instance comprise a growth medium or a culture medium, for instance a cell growth medium or a cell culture medium. The aqueous droplet medium may for instance comprise culture medium for eukaryotic cells or tissue, or a growth medium for bacteria, such as for example M9 minimal medium. Often, the aqueous droplet medium comprisesa growth medium or culture medium which is suitable for supporting the growth of the biological cells or tissue present in the target region of the system of the invention.

[0064] Thus, for instance, the aqueous droplet medium may be an aqueous solution which comprises M9 minimal salts. The aqueous solution which comprises M9 minimal salts may further comprise one or more amino acids. It may additionally further comprise glucose or glycerol. Often, the aqueous solution which comprises M9 minimal salts further comprises at least one of MgSO4, CaCL. one or more amino acids, glucose or glycerol. Typically, the aqueous solution which comprises M9 minimal salts further comprises MgSO4, CaCL, casamino acids, and glucose or glycerol.

[0065] When the aqueous droplet medium comprises a hydrogel, the aqueous medium may, for instance, comprise agarose and water. The concentration of the agarose in water is typically less than or equal to 10% w / v agarose. For instance, the concentration of the agarose in said water may be from 0.25 to 5% w / v agarose. Hydrogels other than agarose may also be used. For instance the aqueous medium may comprise methylcellulose, polyethylene glycol diacrylate, polyacrylamide, matrigel, hyaluronan, polyethylene oxide, poly AMPS (poly(2-acrylamido-2-methyl-l-propanesulfonic acid)), polyvinylpyrrolidone, polyvinyl alcohol, sodium polyacrylate, acrylate polymers or poly(N-isopropylacrylamide). Alternatively, the aqueous medium body may comprise a silicone hydrogel or LB (Luria broth) agar.

[0066] The pH of the aqueous medium can be varied over a wide range. In some embodiments, for instance, the pH of the aqueous medium within the aqueous droplet or droplets may be in the range of from 5 to 9 (or for instance in the range of from 6 to 8) although higher and lower pH values are also possible. The aqueous medium may therefore be an aqueous buffer solution. Any suitable buffer can be employed, depending on the desired pH. The buffer solution may for instance comprise Tris-HCl and / or KC1. In some embodiments the pH of the aqueous buffer solution is from 5 to 9, or for instance from 6 to 8. The nature and concentration of the solutes can be varied to vary the properties of the solution.

[0067] As the skilled person will appreciate, it is possible, when a droplet assembly is made using hydrogel droplets in which the hydrogel has gelled (and the droplets are therefore more rigid and less flexible than droplets in which the droplet medium is an aqueous solution), for any two hydrogel droplets to be physically pushed together to “squeeze out” a layer (such as a bilayer) of amphipathic molecules from between the two hydrogel droplets, so that the respective aqueous media of the adjacent hydrogel droplets are in direct contact with each other. It is also possible for the amphipathic molecules to leach out, over time, from between the hydrogel droplets in a droplet assembly of hydrogel droplets, especially if it has been transferred to a fully aqueous environment and is not in contact with a hydrophobic medium comprising amphipathic molecules. Therefore, as the skilled person will appreciate, in some embodiments of the system of the invention as described herein, the synthetic droplet assembly of the invention as described herein, or the process of theinvention as described herein, one or more of the n droplets in the synthetic droplet assembly may not comprise said outer layer of amphipathic molecules and may not contact at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets. Rather, in some embodiments, in which the aqueous droplet medium comprises a hydrogel, at least one of, or indeed each of, the n droplets may contact at least one other of said n droplets directly, so that the aqueous droplet media of the contacting droplets are in direct contact with each other at the droplet interface. Typically, in such embodiments, no amphipathic molecules are present at the interface between contacting droplets. Rather, the aqueous droplet media of the contacting droplets are in direct contact at the interface between the contacting droplets. Thus, often, in such embodiments, the synthetic droplet assembly comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises an aqueous droplet medium comprising a hydrogel, and the aqueous droplet medium of each of said n droplets is in direct contact with the aqueous droplet medium of at least one other of said n droplets at an interface between the contacting droplets.

[0068] Usually however, the aqueous droplet medium is an aqueous solution. Usually, each of the n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium.

[0069] The aqueous droplet medium of each droplet in the droplet assembly may be the same or different. For instance, if a droplet in the synthetic droplet assembly is an effector droplet as described herein, the aqueous droplet medium typically further comprises an effector molecule or means for producing an effector molecule. However, if a droplet in the synthetic droplet assembly is a mask droplet as described herein, the aqueous droplet medium of the droplet generally does not comprise an effector molecule (or means for producing an effector molecule). Similarly, if a droplet in the synthetic droplet assembly is (i) a target interface outlet droplet as described herein, (ii) a connector droplet as described herein (such as a connector droplet in a connector region), or (iii) an effector droplet as described herein (for instance an effector droplet in a reservoir region, or a connector droplet or target interface outlet droplet which is also an effector droplet), then the aqueous droplet medium of the droplet often comprises a protein pore, suitable for allowing passage of an effector molecule into and / or out of the droplet in question. However, if a droplet in the synthetic droplet assembly is a mask droplet as described herein, the aqueous droplet medium of the droplet generally does not comprise such a protein pore, i.e. it generally does not comprise any pore that allows passage of the, or any, effector molecule into or out of the mask droplet. Thus, if a droplet in the synthetic droplet assembly is a mask droplet as described herein, it typically does not comprise a protein pore.

[0070] Each droplet of the aqueous droplet medium is usually formed initially by dispensing it into a hydrophobic medium in the presence of amphipathic molecules. The amphipathic molecules may, for instance, be disposed in the aqueous medium or in the hydrophobic medium. Typically,the amphipathic molecules are disposed in the hydrophobic medium. The concentration of the amphipathic molecules in the hydrophobic medium may, for instance, be from 0.1 RIM to 10 mM, or, for instance, from 0.2 mM to 5 mM, such as from 1 mM to 3 mM, e.g. about 2 mM. When the aqueous medium is dispensed into the hydrophobic medium in the presence of the amphipathic molecules, an aqueous droplet forms, which droplet comprises (i) an aqueous droplet medium and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium.

[0071] As discussed, the droplet assembly comprises n (at least two, but often much more than two) droplets in contact with each other. The boundary that is shared between contacting droplets, at the point of contact between the droplets, is referred to herein as an interface. An interface is formed when part of the outer layer of one droplet contacts part of the outer layer of another droplet. For instance, when the droplet is brought into contact with the other droplet, a layer of amphipathic molecules typically forms at the interface between the two objects. Depending on the amphipathic molecules, the layer of amphipathic molecules that forms at the interface between two droplets may be a bilayer. Typically, each layer of said amphipathic molecules which is an interface between contacting droplets is a bilayer of said amphipathic molecules. This is typically the case when the amphipathic molecules are lipids, for instance phospholipids or fatty acids. Such a bilayer comprises amphipathic molecules from the outer layer of amphipathic molecules around the surface of the aqueous medium of each droplet at the interface. The bilayer (known in the art as a droplet interface bilayer, or “DIB”) forms as it is an energetically more favourable configuration for the amphipathic molecules to adopt. As the skilled person will appreciate, the contacting droplets will acquire the geometry with the lowest free surface energy.

[0072] The amphipathic molecules may not necessarily comprise lipids but may comprise molecules of a block copolymer instead, for instance a triblock copolymer. In such cases the layer of the amphipathic molecules which is an interface between contacting droplets is not necessarily be a bilayer. It may, for instance, be a monolayer of triblock copolymer molecules, in which two, outer, polar blocks of each triblock molecule contact the respective aqueous media of two adjacent droplets, and a central, apolar block of each triblock molecule resides at the centre of the monolayer (in between the two polar blocks of each molecule). Suitable block copolymers are described in WO 2014 / 064444, and include the triblock copolymer amphipathic molecule poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA). The or each layer of amphipathic molecules which is an interface between contacting droplets may for instance be a layer of a block copolymer, for example a layer of a triblock copolymer such as PMOXA-PDMS-PMOXA.

[0073] Thus, often, the or each layer of said amphipathic molecules which is an interface between contacting droplets is (i) a bilayer of said amphipathic molecules or (ii) a layer of said amphipathic molecules wherein the amphipathic molecules comprise a block copolymer (for instance a triblock copolymer). In case (ii), the layer may, for instance, comprise a monolayer of said amphipathicmolecules, for instance a monolayer of the block (e.g. triblock) copolymer molecules.

[0074] Alternatively, in case (ii) the layer may comprise a bilayer of the amphipathic molecules, for instance a bilayer of the block (e.g. triblock) copolymer molecules.

[0075] Usually, each layer of the amphipathic molecules which is an interface between contacting droplets is a bilayer of the amphipathic molecules.

[0076] Thus, often, each layer of amphipathic molecules which is an interface between contacting droplets is a bilayer of the amphipathic molecules. In other words, in the system of the invention, each of said n droplets may contact at least one other of said n droplets to form a bilayer of said amphipathic molecules as an interface between the contacting droplets.

[0077] The amphipathic molecules of a droplet need not be all of the same type. Rather, the amphipathic molecules may in some embodiments be a mixture of two or more different kinds of amphipathic molecule. Another important example is that the amphipathic molecules in the respective outer layers of different droplets in a droplet assembly may be of different types so that any bilayer(s) formed between the different droplets may be asymmetric.

[0078] The amphipathic molecules may be any suitable amphipathic molecule. Often, the amphipathic molecules will be ones which are capable, when present in a high enough concentration, of forming a bilayer at any one of said interfaces. The type of amphipathic molecule that is capable of forming a bilayer may, for instance, depend on additional components of the contacting droplets. For example, if the droplets are disposed in a hydrophobic medium, the amphipathic molecules may be any suitable amphipathic molecules capable of forming a bilayer within a hydrophobic medium. The type of amphipathic molecules capable of forming a bilayer within the hydrophobic medium would typically depend on the nature of the hydrophobic medium and the aqueous medium of the droplets, but a wide range of amphipathic molecules are possible.

[0079] Amphipathic molecules are molecules which have both hydrophobic and hydrophilic groups. The outer layer of amphipathic molecules usually comprises a monolayer of amphipathic molecules on the surface of the droplet. The monolayer is typically formed and maintained naturally by the interaction of the hydrophilic and hydrophobic groups with the aqueous medium and the bulk medium so that the molecules align on the surface of the droplet with the hydrophilic groups facing inwards towards the aqueous medium and the hydrophobic groups facing outwards, for instance towards a hydrophobic medium.

[0080] The amphipathic molecules may, for instance, be non-polymeric amphipathic molecules. Alternatively, the amphipathic molecules may be polymeric amphipathic molecules.

[0081] An important class of amphipathic molecules which can be used in the droplet assembly is lipid molecules. The lipid molecules may be any of the major classes of lipid, including phospholipids, fatty acids, fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides. Some important examples include phospholipids and fatty acids, for instance phospholipids. The lipid molecules may be naturallyoccurring or synthetic. Whilst the formation of a bilayer from lipid molecules has been demonstrated the method is expected to be appropriate for any amphipathic molecules.

[0082] A common class of hydrophobic group that may be present in an amphipathic molecule is a hydrocarbon group, as for instance in most lipids. However, another suitable kind of hydrophobic group that may be employed is a fluorocarbon group. Thus, a further important class of amphipathic molecule is an amphipathic molecule that comprises at least one fluorocarbon group. An example of such a molecule would be a lipid-like molecule which comprises a hydrophobic fluorocarbon tail and a hydrophilic head group.

[0083] The amphipathic molecules of the droplet need not be all of the same type. Rather, the amphipathic molecules may in some embodiments be a mixture of two or more different kinds of amphipathic molecule. Another important example is that the amphipathic molecules in the respective outer layers of different droplets in a droplet assembly may be of different types so that, if bilayers are formed, the bilayer(s) formed between the different droplets may be asymmetric. In some embodiments, the lipid leaflets of two contacting droplets are different.

[0084] Typically, therefore, the amphipathic molecules comprise lipid molecules. The lipid molecules need not be all of the same type. Thus, the amphipathic molecules may comprise a single type of lipid or a mixture of two or more different types of lipid molecules. Likewise, when the droplet is in contact with another droplet, the lipid compositions of the outer layers of the individual droplets may be the same as or different from one another. Lipid molecules are particularly advantageous because lipid bilayers, or more generally bilayers of amphipathic molecules, are models of cell membranes and the droplet assembly may therefore serve as an excellent platform for a range of experimental studies, including for instance as novel platforms for the fundamental study of membrane proteins, or as multi-compartment protocellular chassis for “bottom-up” synthetic biology.

[0085] The lipid may, for instance, be sensitive to its environments (i.e. be a smart lipid). The lipid may, for instance, be sensitive to changes is pH, light or temperature. Thus the lipid may be a pH-sensitive lipid, a temperature-sensitive lipid or a light-sensitive lipid.

[0086] The lipid may allow a membrane protein (such as a natural, engineered or synthetic membrane protein) to act as a functional component of the minimal tissue.

[0087] Phospholipids are particularly preferred for reasons outlined above and also because they are a major component of all cell membranes, making droplets comprising phospholipids particularly suitable for synthetic biology applications, as well as for drug delivery.

[0088] Accordingly, the amphipathic molecules that form an outer layer on at least part of the surface of the aqueous medium typically comprise phospholipid molecules. The phospholipid molecules may be the same or different, i.e. the amphipathic molecules comprise a single kind of phospholipid, or a mixture of two or more different phospholipids. Phospholipids are well known to the skilled person and many are commercially available, from suppliers such as Avanti PolarLipids. The phospholipid molecules may be glycerophospholipids or phosphosphingolipids or a mixture of the two. The phospholipid molecules may comprise anionic phospholipids, phospholipids comprising primary amines, choline-containing phospholipids and / or glycosphingoplipids. Usually, the amphipathic molecules comprise one or more glycerophospholipids. As the skilled person will appreciate, glycerophospholipids include, but are not limited to glycerophospholipids having a structure as defined in the following formula (I):

[0089] O

[0090] 111

[0091] R— C— CL

[0092] 7

[0093] R— C— O —

[0094] II O

[0095]

[0096] wherein:

[0097] R1and R2, which are the same or different, are selected from C10-C25 alkyl groups and Cw-C25 alkylene groups;

[0098] either R3is absent such that OR3is O', or R3is present and is H, CH2CH2N(R4)3+, a sugar group, or an amino acid group; and

[0099] each R4, which is the same or different, is independently selected from H and unsubstituted C1-C4 alkyl.

[0100] Typically, when R3is CH2CH2N(R4)3+, each R4, which is the same or different, is selected from H and methyl. As the skilled person will appreciate, when each and every R4is methyl, the R3group is a choline group, and when each and every R4is H, the R3group is an ethanolamine group.

[0101] When R3is an amino acid group it may for instance be a serine group, i.e.

[0102] -CH2CH(NH2)(COOH). When R3is a sugar group, it may for instance be glycerol, i.e.

[0103] -CH2CHOHCH2OH, or for instance inositol, i.e. -CH(CH0H)5.

[0104] Typical examples of R1and R2groups are C10-C25 alkyl groups, including, but not limited to linear C10-C25 alkyl groups such as, for instance, CFfi CFLjio-, CFfi CFLjn-, CH3(CH2)i4-, CH3(CH2)i6-, CH3(CH2)i8-, CFfi CFL^- and branched C10-C25 alkyl groups such as for instance -CH2-CH(CH3)-(CH2)3-CH(CH3)-(CH2)3-CH(CH3)-(CH2)3-CH(CH3)2.

[0105] Further typical examples of R1and R2groups are unsubstituted C10-C25 alkylene groups, including, but not limited to, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4(CH=CHCH2)3CH=CH(CH2)3-, and CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-.

[0106] As the skilled person will appreciate, the O' group in the phosphate group adjacent to the OR3group may in some embodiments be protonated, or associated with a suitable cation, for instance a metal cation such as Na+.Thus, the amphipathic molecules may comprise one or more glycerophospholipids having the structure of formula (I) as defined above.

[0107] Thus, the amphipathic molecules may comprise lipid molecules. The lipid molecules may comprise phospholipids. Often, amphipathic molecules comprise one or more glycerophospholipids. The amphipathic molecules may for instance comprise any one or more of the following glycerophospholipids

[0108] For instance, the amphipathic molecules may comprise any one or more of the following glycerophospholipids: l,2-diphytanoyl-5«-glycero-3-phosphocholine (DPhPC), 1 -palmitoyl -2-oleoyl-glycero-3 -phosphocholine (POPC), l,2-distearoyl-5«-glycero-3 -phosphocholine (DSPC), l,2-dipahnitoyl-sw-glycero-3 -phosphocholine (DPPC), l.2-dipalmito l-s77-glyccro-3-|phospho-rac-(1 -glycerol)] (DPPG), 1 -palmitoyl -2 -oleoyl -s'w-glyccro-3 -phosphoglycerol (POPG), or glycerophospholipid l,2-dioleoyl-5«-glycero-3 -phosphoethanolamine (DOPE). Any one or more of the aforementioned glycerophospholipids can be employed as the amphiphilic molecules herein, including as the amphiphilic molecules in the n droplets of the droplet assembly, or a mixture of two or more thereof may be employed. Often, the amphipathic molecules comprise 1,2-diphytanoyl-sw-glycero-phosphatidylcholine (DPhPC). The amphipathic molecules may for instance comprise l,2-diphytanoyl-5«-glycero-phosphatidylcholine (DPhPC) and 1 -palmitoyl -2-oleoyl-glycero-3-phosphocholine (POPC). The amphipathic molecules may for instance comprise DPhPC and POPC in a molar ratio of from 3 : 1 to 1:1. Often, for instance, the amphipathic molecules comprise DPhPC and POPC in a 2: 1 molar ratio.

[0109] Additionally or alternatively, the amphipathic molecules may comprise a steroid, which steroid comprises an alkyl side-chain. The amphipathic molecules may, for instance, comprise cholesterol, P-sitosterol and lanosterol.

[0110] In some embodiments, the amphipathic molecules comprise glycerophospholipids which are a phosphatidylcholine, such as POPC (1 -palmitoyl -2 -oleoyl -s'w-glyccro-3 -phosphocholine) or DPPC ( l.2-dipahnitoyl-s77-glyccro-3-phosphocholinc). or which are a phosphatidylglycerol, such as POPG (l-palmitoyl-2-oleoyl-5«-glycero-3-phosphoglycerol).

[0111] Preferably, the amphipathic molecules comprise DPhPC.

[0112] The amphipathic molecules may, for instance, comprise one or more fatty acids, e.g. oleic acid. Fatty acids are of course well known to the skilled person and a wide range of these are commercially available.

[0113] The amphipathic molecules may for instance comprise a mixture comprising: (a) one or more phospholipids, and (b) one or more fatty acids.

[0114] In addition to the amphipathic molecules, the outer layer of amphipathic molecules of one or more of, or each of, the n droplets of the synthetic droplet assembly, may further comprise a PEGylated lipid. The term “PEGylated lipid”, as used herein, refers to a lipid which has been derivatised with polyethylene glycol). The inclusion of one or more PEGylated lipids maystabilise the droplet assembly in vivo, and in particular prolong the life of the droplet assembly. This means that the inclusion of one or more PEGylated lipids may also have the useful effect of prolonging the plasma half-life of the effector molecule within the droplet assembly. Such effects have been observed previously when PEGylated lipids are used in liposomal drug formulations. PEGylated lipids are known in the art and are commercially available from suppliers such as NOF Corporation, Japan (see http: / / www.phospholipid.jp / phospholipid_2-3.html). Any suitable PEGylated lipid may be employed, including, but not limited to, PEG-phospholipids, diacylglycerol-PEG, cholesterol-PEG derivatives, and mixtures thereof.

[0115] The polyethylene glycol) (PEG) component of the PEGylated lipid may have any one of several different geometries. Thus, it could be substantially linear PEG or branched PEG. The branched PEG may for instance have from three to ten PEG chains emanating from a central core group. Alternatively, the branched PEG could be a star PEG, having from 10 to 100 PEG chains emanating from a central core group. Alternatively, the PEG may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0116] The one or more PEGylated lipids employed may for instance comprise a PEG-phospholipid of the following formula (II)

[0117] O

[0118] 1

[0119] R— C11

[0120] — (k

[0121] 9

[0122] R— C— O —

[0123] II o

[0124] o L iis

[0125] ^O— P— O-R

[0126]

[0127] °’ (II) wherein R1and R2are as defined above for the glycerophospholipids of formula (I), and R5is a group which comprises polyethylene glycol).

[0128] The group which comprises polyethylene glycol) may for instance have the formula -CH2CH2NHC(O)-X, or for instance -CH2CH2NHC(O)(CH2)3C(O)-X wherein X comprises said poly(ethylene glycol). The group X may for instance comprise substantially linear PEG, or for instance a branched PEG, having, for instance, from three to ten PEG chains emanating from a central core group. Alternatively, it can be a star PEG, having, for instance, from 10 to 100 PEG chains emanating from a central core group. Or for instance it may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0129] Thus, R5may for instance be -CFECFENFIC^XOCFECFEjqOCFE, -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOCH3, -CH2CH2NHC(O)-(OCH2CH2)qOH, or -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOH, wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0130] Alternatively, R5may be -(CFECFEOjqCFE or -(CFECFEOjqH, wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.Additionally or alternatively, the one or more PEGylated lipids may comprise a diacylglycerol-PEG of formula (III)

[0131] O

[0132] 1

[0133] R— C11

[0134] — O

[0135] 7

[0136] R— C— O—

[0137] II

[0138] ° ^OUR

[0139]

[0140] K6(III)

[0141] wherein R1and R2are as defined above for the glycerophospholipids of formula (I), and R6is a group which comprises polyethylene glycol).

[0142] The polyethylene glycol) may for instance comprise substantially linear PEG, or for instance a branched PEG, having, for instance, from three to ten PEG chains emanating from a central core group. Alternatively, it can be a star PEG, having, for instance, from 10 to 100 PEG chains emanating from a central core group. Or for instance it may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0143] R6may for instance be -(CEECEEO^CHs, -(CEECEEO^H,

[0144] -CH2CH2NHC(O)-(OCH2CH2)qOCH3, -CH2CH2NHC(O)-(OCH2CH2)qOH, -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOCH3 or

[0145] -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOH wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0146] Additionally or alternatively, the one or more PEGylated lipids may comprise a cholesterol-PEG derivative of formula (IV)

[0147]

[0148] wherein R7is a group which comprises polyethylene glycol).

[0149] Again, the poly(ethylene glycol) may comprise substantially linear PEG, or for instance a branched PEG, having, for instance, from three to ten PEG chains emanating from a central core group. Alternatively, it can be a star PEG, having, for instance, from 10 to 100 PEG chains emanating from a central core group. Or for instance it may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0150] R7may for instance be -(OCEECIDqOH or -(OCEECIDqOCEE wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0151] Polyglycerine may be used instead of polyethylene glycol).The amphipathic molecules may alternatively be polymeric amphipathic molecules. For instance, the amphipathic molecules may comprise a block copolymer, for instance a triblock copolymer. Suitable block copolymers and triblock copolymers are described in WO 2014 / 064444, and include the triblock copolymer PMOXA-PDMS-PMOXA.

[0152] With regard to the size of each of the droplets in the synthetic droplet assembly (in terms of droplet diameter or droplet volume), a variety of droplet sizes may be produced by the droplet printing processes known in the art and disclosed in, for instance, WO 2014 / 087175 Al, WO 2021 / 234394 Al, G. Villar et al. Science 2013, 340, 48, and I. Cazimoglu et al, ACS Nano 2021, 15, 20214. As discussed in WO 2014 / 087175 Al, for instance, droplet size may be tuned by varying the amplitude and duration of voltage pulses applied to a piezoelectric component of a droplet generator of the droplet printer. Similarly, the dimensions of the outlets (nozzles or capillaries) of the droplet generators of the printer may be varied to obtain desired droplet sizes.

[0153] In addition, the inventors have now provided access to droplet assemblies in which droplet sizes are smaller than previously obtainable by 3D droplet printing alone. The Example hereinbelow and Figure 4(d) describe a heat-induced post-printing shrinking process, in which droplet size was reduced post-printing, by release of water from the droplets through evaporation. Thus, a reduction of droplet size post-printing was achieved without adversely affecting the general morphology of the droplet networks. Accordingly, a wide range of droplet sizes are possible in the synthetic droplet assembly which comprises n droplets, either by tuning parameters of a printing process by which the assembly is initially produced, and / or, for instance, by performing a postprinting shrinking process as described herein.

[0154] Thus, typically, each of the n droplets in the synthetic droplet assembly has a diameter of less than or equal to 1 mm. Often, each of the n droplets in the synthetic droplet assembly has a diameter of less than or equal to 500 pm, for instance less than or equal to 200 pm. Typically, for instance, each of the n droplets in the synthetic droplet assembly has a diameter of less than or equal to 150 pm, or less than or equal to 100 pm, for instance less than or equal to 60 pm.

[0155] If the synthetic droplet assembly has not been subjected to a post-printing shrinking process, e.g. of the kind described in the Example hereinbelow, then typically each of the n droplets in the synthetic droplet assembly has a diameter which is equal to or greater than 10 pm. Thus, often, each of the n droplets in the synthetic droplet assembly has a diameter of from 10 pm to 1 mm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of 20 pm to 500 pm, for instance from 30 pm to 200 pm. Often, for instance, each of the n droplets in the synthetic droplet assembly has a diameter of from 50 pm to 150 pm, or for instance from 60 pm to 120 pm.

[0156] However, as mentioned above, significantly smaller droplet diameters are possible via a post-printing shrinking process, e.g. of the kind described in the Example hereinbelow, and thus it is possible, for instance, that each of the n droplets in the synthetic droplet assembly may have adiameter which is equal to or greater than 50 rim. Thus, broad ranges for droplet sizes are available and so it may be the case that each of the n droplets in the synthetic droplet assembly has a diameter of from 50 nm to 1 mm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of 50 nm to 500 pm, for instance from 100 nm to 200 pm. Often, for instance, each of the n droplets in the synthetic droplet assembly has a diameter of from 500 nm to 150 pm, or for instance from 1 pm to 120 pm.

[0157] It is also possible therefore that the droplet sizes in the droplet assembly may be particularly small and, therefore, that each of the n droplets in the synthetic droplet assembly may have a diameter of less than 10 pm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm, or less than or equal to 3 pm, for example less than or equal to 2 pm. Thus, each of the n droplets in the synthetic droplet assembly may have a diameter of from 50 nm to less than 10 pm, for instance from 50 nm to 9 pm, or from 50 nm to 8 pm. Often, each of the n droplets in the synthetic droplet assembly has a diameter of from 50 nm to 6 pm, for instance from 70 nm to 5 pm, or for example from 100 nm to 3 pm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm.

[0158] Similarly, in terms of droplet volume, each of the n droplets in the synthetic droplet assembly typically has a volume of less than or equal to 0.5 pL. Often, each of the n droplets in the synthetic droplet assembly has a volume of less than or equal to 65 nL, for instance less than or equal to 4 nL. Typically, for instance, each of the n droplets in the synthetic droplet assembly has a volume of less than or equal to 2 nL, or less than or equal to 0.5 nL, for instance less than or equal to 113 pL.

[0159] If the synthetic droplet assembly has not been subjected to a post-printing shrinking process, e.g. of the kind described in the Example hereinbelow, then typically each of the n droplets in the synthetic droplet assembly has a volume which is equal to or greater than 0.5 pL. Thus, often, each of the n droplets in the synthetic droplet assembly has a volume of from 0.5 pL to 0.5 pL. For instance, each of the n droplets in the synthetic droplet assembly may have a volume of 4 pL to 65 nL, for instance from 14 pL to 4 nL. Often, for instance, each of the n droplets in the synthetic droplet assembly has a volume of from 65 pL to 1767 pL, or for instance from 113 pL to 904 pL.

[0160] However, as mentioned above, significantly smaller droplet volumes are possible via a post-printing shrinking process, e.g. of the kind described in the Example hereinbelow, and thus it is possible, for instance, that each of the n droplets in the synthetic droplet assembly may have a volume which is as low as 65 zL (zeptolitres), i.e. equal to or greater than 65 zL. Thus, broad ranges for droplet sizes are available and so it may be the case that each of the n droplets in the synthetic droplet assembly has a volume of from 65 zL to 0.5 pL. For instance, each of the ndroplets in the synthetic droplet assembly may have a volume of 65 zL to 65 nL, for instance from 524 zL to 4 nL. Often, for instance, each of the n droplets in the synthetic droplet assembly has a volume of from 65 aL (attolitres) to 1767 pL, or for instance from 524 aL to 904 pL.

[0161] It is also possible therefore that the droplet sizes in the droplet assembly may be particularly small and, therefore, that each of the n droplets in the synthetic droplet assembly may have a volume of less than 0.5 pL. For instance, each of the n droplets in the synthetic droplet assembly may have a volume of less than or equal to 382 fL (femtolitres), less than or equal to 268 fL, or for instance less than or equal to 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL. Thus, each of the n droplets in the synthetic droplet assembly may have a volume of from 65 zL to less than 0.5 pL, for instance from 65 zL to 382 fL, or from 65 zL to 268 fL. Often, each of the n droplets in the synthetic droplet assembly has a volume of from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL. For instance, each of the n droplets in the synthetic droplet assembly may have a volume of from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0162] The synthetic droplet assembly of the system of the invention comprises one or more target interface droplets, each of which, in addition to contacting at least one of the other n droplets in the droplet assembly, contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region.

[0163] Thus, at least one of the n droplets of the synthetic droplet assembly is a target interface droplet, wherein each target interface droplet additionally contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region. The number of said target interface droplets, m, is from 1 to n.

[0164] A purpose of the target interface droplets is to provide a region of contact (one or more regions of contact) between the synthetic droplet assembly and the target region which comprises the biological cells or tissue, so that the effector molecule may pass from the synthetic droplet assembly into the target region.

[0165] Although it is possible for there to be only one target interface droplet in the synthetic droplet assembly, in which case the region of contact between the synthetic droplet assembly and the target region is very small, it is typically the case that the synthetic droplet assembly comprises multiple target interface droplets, to provide a larger region of contact. When the synthetic droplet assembly comprises multiple target interface droplets, these are often grouped together in a row or, more typically, a layer, or they may be grouped together in two or more different rows or layers.

[0166] Thus, often, some of, if not all of, the target interface droplets together form one or more rows of droplets, a layer of droplets, or more than one layer of droplets. The or each row, and the or each layer, of target interface droplets will (by definition) be situated at an outer surface of the synthetic droplet assembly where it interfaces with the target region. The or each row may or maynot be straight (linear), for instance a row may be curved. Similarly, the or each layer may or may not be flat. For instance, a layer may be curved, or undulating.

[0167] It is often the case that some of, if not all of, the target interface droplets together form a layer of droplets. Thus, often, the target interface droplets together form a layer of droplets which is a target interface droplet layer, wherein each target interface droplet in said target interface droplet layer contacts at least one other target interface droplet in the target interface droplet layer to form a layer of said amphipathic molecules as an interface between the contacting target interface droplets. The layer may for instance be flat or curved.

[0168] As will be apparent from the above discussion and the Examples herein, the synthetic droplet assembly typically comprises a plurality of target interface droplets, often multiple target interface droplets. Thus, the number of target interface droplets, m, is often at least two, more typically at least three, and even more typically at least four.

[0169] For instance, the number of target interface droplets, m, may be at least 10, for instance at least 16, at least 25, or for instance at least 64. Often, the number of target interface droplets is at least 100, for instance at least 150. For instance, the number of target interface droplets may be at least 500, or, for instance, at least 1,000. Often, in these cases, the target interface droplets together form a layer of droplets, as discussed above, which may for instance be a flat layer or a curved layer.

[0170] The target interface droplets may for instance together form a first layer of droplets and a second layer of droplets, wherein often the first and second layers are not in contact with each other (e.g. because they are located at different parts of the surface of the synthetic droplet assembly). The target interface droplets may for example form more than two different layers of droplets. Each layer of target interface droplets may be located at a different part of the surface of the synthetic droplet assembly so that it is not in contact with any of the other layers of target interface droplets.

[0171] If the synthetic droplet assembly itself is a single row of droplets or a single layer of droplets, then all n of the droplets can be target interface droplets (in which case m is equal to n) because they may all be in contact with the target region. This can also be the case if the synthetic droplet assembly consists of two layers of droplets, one on top of the other, and if the target region completely surrounds the synthetic droplet assembly so that the target region is in contact with all of the droplets in the two layers. More typically, however, one or more of the droplets in the synthetic droplet assembly is not a target interface droplet, either because it is not at any surface of the droplet assembly, but is instead surrounded by other droplets of the synthetic droplet assembly, or because it is at a surface of the droplet assembly but that particular surface is not in contact with the target region (that surface may instead, for instance, be in contact with a hydrophobic medium).

[0172] Thus, often, the number of target interface droplets, m, is less than n. More typically, the number of target interface droplets, m, is equal to or greater than 2 and less than n.For instance, the number of target interface droplets, m, may be from 2 to n-1. The integer m may for instance be from 2 to n-5, or for instance from 5 to n-8, or from 10 to n-10, or from 10 to n-50 (provided of course that m is always a positive integer).

[0173] Particularly in cases where n is relatively large, e.g. equal to or greater than 10, or more typically, for instance, equal to or greater than 20, equal to or greater than 30, equal to or greater than 50, equal to or greater than 100, equal to or greater than 200, equal to or greater than 500, or equal to or greater than 1,000 (and in these cases n may be as further defined in terms of any of the ranges for n disclosed herein) then it is often the case that the number of target interface droplets, m, is defined as follows:

[0174] The integer m may be at least 2 and less than or equal to three quarters of n. For instance, m may be at least 10 and less than or equal to three quarters of n. Often, for instance, m is at least 50 and less than or equal to three quarters of n, or m is at least 100 and less than or equal to three quarters of n.

[0175] The integer m may for instance be at least 2 and less than or equal to half of n. For instance, m may be at least 10 and less than or equal to half of n. Often, for instance, m is at least 50 and less than or equal to half of n, or at least 100 and less than or equal to half of n.

[0176] The integer m may for instance be at least 2 and less than or equal to a quarter of n. For instance, m may be at least 10 and less than or equal to a quarter of n. Often, for instance, m is at least 50 and less than or equal to a quarter of n, or at least 100 and less than or equal to a quarter of n.

[0177] The integer m may for instance be at least 2 and less than or equal to the square root of n. For instance, m may be at least 10 and less than or equal to the square root of n. Often, for instance, m is at least 50 and less than or equal to the square root of n, or at least 100 and less than or equal to the square root of n.

[0178] The integer m may for instance be at least 2 and less than or equal to the cube root of n. For instance, m may be at least 10 and less than or equal to the cube root of n. Often, for instance, m is at least 50 and less than or equal to the cube root of n, or at least 100 and less than or equal to the cube root of n.

[0179] The integer m may for instance be from 2 to 1,000,000, for instance from 5 to 100,000 or, for example from 10 to 10,000. Often, for instance, m is from 20 to 1,000, for instance from 50 to 500.

[0180] Each target interface droplet contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region. The target region is typically an aqueous region, in that it typically comprises said biological cells or tissue in an aqueous environment. An aqueous environment may be provided because the target region is the biological cells or tissue, and the biological cells or tissue are themselves aqueous in nature. Alternatively, the target region may comprise the biological cells or tissue and an aqueous medium;the biological cells or tissue may for instance be disposed within a hydrogel or an aqueous cell or tissue growth medium. Either way, the target region is an aqueous region. Thus, in the same way as the amphipathic molecules of the aqueous droplets of the synthetic droplet assembly form a layer of the amphipathic molecules as an interface between contacting droplets, the amphipathic molecules of the target interface droplets additionally form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region.

[0181] When they are situated between two aqueous regions, the amphipathic molecules employed in the present invention typically form a bilayer. Thus, often, each layer of amphipathic molecules which is an interface between a target interface droplet and the target region is a bilayer of said amphipathic molecules. In other words, typically, in the system of the invention, each of the target interface droplets (additionally) contacts the target region to form a bilayer of the amphipathic molecules as an interface between the target interface droplet and the target region.

[0182] The synthetic droplet assembly of the system of the invention comprises one or more effector droplets, each of which, in addition to comprising (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, further comprises (iii) an effector molecule or means for producing the effector molecule. The number of said effector droplets in the synthetic droplet assembly, p, is from 1 to n. Thus, at least one of the n droplets of the synthetic droplet assembly is an effector droplet.

[0183] A purpose of each effector droplet may be to house, or to be able to produce, an effector molecule therein. Then, in use, the effector molecule may pass from the effector droplet in question to the target region, either directly (if the effector droplet is also a target interface droplet, for instance a target interface outlet droplet) or via one or more other droplets (if the effector droplet is not also a target interface droplet, for instance if it is not a target interface outlet droplet).

[0184] The effector molecule and / or the means for producing the effector molecule are typically disposed in the aqueous droplet medium of the effector droplet. The effector molecule and / or means for producing the effector molecule are often, for instance, suspended in, or dissolved in, the aqueous droplet medium of the effector droplet. Often, the effector molecule is dissolved in the aqueous medium of the effector droplet.

[0185] An effector droplet may comprise more than one type of effector molecule (i.e. more than one compound which acts an effector), and / or the means for producing more than one type of effector molecule. For instance, an effector droplet may, in addition to said effector molecule (which may be referred to as a “first effector molecule”), further comprise one or more additional effector molecules, each of which is a different compound from the first effector molecule (and from each other). Similarly, an effector droplet may comprise the means for producing one or more additional effector molecules. The one or more additional effector molecules may be referred to as a second effector molecule, and a third effector molecule, and so-on, respectively.Not all of the effector droplets in the synthetic droplet assembly need comprise the same effector molecule. Rather, an effector molecule in one of the effector droplets may be a different compound from the effector molecule in another of the effector droplets in the droplet assembly. Usually, however, the effector molecule of each of the effector droplets in any given reservoir region of the droplet assembly is the same compound. More typically, the effector molecule of each of the effector droplets in the droplet assembly is the same compound.

[0186] As mentioned above, each effector droplet further comprises an effector molecule or a means for producing the effector molecule.

[0187] Usually, each effector droplet comprises an effector molecule. Thus, typically, each effector droplet comprises said effector molecule. Typically, in each effector droplet, the effector molecule is disposed in the aqueous droplet medium of the droplet.

[0188] Usually, when present, the concentration of the effector molecule (for instance its concentration in the aqueous droplet medium of the effector droplet) is equal to or greater than 0.001 mM, for instance, equal to or greater than 0.01 mM. For instance, the concentration of the effector molecule may be equal to or greater than 0.1 mM, for instance, equal to or greater than 0.5 mM. Often, the concentration of effector molecule is equal to or greater than 1 mM, for instance, equal to or greater than 2 mM. For instance, the concentration of the effector molecule may be equal to or greater than 5 mM, for instance, equal to or greater than 10 mM, or equal to or greater than 15 mM. The concentration of the effector molecule may be equal to or greater than 20 mM, for instance, equal to or greater than 25 mM, or equal to or greater than 30 mM.

[0189] The concentration of the effector molecule may, for instance, be from 0.001 mM to 1,000 mM. Typically, the concentration of the effector molecule is from 0.01 mM to 800 mM, for instance from 0.1 mM to 600 mM. More typically, the concentration of the effector molecule is from 0.5 mM to 400 mM, for instance, from 1 mM to 300 mM. In some embodiments, the concentration of the effector molecule is from about 2 mM to about 200 mM, or from about 5 mM to about 100 mM, or about 10 mM to about 80 mM. It may for instance be from 15 mM to 60 mM, or from 20 mM to 50 mM, for instance from 25 mM to 40 mM, or from 30 mM to 35 mM, for instance about 33 mM.

[0190] Such concentrations are particularly suitable for the effector molecule arabinose, especially when employed in combination with the aHU pore in the effector droplet, at any of the concentrations for the protein pore specified herein.

[0191] The concentration of the effector molecule (for instance its concentration in the aqueous droplet medium of the effector droplet) may be equal to or greater than 0.001 pM (1 nM), for instance, equal to or greater than 0.005 pM (5 nM). For instance, the concentration of the effector molecule may be equal to or greater than 0.01 pM (10 nM), for instance, equal to or greater than 1 nM. Often, the concentration of effector molecule is equal to or greater than 5 nM, for instance, equal to or greater than 10 nM. For instance, the concentration of the effector molecule may beequal to or greater than 10 qM, for instance, equal to or greater than 100 qM. or equal to or greater than 200 qM, or equal to or greater than 500 qM.

[0192] The concentration of the effector molecule may, for instance, be from 0.001 qM (1 nM) to 1,000 mM. The concentration of the effector molecule may be from 0.005 qM (5 nM) to 50 mM, or for instance from 0.01 qM (10 nM) to 5 mM. Often, the concentration of the effector molecule is from 1 nM to 1,000 nM, for instance, from 5 nM to 500 nM, or from 10 nM to 100 nM. In some embodiments, the concentration of the effector molecule is from about 10 qM to about 100 mM, or from about 100 qM to about 50 mM, or about 200 qM to about 10 mM. It may for instance be from about 500 qM to about 5 mM.

[0193] Such concentrations are often suitable for small molecule drug effector molecules such as, for instance, isoprenaline or doxorubicin.

[0194] Alternatively, however, each effector droplet may comprise means for producing an effector molecule. When each effector droplet comprises said means for producing an effector molecule, the effector droplet may or may not also comprise the effector molecule itself (this may depend on whether or not the effector molecule has yet been produced in the droplet by the means for producing the effector molecule).

[0195] Often, one or more of the effector droplets, for instance each of the effector droplets, in the synthetic droplet assembly comprises said means for producing the effector molecule. For instance, one or more of the effector droplets, for example each of the effector droplets, may comprise (i) means for producing the effector molecule and (ii) said effector molecule.

[0196] The effector molecule may be natural or synthetic. The effector molecule may be of any size. For instance, it may be a small molecule, an oligomer, or a macromolecule, such as for instance a natural or synthetic polymer. It may for instance be a small molecule drug, a peptide or protein, or a polynucleotide such as DNA or RNA.

[0197] The effector molecule may be any molecule that is desired to be delivered to the biological cells or tissue of the target region, or for instance any molecule that has any kind of effect on the biological cells or tissue.

[0198] The effector molecule may for instance have a beneficial effect on the cells or tissue, for instance it may be a nutrient or other molecule that promotes cell or tissue growth, or it may be a pharmaceutical that treats a disease in the cells or tissue.

[0199] Alternatively, the effector molecule may have a detrimental effect on the target cells or tissue, for example it may cause cell death or tissue necrosis, or it may prevent or retard cell proliferation. This may be useful, for example, in cases where the target cells or tissue are diseased cells or tissue, for instance cancer cells or tumour tissue, and destruction of those cells may be an objective.

[0200] Often the effector molecule is a molecule that is suitable for modulating a biochemical process in said biological cells or tissue. Said biochemical process may for instance be theexpression of a gene. Thus, the effector molecule may be a molecule capable of inducing protein expression in said biological cells or tissue. The protein expression may be protein expression in eukaryotic cells or tissue, or bacterial protein expression. For instance, the target region may comprise biological cells which are bacteria, and the effector molecule may be a molecule capable of inducing protein expression in said bacteria. Once possibility, as described in the Example herein, is that the effector molecule may be L-(+)-arabinose, which is capable of inducing expression of the mCherry gene in the described bacteria. Thus, the effector molecule may for instance be L-(+)-arabinose.

[0201] Other possibilities are that the effector molecule may be a pharmaceutical, or, for instance, a signalling molecule.

[0202] The effector molecule is often a small molecule pharmaceutical, for instance doxorubicin or isoprenaline. The effector molecule may be a molecule suitable for modulating the frequency of contraction of myocytes, for instance cardiomyocytes. The effector molecule may be suitable for modulating the frequency of contractions in muscle tissue, for instance in heart muscle. The effector molecule may for instance be a drug suitable for modulating a heartbeat in vivo. The effector molecule may for instance be a drug for treating bradycardia. The effector molecule may be isoprenaline. The effector molecule may be doxorubicin. The effector molecule may be a chemotherapy medication. The effector molecule may be a cancer medication. The effector molecule may be an anticancer agent, for instance an antiproliferative drug.

[0203] The effector molecule may be suitable for triggering a signalling response in cells. The effector molecule may be suitable for causing a change in intracellular calcium levels. The effector molecule may for instance be a molecule or reagent suitable for causing a change in pH. The effector molecule may for instance be an acid or a base. The effector molecule may for instance be an acid, such as, for instance, an inorganic acid, such as a hydrohalic acid, or alternatively an organic acid. The effector molecule may for instance be hydrochloric acid (HC1).

[0204] As discussed above, the effector molecule may be of any size. In some embodiments, however, the effector molecule has a molecular weight of equal to or less than 2,000 Da. An advantage of an effector molecule having a molecular weight of equal to or less than 2,000 Da is that it may pass through an a -hemolysin (aHL) protein pore. Such a membrane protein (protein pore) may be present in a layer of the amphipathic molecules which is an interface between contacting droplets in the droplet assembly, or in a layer of the amphipathic molecules which is an interface between a target interface droplet and the target region. Such a protein pore may thereby allow passage of the effector molecule from one droplet to another (adjacent) droplet in the synthetic droplet assembly, or from a target interface droplet (a target interface outlet droplet) of the droplet assembly to the target region. Other protein pores may be employed instead of aHL if the effector molecule is larger, for instance if it has a molecular weight greater than 2,000 Da. Indeed, whereas the narrowest inner diameter of aHL is 1.4 nm, other membrane protein poressuch as perfringolysin O have larger internal diameters (between 25 nm and 30 nm in the case of perfringolysin O) that facilitate passage of larger molecules.

[0205] As discussed above, each effector droplet may comprise means for producing the effector molecule. Such means may be a molecule which is a precursor to the effector molecule, that can easily be converted into the effector molecule by chemical or biochemical reaction. Alternatively, the means may be two or more molecules that can be reacted together, for instance coupled together, to produce the effector molecule. Thus, the means for producing the effector molecule may comprise one or more precursors to the effector molecule. The one or more precursors to the effector molecule comprise one or more reactants capable of conversion into the effector molecule.

[0206] Alternatively, the means for producing the effector molecule may comprise one or more molecules which enable production of the effector molecule. The one or more molecules that enable production of the effector molecule may for example be molecular machinery that enables production of the effector molecule by cell-free protein expression, for instance an in vitro transcription-translation system. Alternatively, they may be a catalyst that catalyses the production of the effector molecule. The catalyst may for instance be a small molecule or an enzyme.

[0207] The means for producing the effector molecule may comprise any combination of the above; for instance, the means may comprise one or more precursors to the effector molecule and a catalyst for catalysing a reaction which converts said one or more precursors into the effector molecule.

[0208] Thus, the means for producing the effector molecule may comprise one or more precursors to the effector molecule and / or one or more molecules which enable production of the effector molecule. The one or more precursors to the effector molecule may for instance comprise one or more reactants capable of conversion into the effector molecule. The one or more molecules which enable production of the effector molecule comprise a catalyst, an enzyme, or an in vitro transcription-translation system, for producing the effector molecule.

[0209] One or more of the effector droplets, for instance each of the effector droplets, may comprise both an effector molecule and means for producing the effector molecule. Often, each effector droplet comprises an effector molecule.

[0210] The number of said effector droplets in the synthetic droplet assembly, p, is from 1 to n. A purpose of the effector droplets is to house the effector molecule therein, and / or to be able to produce the effector molecule therein, so that the effector molecule can be delivered to the target region.

[0211] Thus, although it is possible for there to be only one effector droplet in the synthetic droplet assembly, in which case the droplet assembly may only contain a relatively small amount of the effector molecule, it is typically the case that the synthetic droplet assembly comprises multiple effector droplets, to provide more of the effector molecule for delivery to the target region. Whenthe synthetic droplet assembly comprises multiple effector droplets, these are often grouped together in one or more rows of effector droplets or in one or more layers of effector droplets.

[0212] Thus, often, some of, if not all of, the effector droplets together form one or more rows of effector droplets, a layer of effector droplets, or more than one layer of effector droplets. The or each row may or may not be straight (linear), for instance a row may be curved. Similarly, the or each layer may or may not be flat. For instance, a layer may be curved, or undulating. It is often the case that some of, if not all of, the effector droplets together form a plurality of layers of effector droplets on top of each other, for instance a cube or cuboid of effector droplets. In this way a large reservoir or source of the effector molecule may be provided in the synthetic droplet assembly. Each effector droplet in each row, layer or plurality of layers (e.g. cube or cuboid) of effector droplets contacts at least one other effector droplet in the same row, layer or plurality of layers (e.g. cube or cuboid), to form a layer of said amphipathic molecules as an interface between the contacting effector droplets.

[0213] As will be apparent from the above discussion and the Examples herein, the synthetic droplet assembly typically comprises a plurality of effector droplets, often multiple effector droplets. Thus, the number of effector droplets, p, is often from two to n.

[0214] Thus, the number of effector droplets, p, is often at least two, more typically at least three, and even more typically at least four.

[0215] For instance, the number of effector droplets, p, may be at least 10, for instance at least 16, at least 25, or for instance at least 64. Often, the number of effector droplets is at least 100, for instance at least 150. For instance, the number of effector droplets, p, may be at least 500, or, for instance, at least 1,000, or at least 10,000. Often, in these cases, some or all of the effector droplets together form one or more rows of effector droplets, a layer of effector droplets, or a plurality of layers of effector droplets, as discussed above.

[0216] It is possible for all of the droplets in the synthetic droplet assembly, including each of the target interface droplets, to be effector droplets. In this case, p is equal to n. Usually however, not all of the droplets in the synthetic droplet assembly are effector droplets.

[0217] Thus, often, the number of effector droplets, p, is less than n. More typically, the number of effector droplets, p, is often equal to or greater than 2 and less than n. For instance, the number of effector droplets, p, may be from 2 to n-1. The integer p may for instance be from 5 to n-5, or for instance from 10 to n-8, or from 20 to n-10, or from 100 to n-50 (provided of course that p is always a positive integer).

[0218] Particularly in cases where n is relatively large, e.g. equal to or greater than 10, or more typically, for instance, equal to or greater than 20, equal to or greater than 30, equal to or greater than 50, equal to or greater than 100, equal to or greater than 200, equal to or greater than 500, or equal to or greater than 1,000, or equal to or greater than 10,000 (and in these cases n may be asfurther defined in terms of any of the ranges for n disclosed herein) then it is often the case that the number of effector droplets, p, is defined as follows:

[0219] The integer p may be at least 2 and less than or equal to n. For instance, p may be at least 10 and less than or equal to n. Often, for instance, p is at least 50 and less than or equal to n, or p is at least 100 and less than or equal to n.

[0220] The integer p may for instance be at least 2 and less than or equal to 90% of n. For instance, p may be at least 10 and less than or equal to 90% of n. Often, for instance, p is at least 50 and less than or equal to 90% of n, or p is at least 100 and less than or equal to 90% of n.

[0221] The integer p may be at least 2 and less than or equal to three quarters of n. For instance, p may be at least 10 and less than or equal to three quarters of n. Often, for instance, p is at least 50 and less than or equal to three quarters of n, or p is at least 100 and less than or equal to three quarters of n.

[0222] The integer p may for instance be greater than or equal to a quarter of n and less than or equal to n. For instance, p may be greater than or equal to a quarter of n and less than or equal to 90% of n. Often, for instance, p is greater than or equal to a quarter of n and less than or equal to three quarters of n.

[0223] The integer p may for instance be from 2 to 1,000,000, for instance from 5 to 100,000 or, for example from 10 to 10,000. Often, for instance, p is from 20 to 5,000, for instance from 100 to 2,000, or from 200 to 1,500, or for instance from 400 to 1,200.

[0224] Often, at least one, and typically two or more, of the target interface droplets are also effector droplets. In some embodiments all of the target interface droplets are also effector droplets. Often however, fewer than all of the target interface droplets are also effector droplets, for instance often less than three quarters of the target interface droplets are also effector droplets. Often, for instance, less than half of the target interface droplets are also effector droplets. For instance, typically less than a quarter of the target interface droplets are also effector droplets. In other embodiments, none of the target interface droplets is also an effector droplet. This may be because no amount of the effector molecule has yet passed from the one or more effector droplets to any of the target interface droplets and because none of the target interface droplets has means for producing the effector molecule.

[0225] Each target interface droplet which is also an effector droplet typically further comprises a protein pore, for allowing passage of the effector molecule from the droplet to the target region. Typically, each layer of amphipathic molecules which is an interface between (i) a target interface droplet which is an effector droplet and (ii) the target region, further comprises a protein pore. The protein pore may be as further defined herein, for instance it may be a-hemolysin (aHL) or a different pore, for instance a protein pore with a larger internal diameter such as perfringolysin O.

[0226] The target region comprises biological cells or tissue.The target region may comprise biological cells. The target region may consist of biological cells. The biological cells may be living cells. The biological cells may comprise eukaryotic cells. The eukaryotic cells may be cells taken from a living organism, for instance a mammal, such as for example a human. The biological cells may be mammalian cells, for instance human cells. The biological cells may for instance comprise nerve cells, for instance astrocytes. The biological cells may for instance comprise muscle cells. The biological cells may for instance comprise myocytes, such as cardiomyocytes. The biological cells may for instance comprise a mammalian cell line, for instance a human cell line. The biological cells may for instance comprise cancer cells, for instance ovarian cancer cells. The biological cells may comprise prokaryotic cells, for instance bacteria, for example Escherichia coli.

[0227] The target region may comprise tissue, i.e. biological tissue. The target region may consist of tissue. The tissue may be a tissue sample, for example a biopsy. The tissue may be living tissue. The living tissue may be part of a living organism, for instance a mammal, such as for example a human. Thus, the synthetic droplet assembly may be attached to, or implanted within, said living tissue. The living tissue may comprise healthy tissue, and / or it may comprise diseased tissue. The living tissue may for instance comprise cells or tissue to be treated with the effector molecule, e.g. in order to rid the cells or tissue of a disease. The living tissue may alternatively comprise diseased cells or tissue, for instance tumour cells, to be killed or destroyed by the effector molecule.

[0228] Thus, in the system of the invention the target region may be living tissue. The target region may comprise cells to undergo therapeutic treatment by exposure to the effector molecule. The target region may comprise cells to be killed by the effector molecule, for instance tumour cells.

[0229] The tissue in the target region may for instance comprise nerve tissue, for instance tissue comprising astrocytes. The tissue may for instance comprise muscle tissue, for instance cardiac muscle tissue. The tissue may for instance comprise myocytes, such as cardiomyocytes. The tissue may for instance comprise cancerous tissue, for instance tumour cells. The tissue may comprise cancer cells, for instance ovarian cancer cells.

[0230] As discussed above the target region is generally aqueous in nature, because the biological cells or tissue are themselves aqueous in their nature and / or because the target region further comprises an aqueous medium, e.g. a bulk aqueous medium, in addition to said biological cells or tissue. Such a bulk aqueous medium may, for instance, be a hydrogel. When the target region does further comprise an aqueous medium, the biological cells or tissue are generally in contact with the aqueous medium. For instance, the biological cells or tissue may be disposed in the aqueous medium, or the aqueous medium may be disposed between the biological cells or tissue and the synthetic droplet assembly. In these cases, each of the target interface droplets may contact the aqueous medium of the target region and each layer of the amphipathic molecules which is an interface between a target interface droplet and the target region may be an interface between thetarget interface droplet and the aqueous medium of the target region. Often, each layer of the amphipathic molecules which is an interface between a target interface droplet and the aqueous medium of the target region is a bilayer of the amphipathic molecules.

[0231] The aqueous medium may be a hydrogel. The biological cells or tissue may be in contact with the hydrogel. For instance, the biological cells or tissue may be disposed within the hydrogel, or the hydrogel may be disposed between the droplet assembly and the biological cells or tissue. In these cases, each of the target interface droplets may contact the hydrogel of the target region and each layer of the amphipathic molecules which is an interface between a target interface droplet and the target region may be an interface between the target interface droplet and the hydrogel of the target region. Often, each layer of the amphipathic molecules which is an interface between a target interface droplet and the hydrogel of the target region is a bilayer of the amphipathic molecules.

[0232] Thus, often, the target region comprises a volume of a hydrogel, and the biological cells or tissue are in contact with the hydrogel. Typically, the biological cells or tissue are disposed within the volume of hydrogel. Additionally, or alternatively, the volume of hydrogel may be disposed between the droplet assembly and the biological cells or tissue. Typically, each of the target interface droplets contacts the hydrogel. Usually, each layer of the amphipathic molecules which is an interface between a target interface droplet and the target region is an interface between the target interface droplet and the hydrogel. Often, each layer of the amphipathic molecules which is an interface between a target interface droplet and the hydrogel is a bilayer of the amphipathic molecules.

[0233] In cases where the target region comprises said biological cells or tissue but does not comprise a hydrogel (and for instance does not comprise any other kind of bulk aqueous medium), then each of the target interface droplets may contact the biological cells or tissue directly and each layer of the amphipathic molecules which is an interface between a target interface droplet and the target region may be an interface between the target interface droplet and the biological cells or tissue. Each layer of the amphipathic molecules which is an interface between a target interface droplet and the biological cells or tissue of the target region may be a bilayer of the amphipathic molecules.

[0234] For instance, the target region may comprise, or consist of, tissue (e.g. living tissue) and each layer of the amphipathic molecules which is an interface between a target interface droplet and the target region may be an interface between the target interface droplet and the tissue (e.g. living tissue). Each layer of the amphipathic molecules which is an interface between a target interface droplet and the tissue may be a bilayer of the amphipathic molecules.

[0235] It will be understood that the target region is generally significantly larger in size (e.g. in volume) than any individual droplet in the synthetic droplet assembly, especially given that the assembly often comprises multiple target interface droplets (as discussed above) in contact withone and the same target region. The target region is also often larger in size (e.g. in volume) than the synthetic droplet assembly. In particular, the target region often has a total volume which is equal to or greater than the total volume of the synthetic droplet assembly. Thus, for instance, the above-defined bulk aqueous medium of the target region may have a total volume which is equal to or greater than the total volume of the synthetic droplet assembly. Similarly, the above-defined volume of hydrogel may have a total volume which is equal to or greater than the total volume of the synthetic droplet assembly.

[0236] When the target region comprises a volume of a hydrogel, the hydrogel may be a hydrogel comprising agarose. Thus, the hydrogel may comprise agarose and water. The concentration of the agarose in said water may for instance be less than or equal to 10% w / v agarose. It is often, for instance, from 0.25 % to 5% w / v agarose. Hydrogels other than agarose may also be used. For instance the hydrogel may comprise methylcellulose, polyethylene glycol diacrylate, polyacrylamide, matrigel, hyaluronan, polyethylene oxide, poly AMPS (poly(2-acrylamido-2-methyl-1 -propanesulfonic acid)), polyvinylpyrrolidone, polyvinyl alcohol, sodium polyacrylate, acrylate polymers or poly(N-isopropylacrylamide). Alternatively, the hydrogel may comprise a silicone hydrogel or LB (Luria broth) agar.

[0237] The target region may comprise biological cells disposed in the volume of the hydrogel. The biological cells may comprise eukaryotic cells. The eukaryotic cells may be cells taken from a living organism, for instance a mammal, such as for example a human. The biological cells may comprise prokaryotic cells, for instance bacteria, for example Escherichia coli. The density of the biological cells in the volume of hydrogel is often, for instance, at least 1.0 x 106cells mL1, and is more typically at least 1.0 x 106cells mL1. The density of the biological cells may for instance be at least 1.5 x 107cells mL1.

[0238] The system of the invention may or may not further comprise a hydrophobic medium. The system of the invention often comprises a hydrophobic medium in embodiments where not all of the external surface of the synthetic droplet assembly is in contact with the target region. In those embodiments, a hydrophobic medium may be in contact with an external surface of the droplet assembly that is not in contact with the target region. The presence of the hydrophobic medium in such cases may have a stabilising effect on the synthetic droplet assembly. For instance, the outer layer of amphipathic molecules of each of the droplets of the droplet assembly that is at said external surface may form a stable monolayer at the interface between the droplet and the hydrophobic medium. In such a monolayer, polar head groups of the amphipathic molecules may be in contact with the aqueous droplet medium of the droplet and non-polar tail groups of the amphipathic molecules may be in contact with the hydrophobic medium.

[0239] In some embodiments, however, the synthetic droplet assembly is embedded or implanted within the target region. For instance, the synthetic droplet assembly may be embedded or implanted within living tissue of a living organism, e.g. for delivering a therapeutic to the tissue.Usually, in such cases, no external surface of the droplet assembly is exposed to the outside environment. Often, in such cases, all of the external surface of the synthetic droplet assembly is in contact with the target region. Therefore usually, in such cases, there is no need for a hydrophobic medium and thus the system of the invention often does not further comprise a hydrophobic medium.

[0240] Thus, typically, in the system of the invention, the synthetic droplet assembly is embedded or implanted within the target region. Usually, in such embodiments, the number of target interface droplets, m, is equal to or greater than two (and m may be as further defined herein) and the entire external surface of the droplet assembly is formed by the layers of the amphipathic molecules that are the interfaces between the m target interface droplets and the target region. Typically, in such cases, the system does not comprise a hydrophobic medium.

[0241] In other embodiments, however, the system of the invention further comprises a hydrophobic medium. Usually, in such embodiments, the hydrophobic medium contacts an external surface of the droplet assembly that is not in contact with the target region. Thus, often, the outer layer of amphipathic molecules of each of the droplets at said external surface contacts the hydrophobic medium. For instance, the hydrophobic medium may contact the whole of the external surface of the droplet assembly that is not in contact with the target region. Thus, it may be that every droplet which is at an external surface of the droplet assembly that is not a target interface droplet is in contact with a hydrophobic medium; the outer layer of amphipathic molecules of each of those droplets may be in contact with the hydrophobic medium.

[0242] The hydrophobic medium may be selected from a wide range of materials. It is often the same hydrophobic medium as a hydrophobic medium in which the droplet assembly was produced by 3D-droplet printing (e.g. as described in any of WO 2014 / 087175 Al, WO 2021 / 234394 Al, G. Villar et al. Science 2013, 340, 48, and I. Cazimoglu et al, ACS Nano 2021, 15, 20214). The hydrophobic medium can, for instance, be selected to affect the buoyancy of the droplets and the speed of formation of the layer of amphipathic molecules around the droplets after the droplets are first introduced into the hydrophobic medium during the droplet printing process.

[0243] The hydrophobic medium may comprise a single hydrophobic compound. Alternatively, it may comprise a mixture of two or more different hydrophobic compounds. The hydrophobic medium employed may, for instance, have a density close to that of water, for instance a density of less than or equal to about 1 g cm'3.

[0244] The hydrophobic medium is typically an oil. The oil may be a single, pure, compound, or the oil may comprise a mixture of two or more compounds. It is usually desirable that the oil does not significantly destabilize any layers of amphipathic molecules (e.g. bilayers of amphipathic molecules) formed.

[0245] The oil may for instance comprise silicone oil, for instance poly(methylphenylsiloxane). Silicone oil is advantageous on account of its density being close to that of water, which ensuresthat the droplet is approximately neutrally buoyant in water. The silicone oil may for instance be poly(methylphenylsiloxane), which has a density of about 1 g cm'3. The oil may consist of a single silicone oil, for instance poly(methylphenylsiloxane), such as AR20. Alternatively, the oil may comprise a mixture of two or more different silicone oils. Any suitable silicone oil may be used. For instance, the oil may comprise silicon oil DC200 (a polymer comprising monomer units of-O-Si(CHs)2-), poly(dimethylsiloxane) (PDMS), hydroxy terminated, or PDMS 200. Often, however, the silicone oil is a poly(methylphenylsiloxane), such as AR20.

[0246] Additionally, or alternatively, the oil may comprise a hydrocarbon. The hydrocarbon typically has from 5 to 40 carbon atoms (a C5-C40 hydrocarbon), more typically from 10 to 30 carbon atoms (a C10-C30 hydrocarbon). Typically, it is an alkane or an alkene. Thus, the hydrocarbon may be a C5-C30 alkane, or a C10-C20 alkane. In another embodiment, the hydrocarbon may be a C5-C20 alkene, or a C10-C20 alkene. The hydrocarbon is typically unsubstituted. In one embodiment it is undecane. In a preferred embodiment, the hydrocarbon is an unsubstituted C5-C20 alkane, preferably an unsubstituted C10-C20 alkane. The hydrocarbon may for instance be squalene, hexadecane, undecane or decane. However, in some embodiments the hydrocarbon may be substituted with a halogen atom. For example the oil may be a fluorocarbon, or a bromo-substituted C10-C30 alkane, or for instance a bromo-substituted C10-C20 alkane, e.g. bromododecane. When the oil comprises a hydrocarbon, it may comprise a single hydrocarbon compound, or a mixture of two or more hydrocarbons.

[0247] In some embodiments, the oil is a mixture comprising: (a) one or more hydrocarbons, and (b) one or more silicone oils. The one or more silicone oils may be as defined above, for instance they may comprise poly(methylphenylsiloxane) (for instance AR20). The one or more hydrocarbons may, for instance, comprise any suitable liquid hydrocarbon. Whether a particular hydrocarbon is liquid will depend upon the temperature of the hydrophobic medium. Thus, the term liquid hydrocarbon refers to a hydrocarbon that is a liquid at the temperature that the hydrophobic medium is at. Typically, the hydrophobic medium will be at room temperature. However, in some embodiments, the hydrophobic medium may be above or below room temperature. In some embodiments, the oil may comprise a solid. A solid hydrocarbon may, for instance, be used in combination with a silicone oil. The oil may, for instance, be a mixture of solids that dissolve to form a liquid. The hydrocarbon may be branched or unbranched, for example a hydrocarbon having from 5 to 40 carbon atoms, or from 5 to 30 carbon atoms (although hydrocarbons of lower molecular weight would require control of evaporation). Preferably, the hydrocarbon is a liquid at the operating temperature of the synthetic droplet assembly employed in the invention. Suitable examples include alkanes or alkenes, such as hexadecane, undecane, decane, pentane or squalene. Often, it is a liquid alkane hydrocarbon, for instance a C10-C20 alkane such as hexadecane, undecane, decane. Undecane may be preferred. Usually, the oil comprises a hydrocarbon and thehydrocarbon comprises undecane. Often the hydrocarbon is undecane. Thus, the oil may comprise undecane.

[0248] In one embodiment, the hydrophobic medium comprises a silicone oil and an unsubstituted C10-C20 alkane, and the amphipathic molecules employed comprise one or more glycerophospholipids. For instance, the hydrophobic medium may comprise poly(methylphenylsiloxane) (for instance AR20) and undecane, and the outer layer of amphipathic molecules may comprise DPhPC and / or POPC.

[0249] In some embodiments, the hydrophobic medium comprises a silicone oil and a hydrocarbon. Such mixtures have been found to provide advantageously short incubation times required for stable bilayers to be formed during the printing process for producing synthetic droplet assemblies. The silicone oil and hydrocarbon in the mixture may be as further defined above. Typically, the hydrocarbon is an unsubstituted C10-C20 alkane, preferably undecane. The silicone oil usually has a density close to, but less than, that of water, to control the sinking rate of droplets during printing. The silicon oil may be as further defined above, and is often

[0250] poly(m ethylphenylsiloxane) (for instance AR20). Thus, often, the hydrophobic medium is an oil, and the oil comprises a hydrocarbon and a silicone oil, wherein the hydrocarbon is undecane and the silicone oil is a poly(methylphenylsiloxane) (for instance AR20). Usually, the volume:volume ratio of the hydrocarbon to the silicone oil is from 1 : 3 to 3 : 1. It is often, for instance from 1 : 2 to 1:1. The volume ratio of the hydrocarbon to the silicone oil may for instance be about 35:65.

[0251] Usually, amphipathic molecules are disposed in the hydrophobic medium. The amphipathic molecules disposed in the hydrophobic medium are typically the same as those employed in the synthetic droplet assembly itself, and may therefore be as further defined anywhere herein. The concentration of the amphipathic molecules in the hydrophobic medium may be any suitable concentration. For instance, often, the concentration of the amphipathic molecules in the hydrophobic medium is less than or equal to 15 mg ml / 1. For instance, the concentration of amphipathic molecules may be from 0 to 10 mg ml / 1. Usually, the concentration of amphipathic molecules is from 0.05 mg m1to 10 mg ml / 1, for instance, from 0.05 mg ml / 1to 5 mg ml / 1. More typically, the concentration of amphipathic molecules is from 0.1 mg ml / 1to 2.5 mg ml / 1, for instance, from 0.2 mg ml / 1to 0.5 mg ml / 1. In some embodiments, the concentration of the amphipathic molecules in the hydrophobic medium is, for instance, from 0.1 RIM to 10 mM, or, for instance, from 0.2 mM to 5 mM, such as from 1 mM to 3 mM, e.g. about 2 mM.

[0252] Typically, in the system of the invention, at least one target interface droplet is a target interface outlet droplet. A target interface outlet droplet is a target interface droplet which further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region. The layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore.The protein pore is a membrane protein which is able to form a pore, channel or pump in a layer of amphipathic molecules. Thus, the protein pore is a membrane protein able to form a channel, pore or pump in any of the layers (e.g. bilayers) of amphipathic molecules at the interface between two droplets of the synthetic droplet assembly described herein, or between a target interface droplet of the synthetic droplet assembly described herein and the target region. Such a target interface droplet, which has a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, which pore is situated in the layer of amphipathic molecules at the interface between the droplet and the target region, may be referred to as a target interface outlet droplet; this is because it provides an outlet for the effector molecule, which allows the effector molecule to pass from the synthetic droplet assembly to the target region. Often said interfaces between droplets or between a droplet and the target region are bilayers of the amphipathic molecules, and thus the protein pore is a membrane protein which is able to form a channel, pore or pump in such a bilayer of amphipathic molecules. The use of integral membrane proteins in droplet interface layers of amphipathic molecules has been demonstrated, but it is equally expected that peripheral membrane proteins could be used.

[0253] Membrane pumps, channels and pores can allow for precise control over the exchange of the effector molecule and / or other materials, between (i) individual droplets within the synthetic assembly and (ii) the synthetic droplet assembly and the target region. The protein pore could for instance be an a-hemolysin (aHL) pore, such as a staphylococcal a-hemolysin pore. However, any suitable membrane protein can be used including one from the two major classes, that is, P-barrels or a-helical bundles, to form the protein pore. An example of another protein pore which may be employed, which has a larger internal diameter than the aHL pore is perfringolysin O. The protein pore may also, for instance be a channel, which may be a voltage-gated ion channel, a lightsensitive channel such as bacteriorhodopsin, a ligand-gated channel or a mechano-sensitive channel.

[0254] Suitable protein pores which allow for exchange of materials are known and readily available to the skilled person; many such proteins are either commercially available or can be prepared by known methods. For instance, wild type (WT) aHL monomers can be prepared by in vitro transcription-translation (IVTT), and heptamerised by incubation with rabbit red blood cell membranes. The heptamers are typically purified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Maglia, G. et al., Method. Enzymol. 475, 591-623, (2010). Also, Bayley, H. et al., Droplet interface bilayers. Mol. BioSyst. 4, 1191-1208 (2008) lists several proteins that were tested for insertion into droplet interface bilayers made in bulk oil. Other suitable membrane proteins include, but are not limited to, bacterial peptides and ionophores.

[0255] The protein pore may, alternatively, be an engineered protein pore or a synthetic protein pore. The engineered protein pore may, for instance, be a genetically engineered protein pore, or a covalent or non-covalent chemically engineering protein pore. The synthetic protein pore may, forinstance, be a peptide or an organic molecule. The protein pore need not comprise only protein or peptide molecules but may comprise other molecules in addition to the protein component. For instance, the protein pore may be a hybrid nucleic acid-protein pore of the kind described in WO 2020 / 025909. Droplets can exchange chemical species with each other through protein pores incorporated in the layer of amphipathic molecules at the interface between the droplets.

[0256] Often, the protein pore is a-hemolysin (aHL).

[0257] Usually, when present, the concentration of protein pore is equal to or greater than 0.1 ng mL1, for instance, equal to or greater than 1 ng mL1. For instance, the concentration of the protein pore may be equal to or greater than 2 ng mL1, for instance, equal to or greater than 10 ng mL1. Often, the concentration of protein pore is equal to or greater than 100 ng mL1, for instance, equal to or greater than 1 pg mL'1. For instance, the concentration of the protein pore may be equal to or greater than 2 pg mL1, for instance, equal to or greater than 5 pg mL1, or equal to or greater than 10 pg mL1.

[0258] The concentration of the protein pore may, for instance, be from 0.1 ng mL1to 100 mg mL1. Typically, the concentration of the protein pore is from 1 ng mL1to 1 mg mL1, for instance from 100 ng mL'1to 100 pg mL1. More typically, the concentration of the protein pore is from 1 pg mL1to 100 pg mL1, for instance, from 2 pg mL1to 80 pg mL1. In some embodiments, the concentration of the protein pore is from about 1 pg mL'1to about 60 pg mL1, or from about 5 pg mL1to about 50 pg mL1, or about 10 pg mL'1to about 50 pg mL1. It may for instance be from 10 pg mL'1to 40 pg mL1, or from 20 pg mL'1to 40 pg mL1, for instance from 25 pg mL'1to 35 pg mL1. In other embodiments, the concentration of the protein pore is from about 10 pg mL'1to about 80 pg mL1, or from about 20 pg mL'1to about 70 pg mL1, or about 20 pg mL'1to about 60 pg mL1. It may for instance be from 30 pg mL'1to 60 pg mL1, or from 40 pg mL'1to 60 pg mL1, for instance about 50 pg mL1.

[0259] Such concentrations are particularly suitable for the aHL pore, especially when employed in combination with the effector molecule arabinose, at any of the concentrations for the effector molecule specified herein.

[0260] Typically, the concentration of the protein pore is the concentration of the protein pore (i.e. the pore-forming protein) in the aqueous droplet medium of the droplet, when the droplet is formed. When a droplet comprising a protein pore (pore-forming protein) is contacted with another droplet (which may or may not comprise a protein pore), a layer of amphipathic molecules (often a bilayer of amphipathic molecules) is formed at the interface. That interface layer of amphipathic molecules typically then comprises the protein pore. Therefore a protein pore initially in the aqueous droplet medium may move to the layer of amphipathic molecules (often a bilayer of amphipathic molecules) at the interface of the droplet with another droplet and / or the target region.Suitable concentrations of the protein pore may depend on a number of factors. The rate of insertion of the membrane protein into the bilayer may, for instance, decrease with time. Typically, this will put a lower limit on the concentrations of the protein pore that may be used.

[0261] The concentration of protein pore in one droplet of the droplet assembly and another droplet may be the same or different. Thus, the concentration of protein pores in each droplet of the droplet assembly may be the same or different.

[0262] Typically, at least one layer (e.g. bilayer) of amphipathic molecules in the synthetic droplet assembly comprises a protein pore. A layer (e.g. bilayer) of amphipathic molecules at an interface between contacting droplets, or at an interface between a target interface droplet and the target region, may comprise more than one protein pore. For instance, a particular layer (e.g. bilayer) of amphipathic molecules may contain multiple copies of the same protein pore, or two or more different classes of protein pores. Where more than one class is present, the layer (e.g. bilayer) of amphipathic molecules may contain multiple copies of each different class.

[0263] The protein pore may be as further defined herein, for instance it may be a-hemolysin (aHL) or a different pore, for instance a protein pore with a larger internal diameter such as perfringolysin O.

[0264] Thus typically at least one target interface droplet is a target interface outlet droplet, wherein the target interface outlet droplet further comprises a protein pore as defined above (optionally at a concentration as defined above) wherein the layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore. The protein pore usefully allows passage of the effector molecule from the target interface outlet droplet to the target region.

[0265] Typically, the number of target interface droplets, m, is at least 10 and at least 2, more typically at least 4, and even more typically at least 5 of the target interface droplets are target interface outlet droplets. More typically, the number of target interface droplets is at least 100 and at least 5, more typically at least 10, and even more typically at least 20 of the target interface droplets are target interface outlet droplets.

[0266] The integers n and m, may be as further defined above, and it is typically the case that at least 5 % of the target interface droplets of the synthetic droplet assembly are target interface outlet droplets (i.e. the number of target interface outlet droplets is at least 5 % of m). For instance at least 10 % of the target interface droplets of the synthetic droplet assembly may be target interface outlet droplets (i.e. the number of target interface outlet droplets may be at least 10 % of m). For example, the number of target interface droplets may be at least 100 and at least 5, for instance at least 10, or at least 20 of the target interface droplets may be target interface outlet droplets. More of the target interface droplets may be target interface outlet droplets. For instance, at least 25 %, at least 50 %, for instance at least 75 %, of the target interface droplets may be target interface outlet droplets (i.e. the number of target interface outlet droplets may be at least 25 %, at least 50%, for instance at least 75 %, of m). In some embodiments, all of the target interface droplets of the synthetic droplet assembly are target interface outlet droplets. However, in other embodiments, from 2 % to 90 % of the target interface droplets are target interface outlet droplets (i.e. the number of target interface outlet droplets is between 2 % and 90 % of m), for instance from 5 % to 75 %, or from 5 % to 50 % the target interface droplets may be target interface outlet droplets (i.e. the number of target interface outlet droplets is between 5 % and 75 % of m, or between 5 % and 50 % of m). Often, for instance, from 5 % to 25 %, or from 10 % to 20 %, of the target interface droplets are target interface outlet droplets (i.e. the number of target interface outlet droplets may be between 5 % and 25 % of m, or between 10 % and 20 % of m).

[0267] Often, at least one, and typically two or more, of the target interface outlet droplets are also effector droplets (i.e. they contain the effector molecule or means for producing it). In some embodiments all of the target interface outlet droplets are also effector droplets. Often however, fewer than all of the target interface outlet droplets are also effector droplets, for instance often less than three quarters of the target interface outlet droplets are also effector droplets. Often, for instance, less than half of the target interface outlet droplets are also effector droplets. For instance, less than a quarter of the target interface outlet droplets may also be effector droplets. Indeed, it may be that none of the target interface outlet droplets is also an effector droplet. This may be because no amount of the effector molecule has yet passed (from one or more effector droplets elsewhere in the droplet assembly) into any of the target interface outlet droplets and because none of the target interface outlet droplets has means for producing the effector molecule.

[0268] Each target interface outlet droplet which is also an effector droplet typically comprises said effector molecule and said protein pore, for allowing passage of the effector molecule from the droplet to the target region. Typically, each layer of amphipathic molecules which is an interface between (i) a target interface outlet droplet which is an effector droplet and (ii) the target region, further comprises a protein pore. The protein pore may be as further defined herein, for instance it may be a-hemolysin (aHL) or a different pore, for instance a protein pore with a larger internal diameter such as perfringolysin O.

[0269] Target interface outlet droplets may be thought of as “gateways” that allow passage of effector molecules to from the synthetic droplet assembly to the target region, and it will be apparent from the discussion above that not all of the target interface droplets need be target interface outlet droplets. The target interface outlet droplets may be arranged amongst the target interface droplets in a particular pattern, so that the effector molecule can be released from the synthetic droplet assembly in that particular pattern. An example of this is shown in Figure 3(e), labelled “bottom view”, where all the droplets shown are target interface droplets but only the shaded droplets that are arranged in a cross-shape are target interface outlet droplets. Thus the particular pattern in Figure 3(e) is a cross-shape and it permits the release of the effector molecule from the droplet assembly into the target region in a cross shape, in turn causing an effect in thecells in the target region in that same cross shape. Thus, the patterning of target interface outlet droplets in the target interface droplets permits patterned release of an effector, and a patterned effect to be caused in the cells or tissue of the target region. This is of course very useful in situations when it is desirable selectively to target only certain regions of the tissue in the target region, or only certain cells in the target region, as opposed to all cells or tissue. The particular cells or tissue to be targeted selectively may for instance be diseased tissue or cells that need treating or killing, as opposed to healthy cells or tissue which need not be, or are desirably not, exposed to the effector molecule. Thus, the pattern may match cells to be targeted and avoid cells not to be targeted.

[0270] Thus, the target interface outlet droplets may together form a pattern of target interface outlet droplets. The pattern of target interface outlet droplets in the target interface droplet layer typically corresponds to the shape of an area of the target region to be exposed to the effector molecule. The pattern of target interface outlet droplets may for instance be a ring, triangle, heart, cross, arrow or smiley face, or any other pattern which is desirable for the effector molecule to adopt when it is released into the target region.

[0271] Typically, the target interface droplets comprise mask droplets. A mask droplet is a droplet that does not comprise the protein pore (so that the effector molecule cannot pass into or out of the mask droplet) and does not comprise the effector molecule or a means for producing the effector molecule. The purpose of the mask droplets is to block passage of the effector molecule though the volume occupied by the mask droplets, i.e. to act as a mask. Thus, by employing mask droplets as target interface droplets, in combination with target interface outlet droplets, patterned release can efficiently be achieved from the synthetic droplet assembly into the target area. Thus, often, the target interface droplets of the synthetic droplet assembly comprise target interface outlet droplets and mask droplets. Often, all of the target interface droplets which are not target interface outlet droplets are mask droplets, in other words, each target interface droplet is either a mask droplet or a target interface outlet droplet.

[0272] Thus, often, in the system of the invention, the number of target interface droplets, m, in the synthetic droplet assembly is equal to or greater than two (and m may optionally be as further defined herein) and:

[0273] at least one of the target interface droplets is a target interface outlet droplet, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore; and

[0274] at least one other of the target interface droplets is a mask droplet, wherein each mask droplet does not comprise said protein pore and does not comprise the effector molecule.Often, the number of target interface droplets, m, is at least 4, preferably at least 16, and more preferably at least 64. Indeed, the number of target interface droplets, m may be still higher, as defined further herein. It such cases, is typically the case that at least two of the target interface droplets are target interface outlet droplets, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore; and at least two other of the target interface droplets are mask droplets, wherein each mask droplet does not comprise said protein pore and does not comprise the effector molecule.

[0275] Often, the target interface droplets together form a flat or curved layer of droplets which is a target interface droplet layer, wherein each target interface droplet in said target interface droplet layer contacts at least one other target interface droplet in the target interface droplet layer to form a layer of said amphipathic molecules as an interface between the contacting target interface droplets. Often, at least two of the target interface droplets are target interface outlet droplets, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore, and the at least two target interface outlet droplets together form a pattern of target interface outlet droplets in the target interface droplet layer. Often, the target interface droplets which are not target interface outlet droplets are mask droplets.

[0276] An example of such a target interface droplet layer is shown in Figure 3(e), labelled “bottom view”, where all the droplets shown form part of a target interface droplet layer. In that layer, which is made up of 169 target interface droplets, only the 25 shaded droplets that are arranged in a cross-shape are target interface outlet droplets. All the other target interface droplets in that layer are mask droplets. Thus the particular pattern of target interface outlet droplets in Figure 3(e) is a cross-shape and it permits the release of the effector molecule into the target region in the cross shape, with the mask droplets blocking release elsewhere. The release of the effector in the cross pattern in turn causes an effect in the cells in the target region in that same cross-shape pattern.

[0277] The integers n and m, may be as further defined above; and the integer m, for the number of target interface droplets, may for instance be at least 4, preferably at least 16, and more preferably at least 64, for instance at least 100, or even higher as defined above. These m target interface droplets may together form said flat or curved layer of droplets which is a target interface droplet layer. In said target interface droplet layer, often at least 5 % of the target interface droplets are target interface outlet droplets (i.e. the number of target interface outlet droplets is at least 5 % of m), and the remaining target interface droplets may be mask droplets. For instance at least 10 %of the target interface droplets of the layer may be target interface outlet droplets (i.e. the number of target interface outlet droplets may be at least 10 % of m), and the remaining target interface droplets may be mask droplets. For example, the number of target interface droplets, m, may be at least 100 and at least 5, for instance at least 10, or at least 20 of the target interface droplets may be target interface outlet droplets, and the remaining target interface droplets may be mask droplets. More of the target interface droplets may be target interface outlet droplets. For instance, at least 25 %, at least 50 %, for instance at least 75 %, of the target interface droplets may be target interface outlet droplets (i.e. the number of target interface outlet droplets may be at least 25 %, at least 50 %, for instance at least 75 %, of m), and the remaining target interface droplets may be mask droplets. In some embodiments, all of the target interface droplets of the synthetic droplet assembly are target interface outlet droplets. However, in other embodiments, from 2 % to 90 % of the target interface droplets are target interface outlet droplets (i.e. the number of target interface outlet droplets is between 2 % and 90 % of m), for instance from 5 % to 75 %, or from 5 % to 50 % the target interface droplets may be target interface outlet droplets (i.e. the number of target interface outlet droplets is between 5 % and 75 % of m, or between 5 % and 50 % of m) , and the remaining target interface droplets may be mask droplets. Often, for instance, from 5 % to 25 %, or from 10 % to 20 %, of the target interface droplets are target interface outlet droplets (i.e. the number of target interface outlet droplets may be between 5 % and 25 % of m, or between 10 % and 20 % of m), and the remaining target interface droplets may be mask droplets.

[0278] The pattern of the target interface outlet droplets in the target interface droplet layer typically corresponds to the shape of an area of the target region to be exposed to the effector molecule. The pattern of target interface outlet droplets in the target interface droplet layer may for instance be a ring, triangle, heart, cross, arrow or smiley face, or any other pattern which is desirable for the effector molecule to adopt when it is released into the target region.

[0279] As discussed above, in the system of the invention it is often the case that each target interface outlet droplet is also an effector droplet. Indeed, typically each target interface outlet droplet further comprises the effector molecule.

[0280] Often, the synthetic droplet assembly comprises a reservoir region. The purpose of the reservoir region is to provide a reservoir of the effector molecule within the synthetic droplet assembly which can supply the effector molecule to the target interface outlet droplets (either directly or via a passage of connector droplets) where it is released into the target region. The reservoir region is made up of effector droplets. Typically, the effector droplets in the reservoir region comprises the effector molecule and a protein pore.

[0281] Thus, often, the synthetic droplet assembly comprises a reservoir region which is a plurality of said effector droplets, wherein each effector droplet in the reservoir region: (i) contacts at least one other effector droplet in the reservoir region to form a layer of said amphipathic molecules as an interface between the contacting effector droplets, and (ii) further comprises aprotein pore for allowing passage of the effector molecule between the effector droplets in the reservoir region, wherein each layer of amphipathic molecules which is an interface between contacting effector droplets further comprises said protein pore. An effector droplet in a reservoir region may also be referred to herein as a “reservoir droplet”.

[0282] Such a reservoir region is shown in Figs. 3(d)-(f) and 8(a)-(d), which contains four 13 x 13 layers of effector droplets containing the arabinose and the aHL pore, i.e. a block of 676 effector droplets, making up a reservoir of the arabinose effector molecule.

[0283] Thus, often, the number of the effector droplets in the reservoir region is at least 20, preferably at least 50, and more preferably at least 100. The number of the effector droplets in the reservoir region may for instance be at least 500. The number of the effector droplets in the reservoir region may for instance be from 20 to 10,000, for instance from 50 to 5,000, and is more typically from 100 to 3,000. The number of the effector droplets in the reservoir region may for instance be from 300 to 2,000, or from 500 to 1,000.

[0284] The reservoir region may be connected to the target interface outlet droplets directly or indirectly. The reservoir region may (i) be connected to the target interface outlet droplets directly by being adjacent to and in contact with the target interface outlet droplets. Another possibility is (ii) that the target interface outlet droplets are themselves effector droplets that are part of the reservoir region. Alternatively, (iii) the reservoir region may be connected to the target interface outlet droplets indirectly, via one or more connecting passages of one or more “connector” droplets.

[0285] Regarding the first possibility mentioned above, typically at least one of the effector droplets in the reservoir region contacts a target interface outlet droplet, to form a layer of said amphipathic molecules as an interface between the contacting effector droplet and target interface outlet droplet, wherein each layer of said amphipathic molecules between an effector droplet and a target interface outlet droplet further comprises said protein pore.

[0286] Regarding the second possibility mentioned above, typically at least one of the effector droplets in the reservoir region is a target interface outlet droplet.

[0287] Regarding the third possibility mentioned above, typically at least one of the n droplets in the synthetic droplet assembly is a connector droplet, wherein each connector droplet further comprises a protein pore for allowing passage of the effector molecule into and out of the connector droplet. Each connector droplet may contact at least two other of the n droplets in the synthetic droplet assembly, to form a layer of said amphipathic molecules as an interface between the connector droplet and each of the at least two other droplets, wherein each layer of said amphipathic molecules further comprises said protein pore. In this way, each connector droplet, allows passage of the effector molecule through it. Larger “passages” of multiple connector droplets linked together by pore-containing layers of amphipathic molecules may be present in the droplet assembly, to provide passages, or “connector regions” for the effector molecule to travelthrough. Such passages, or “connector regions”, often connect one or more reservoir regions to one or more target interface outlet droplets.

[0288] It may be the case that a single connector droplet connects a reservoir region to a target interface outlet droplet. Thus, in some embodiments, the connector droplet contacts an effector droplet in the reservoir region and a target interface outlet droplet.

[0289] Usually, however, the droplet assembly comprises a connector region, comprising multiple connector droplets.

[0290] Thus, often, in the system of the invention, the synthetic droplet assembly comprises a connector region which is a plurality of said connector droplets, wherein each connector droplet in the connector region: (i) contacts at least one other connector droplet in the connector region to form a layer of said amphipathic molecules as an interface between the contacting connector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the connector droplets in the connector region, wherein each layer of amphipathic molecules which is an interface between contacting connector droplets further comprises said protein pore.

[0291] Often, at least one of the connector droplets in the connector region contacts an effector droplet in the reservoir region, and at least one other of the connector droplets in the connector region contacts a target interface outlet droplet. Often, a plurality of the connector droplets in the connector region contact a plurality of effector droplets in the reservoir region, and another plurality of the connector droplets in the connector region contact a plurality of target interface outlet droplets.

[0292] Such a connector region is shown in Figs. 3(d)-(f) and 8(a)-(c). It is present within the three layers of droplets that are in between, (i) the reservoir region (the four 13 x 13 layers of effector droplets containing the arabinose and the aHL pore) and (ii) the target interface droplet layer shown in Figure 3(e) labelled “bottom view”, containing 169 target interface droplets. The connector region in Figs. 3(d)-(f) and 8(a)-(c) is a three-dimensional cross-shape of connector droplets which, at one end, contact the reservoir region, and at the other end, contact the (25) target interface outlet droplets which form the cross-shape in the target interface droplet layer.

[0293] Often, the synthetic droplet assembly comprises at least one said connector region, wherein each target interface outlet droplet in the synthetic droplet assembly contacts a connector droplet in a connector region, and wherein at least one of the connector droplets in each connector region contacts an effector droplet in the reservoir region. Thus, the synthetic droplet assembly may comprise two or more connector regions, wherein each target interface outlet droplet in the synthetic droplet assembly contacts a connector droplet in a connector region, and wherein at least one of the connector droplets in each connector region contacts an effector droplet in the reservoir region.As will be understood by the skilled person from the discussion above, the effector molecule is able to enter and pass through the connector droplets, and indeed the connector droplets in the connector region shown in Figs. 3 (d)-(f) and 8(a)-(c) are shown as containing the effector molecule. Those connector droplets are therefore by definition also effector droplets. Thus, in the synthetic droplet assembly it is often, but not necessarily, the case, that at least one of, or at least a plurality of, or for instance all of, the connector droplets are effector droplets. In other embodiments of the synthetic droplet assembly, the effector molecule may not yet have entered the connector droplets or connector regions, in which case the connector droplets will not be effector droplets at that point in time.

[0294] Often, a plurality of the n droplets in the synthetic droplet assembly are mask droplets, wherein each mask droplet does not comprise a protein pore and does not comprise the effector molecule. The synthetic droplet assembly may comprise mask regions comprising multiple rows, or more typically, multiple layers, of mask droplets, which mask droplets are connected to one another by a layer (for instance a bilayer) of said amphipathic molecules.

[0295] The synthetic droplet assembly often comprises at least one mask region, wherein a mask region is a plurality of said mask droplets, wherein each mask droplet in the mask region contacts at least one other mask droplet in the mask region to form a layer of said amphipathic molecules as an interface between the contacting mask droplets. The synthetic droplet assembly often comprises a plurality of mask regions. Such a droplet assembly is shown in Figs. 3 (d)-(f) and 8(a)-(c). Those figures show four mask regions, in the four recesses of the three-dimensional cross shape that is formed by the cross-shaped connector region and target interface outlet droplets. Each mask region is a block comprising several 6 x 6 layers of mask droplets stacked on top of one another. The bottom layer of mask droplets, intended to be in contact with the target region, are target interface droplets which are in the target interface droplet layer. If these “target interface mask droplets” are not considered to be part of the mask region then each mask region shown in Figs. 3(d)-(f) and 8(a)-(c) is a block comprising 108 mask droplets in the form of three 6 x 6 layers of mask droplets stacked on top of one another, each of these blocks being disposed in between the mask droplets of the target interface droplet layer and the reservoir region.

[0296] Often, in preferred embodiments, several of the aforementioned features of the synthetic droplet assembly are combined. Thus, typically, in the synthetic droplet assembly of the system of the invention:

[0297] • the target interface droplets together form a flat or curved layer of droplets which is a target interface droplet layer, wherein each target interface droplet in said target interface droplet layer contacts at least one other target interface droplet in the target interface droplet layer to form a layer of said amphipathic molecules as an interface between the contacting target interface droplets. The integers n and m, may optionally both be as further defined herein, in this embodiment, and are often both relatively large. In addition:• at least two of the target interface droplets in the target interface droplet layer are target interface outlet droplets, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore. Often, more than two of the target interface droplets in the target interface droplet layer are target interface outlet droplets (for instance m may be at least 60, or say, at least 100, and the number of target interface outlet droplets may be at least 5 % of m, for instance at least 10 % of m). In addition:

[0298] • optionally the target interface droplets in the target interface droplet layer which are not target interface outlet droplets are mask droplets;

[0299] • the synthetic droplet assembly comprises a reservoir region which is a plurality of said effector droplets, wherein the integer p may be as further defined herein, the number of the effector droplets in the reservoir region may be as further defined herein and wherein each effector droplet in the reservoir region: (i) contacts at least one other effector droplet in the reservoir region to form a layer of said amphipathic molecules as an interface between the contacting effector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the effector droplets in the reservoir region, wherein each layer of amphipathic molecules which is an interface between contacting effector droplets further comprises said protein pore; and

[0300] • the synthetic droplet assembly comprises at least one connector region wherein the or each connector region is a plurality of said connector droplets, wherein each connector droplet in the connector region: (i) contacts at least one other connector droplet in the connector region to form a layer of said amphipathic molecules as an interface between the contacting connector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the connector droplets in the connector region, wherein each layer of amphipathic molecules which is an interface between contacting connector droplets further comprises said protein pore; and

[0301] • each target interface outlet droplet in the synthetic droplet assembly contacts a connector droplet in a connector region, and wherein at least one of the connector droplets in the or each connector region contacts an effector droplet in the reservoir region; and

[0302] • the synthetic droplet assembly comprises at least one mask region, wherein the or each mask region may be disposed between mask droplets in the target interface droplet layer and effector droplets in the reservoir region.

[0303] Often, the or each connector region is disposed between target interface outlet droplets in the target interface droplet layer and effector droplets in the reservoir region. Indeed, often, the target interface outlet droplets together form a pattern of target interface outlet droplets in the targetinterface droplet layer, and it is typically the case that the same pattern is formed by the connector region or regions disposed between the target interface outlet droplets in the target interface droplet layer and the effector droplets in the reservoir region. The pattern typically corresponds to the shape of an area of the target region to be exposed to the effector molecule. It may be any pattern which is desirable for the effector molecule to adopt when it is released into the target region. The pattern may be a ring, triangle, heart, cross, arrow or smiley face, as demonstrated experimentally herein.

[0304] Often, the connector droplets are effector droplets and the target interface outlet droplets are effector droplets. Indeed, the effector molecule is able to enter and pass through the connector droplets, and enter and pass out of the target interface outlet droplets and into the target region. In line with this, the connector droplets and the target interface outlet droplets shown in Figs. 3 (d)-(f) and 8(a)-(c) contain the effector molecule arabinose. Thus, those connector droplets and target interface outlet droplets are also effector droplets.

[0305] In the system of the invention, typically each effector droplet comprises the effector molecule. More typically, each effector droplet comprises the effector molecule and a protein pore. The protein pore is typically the same protein pore that is defined hereinbefore in any one of the preceding paragraphs.

[0306] The flux of the effector molecule from the or each target interface outlet droplet into the target region can be controlled by adjusting effector molecule and pore concentrations within the synthetic droplet assembly, to produce a desired effect in the cells or tissue of the target region. Thus, often, the concentration of the effector molecule and the concentration of the protein pore are selected to achieve a desired rate of release of the effector molecule from the synthetic droplet assembly into the target region. For instance, often, (i) the concentration of the effector molecule in each effector droplet and (ii) the concentration of the protein pore in each target interface outlet droplet (and optionally in each reservoir droplet, connector droplet and / or effector droplet), are selected to achieve a desired rate of release of the effector molecule from the or each target interface outlet droplet into the target region. The desired rate of release (or flux) of the effector molecule into the target region is typically selected in order to achieve a desired effect in said biological cells or tissue in the target region. Indeed, the Example herein describes tuning the concentrations of arabinose and aHL in the aqueous droplet medium employed in the synthetic droplet assembly, in order to achieve a desired flux of arabinose into the target region and consequently a desired gene expression in the bacterial population in the target region.

[0307] Often, the concentration of protein pore (for instance its concentration in the aqueous droplet medium of the target interface outlet droplet) is equal to or greater than 0.1 ng mL1, and the concentration of the effector molecule (for instance its concentration in the aqueous droplet medium of the effector droplet) is equal to or greater than 0.001 HIM. Typically, for instance, the respective concentrations of the protein pore and the effector molecule are equal to or greater than1 ng mL'1and equal to or greater than 0.001 mM, for instance equal to or greater than 2 ng ml / 1and equal to or greater than 0.01 DIM, respectively. Often, the respective concentrations of the protein pore and the effector molecule are equal to or greater than 10 ng ml / 1and equal to or greater than 0.1 mM, for instance equal to or greater than 1 pg ml / 1and equal to or greater than 0.5 mM, respectively. For instance, the respective concentrations of the protein pore and the effector molecule may be equal to or greater than 2 pg ml / 1and equal to or greater than 1 mM, or equal to or greater than 5 pg mL'1and equal to or greater than 2 mM, or equal to or greater than 10 pg mL'1and equal to or greater than 5 mM, respectively. Often, the respective concentrations of the protein pore and the effector molecule are equal to or greater than 10 pg mL'1and equal to or greater than 10 mM, or equal to or greater than 10 pg mL'1and equal to or greater than 15 mM, or equal to or greater than 10 pg mL'1and equal to or greater than 20 mM, 25 mM, or 30 mM, respectively.

[0308] The concentration of protein pore (for instance its concentration in the aqueous droplet medium of the target interface outlet droplet) is often from 0.1 ng mL'1to 100 mg mL1, and the concentration of the effector molecule (for instance its concentration in the aqueous droplet medium of the effector droplet) is often from 0.001 mM to 1,000 mM. Typically, for instance, the respective concentrations of the protein pore and the effector molecule are from 1 ng mL'1to 1 mg mL'1and from 0.01 mM to 800 mM, for instance from 100 ng mL'1to 100 pg mL'1and from 0.1 mM to 600 mM, respectively. Often, the respective concentrations of the protein pore and the effector molecule are from 1 pg mL'1to 100 pg mL'1and from 0.5 mM to 400 mM, for instance from 2 pg mL'1to 80 pg mL'1and from 1 mM to 300 mM, respectively. For instance, the respective concentrations of the protein pore and the effector molecule may be from about 1 pg mL'1to about 60 pg mL'1and from about 2 mM to about 200 mM, or from about 5 pg mL'1to about 50 pg mL'1and from about 5 mM to about 100 mM, or from about 10 pg mL'1to about 50 pg mL'1and from about 10 mM to about 80 mM, respectively. Often, the respective concentrations of the protein pore and the effector molecule are from 10 pg mL'1to 40 pg mL'1and from 15 mM to 60 mM, or from 10 pg mL'1to 40 pg mL'1and from 20 mM to 50 mM, or from 25 pg mL'1to 35 pg mL'1and from 25 mM to 40 mM, respectively.

[0309] Concentrations of the protein pore and the effector molecule in the above ranges were found to achieve excellent desired flux into the target region and consequently the desired effect on the cells of the target region.

[0310] By attaching “magnetic handles” to the synthetic droplet assembly (e.g. hydrogel droplets containing magnetic particles) the droplet assembly is able to be guided precisely to interface with the target region with the aid of a magnet and can be held in position (see Figures 3(f) and 8).

[0311] Thus, the system of the invention may further comprise magnetic particles attached to the synthetic droplet assembly, suitable for moving the synthetic droplet assembly relative to the target region using a magnet. Typically, the magnetic particles are particles which comprise nickel, ironor cobalt, or an alloy of one or more of nickel, iron and cobalt. Magnetic particles which comprise nickel (Ni) may for instance be employed, for instance MagneHis™ Ni-particles. Often, the magnetic particles are disposed in a plurality of hydrogel droplets, wherein each hydrogel droplet is attached to the synthetic droplet assembly, usually via a droplet interface layer (e.g. bilayer) of amphipathic molecules at an interface between the hydrogel droplet and one or more droplets of the synthetic droplet assembly. The hydrogel of the droplets containing the magnetic particles may be as defined above for the aqueous droplet medium; the hydrogel may for instance be agarose (for example ultra-low gelling agarose), optionally at a concentration as defined above for agarose (for instance 1.5% w / v). The synthetic droplet assembly may comprise one or more mask regions, as defined above, in which case each hydrogel droplet may be attached to a mask region of the synthetic droplet assembly.

[0312] The system optionally further comprises a magnet, for orienting, positioning and / or holding in place the synthetic droplet assembly relative to the target region.

[0313] The present invention additionally provides a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets. At least one of the n droplets in the synthetic droplet assembly of the invention is a target interface droplet. Each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region. The number of said target interface droplets, m, is from 1 to n. Furthermore, at least one of the n droplets in the synthetic droplet assembly of the invention is an effector droplet. Each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.

[0314] Any and all features of the synthetic droplet assembly of the invention may be as further defined herein for the synthetic droplet assembly of the system of the invention. Thus, for instance, the features of each of the n droplets of the synthetic droplet assembly of the invention, such as the number of the droplets, n, the aqueous medium of the droplets, the amphipathic molecules, and the layer of the amphipathic molecules around the surface of the aqueous medium, may be as further defined herein for the synthetic droplet assembly of the system of the invention. Similarly, the layer of the amphipathic molecules that is an interface between contacting droplets that is formed due to each of the n droplets contacting at least one other of the n droplets, may be as further defined herein for the synthetic droplet assembly of the system of the invention. The number, m, of the target interface droplets in the synthetic droplet assembly of the invention, and the features of the target interface droplets themselves, may also be as further defined herein for the syntheticdroplet assembly of the system of the invention, including the feature that target interface droplets may comprise target interface outlet droplets as defined herein, and / or mask droplets as defined herein, and that such target interface droplets may be present within a target interface droplet layer in which the target interface outlet droplets form a pattern amongst the mask droplets. The number, p, of the effector droplets in the assembly, and the nature of the effector molecule and the means for producing the effector molecule, may also be as further defined herein for the synthetic droplet assembly of the system of the invention, including the optional presence of a reservoir region of effector droplets or connector regions that connect the reservoir region to the target interface outlet droplets, optional mask regions which may be disposed between mask droplets in the target interface droplet layer and effector droplets in the reservoir region. Thus, the synthetic droplet assembly of the invention may be as further defined anywhere herein for the synthetic droplet assembly of the system of the invention.

[0315] As mentioned above, in the synthetic droplet assembly of the invention, each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region. Said target region comprising biological cells or tissue, including the cells or tissue of the target region, may be as further defined anywhere herein for the system of the invention. For instance, the target region which each target interface droplet is suitable for contacting may be living tissue. Often, said target region comprises cells to undergo therapeutic treatment by exposure to the effector molecule, or the target region may comprise cells to be killed by the effector molecule, for instance tumour cells. The target region which each target interface droplet is suitable for contacting may alternatively for instance comprise a volume of a hydrogel, wherein the biological cells or tissue are in contact with the hydrogel. The biological cells or tissue may be disposed within the volume of hydrogel, or the volume of hydrogel may be disposed between the droplet assembly and the biological cells or tissue. These features may be as further defined herein for the system of the invention.

[0316] Often, the synthetic droplet assembly of the invention is disposed in a hydrophobic medium. The hydrophobic medium may be as further defined hereinbefore in relation to the system of the invention.

[0317] The synthetic droplet assembly of the invention may be produced using a droplet printing apparatus and process for producing droplet assemblies (for instance as described in any of WO 2014 / 087175 Al, WO 2021 / 234394 Al, G. Villar et al. Science 2013, 340, 48, and I. Cazimoglu et al, ACS Nano 2021, 15, 20214) and as further described in the Example herein.

[0318] The present invention further provides the use of the synthetic droplet assembly of the invention for delivering an effector molecule from the synthetic droplet assembly to biological cells or tissue.The present invention further provides a process for delivering an effector molecule from a synthetic droplet assembly to biological cells or tissue. The synthetic droplet assembly comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets, wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n. At least one of the n droplets is an effector droplet, wherein each effector droplet further comprises the effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n. The process comprises contacting each target interface droplet with a target region which comprises the biological cells or tissue, so that each target interface droplet contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region.

[0319] The synthetic droplet assembly employed in the process of the invention may be as further defined anywhere herein for the synthetic droplet assembly of the system of the invention or the synthetic droplet assembly of the invention. Similarly, the target region employed in the process of the invention may be as further defined anywhere herein for the system of the invention. Similarly, the layer of the amphipathic molecules that is formed during the process, as an interface between the target interface droplet and the target region, may be as further defined anywhere herein for the system of the invention.

[0320] As mentioned above, each effector droplet comprises the effector molecule or means for producing the effector molecule.

[0321] Usually, each effector droplet comprises an effector molecule. In other embodiments, however, an effector droplet, or each effector droplet, comprises said means for producing the effector molecule, in which case the process may further comprise producing the effector molecule in the or each effector droplet using said means. Thus, for instance, the means for producing the effector molecule may comprise one or more precursors to the effector molecule, in which case producing the effector molecule may comprise converting the one or more precursors into the effector molecule. Additionally or alternatively, the means for producing the effector molecule may comprise one or more molecules which enable production of the effector molecule, and producing the effector molecule may comprise using said one or more molecules to produce the effector molecule. For instance, the one or more molecules which enable production of the effector molecule comprise a catalyst, an enzyme, or a cell-free expression system, for instance an in vitro transcription-translation system. Producing the effector molecule may comprise converting the oneor more precursors into the effector molecule, using the one or more molecules which enable production of the effector molecule. For instance, producing the effector molecule may comprise converting the one or more precursors into the effector molecule, using a catalyst, an enzyme, or a cell -free expression system, for instance an in vitro transcription-translation system.

[0322] Prior to the contacting the target interface droplets of the synthetic droplet assembly with the target region, it is often the case that the synthetic droplet assembly is disposed in a hydrophobic medium and the hydrophobic medium is also in contact with said target region. In that case, contacting the target interface droplets of the synthetic droplet assembly with the target region typically comprises moving the synthetic droplet assembly within the hydrophobic medium in order to bring the target interface droplets of the synthetic droplet assembly into contact with the target region.

[0323] Often, for instance, the target region further comprises a bulk aqueous medium, such as a hydrogel, in addition to the biological cells or tissue. The biological cells or tissue may be disposed in the bulk aqueous medium, for instance the hydrogel. In that case, contacting the target interface droplets of the synthetic droplet assembly with the target region comprises contacting the target interface droplets of the synthetic droplet assembly with the bulk aqueous medium (for instance the hydrogel) of the target region. This may comprise moving the synthetic droplet assembly within the hydrophobic medium in order to bring the target interface droplets of the synthetic droplet assembly into contact with bulk aqueous medium (for instance the hydrogel) of the target region. In these cases, bringing the target interface droplets of the droplet assembly into contact with the bulk aqueous medium (for instance the hydrogel) of the target region forms said layer of the amphipathic molecules as an interface between the target interface droplets and the bulk aqueous medium (for instance the hydrogel) of the target region. Often, said layer of the amphipathic molecules is a bilayer of the amphipathic molecules. If the bulk aqueous medium is a hydrogel, such a bilayer may be referred to as a droplet-hydrogel bilayer (DHB). Any protein pore that is present (e.g. in the aqueous droplet medium of the target interface droplets, or in a bulk aqueous medium, such as a hydrogel, of the target region), may then insert into the layer of the amphipathic molecules which is said interface between the target interface droplets and the target region, e.g. into said DHB.

[0324] Magnetic particles, for instance hydrogel droplets which comprise magnetic particles, may be attached to the synthetic droplet assembly as described further herein. In that case, the process of the invention may further comprise moving the synthetic droplet assembly relative to the target region using a magnet. In particular, when the process comprises moving the synthetic droplet assembly within a hydrophobic medium in order to bring the target interface droplets of the synthetic droplet assembly into contact with the target region, the process may comprise moving the synthetic droplet assembly within said hydrophobic medium using a magnet. After contacting each target interface droplet with the target region, to form said layer of the amphipathic moleculesbetween each target interface droplet and the target region, the process may, if necessary, further comprise holding the synthetic droplet assembly in place using said magnet. More generally, the process may further comprise orienting, positioning and / or holding in place the synthetic droplet assembly relative to the target region using said magnet.

[0325] The target region may not further comprise a bulk aqueous medium such as a hydrogel; rather, as discussed above, the target region may be biological tissue. The tissue may be a tissue sample, for example a biopsy. The tissue may be living tissue. The living tissue may be part of a living organism, for instance a mammal, such as for example a human. Thus, in the process of the invention, contacting the target interface droplets of the synthetic droplet assembly with the target region may comprise embedding or implanting the synthetic droplet assembly within the target region. In particular, contacting the target interface droplets of the synthetic droplet assembly with the target region may comprise embedding or implanting the synthetic droplet assembly within tissue. The tissue may for instance be living tissue of a living organism, for instance a mammal, such as for example a human.

[0326] Prior to the contacting the target interface droplets of the synthetic droplet assembly with the target region, the synthetic droplet assembly may disposed in a hydrophobic medium, which hydrophobic medium may be as further defined herein. Thus, embedding or implanting the synthetic droplet assembly within the target region may comprise transferring the synthetic droplet assembly from the hydrophobic medium into the target region.

[0327] The process of the invention typically further comprises allowing the effector molecule to pass from a target interface droplet to the biological cells or tissue of the target region. Often, this is achieved by providing a protein pore in the layer of amphipathic molecules which forms as an interface between the target interface droplet and the target region, which protein pore is suitable for allowing passage of the effector molecule from the target interface droplet to the target region. Such protein pores are discussed in detail hereinbefore, and thus the protein pore may be as further defined anywhere herein. A protein pore can easily be provided in the layer of amphipathic molecules at the interface between the target interface droplet and the target region by, for instance, including the pore in the target interface droplet (typically in the aqueous droplet medium of the target interface droplet) so that, when the layer of the amphipathic molecules at that interface is formed, the protein pore inserts into that layer. The target interface droplet in question may be referred to as a target interface outlet droplet.

[0328] Typically, therefore, in the process of the invention, at least one target interface droplet of the synthetic droplet assembly is a target interface outlet droplet, wherein the target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which forms as an interface between (i) the target interface outlet droplet and (ii) the target region, further comprises said protein pore.Such synthetic droplet assemblies in which at least one target interface droplet is a target interface outlet droplet are further defined herein in relation to the system of the invention, and any of those synthetic droplet assemblies may be employed in the process of the invention. Typically, in such cases, the process further comprises allowing the effector molecule to pass from the target interface outlet droplet(s) to the target region, by passing through said protein pore of each layer of amphipathic molecules which is an interface between (i) a target interface (outlet) droplet and (ii) the target region.

[0329] As discussed in detail above, the effector molecule may already be present in the target interface outlet droplets, for instance if the target interface outlet droplets are also effector droplets, in which case the effector molecule can immediately pass from the target interface outlet droplets to the target region, by passing through said protein pore of each layer of amphipathic molecules between a target interface outlet droplet and the target region. Alternatively, the synthetic droplet assembly may comprise a reservoir region as defined herein, which may be connected to the target interface outlet droplets directly (e.g. if the reservoir region itself is in contact with the target interface outlet droplets) or indirectly (e.g. if the reservoir region is connected to the target interface outlet droplets indirectly, via one or more connector droplets, or via one or more connector regions as defined above). In that case, the effector molecule may first pass from the reservoir region to the target interface outlet droplets (optionally via one or more connector droplets or via one or more connector regions) and may then pass from the target interface outlet droplets to the target region, through the protein pores of the layers of amphipathic molecules between the target interface outlet droplets and the target region.

[0330] Thus, the process may further comprise allowing the effector molecule to pass from a reservoir region to the target interface outlet droplet(s) and allowing the effector molecule to pass from the target interface outlet droplet(s) to the target region, by passing through said protein pore of each layer of amphipathic molecules which is an interface between (i) a target interface (outlet) droplet and (ii) the target region. The process may for instance comprise allowing the effector molecule to pass from a reservoir region to the target interface outlet droplet(s) via one or more connector droplets or via one or more connector regions, and allowing the effector molecule to pass from the target interface outlet droplet(s) to the target region, by passing through said protein pore of each layer of amphipathic molecules which is an interface between (i) a target interface (outlet) droplet and (ii) the target region.

[0331] As shown in the Example herein, the flux of the effector molecule from the or each target interface outlet droplet into the target region can be controlled by adjusting effector molecule and pore concentrations within the synthetic droplet assembly, to produce a desired effect in the cells or tissue of the target region. Thus, the process of the invention typically further comprises selecting (i) concentration of the effector molecule in each effector droplet and the (ii) concentration of the protein pore in each target interface outlet droplet, and thereby achieving a desired rate of releaseof the effector molecule from the or each target interface outlet droplet into the target region. Typically, achieving said desired rate of release of the effector molecule achieves a desired effect in said biological cells or tissue in the target region.

[0332] Often, the synthetic droplet assembly comprises a reservoir region as defined herein and typically also a connector region as defined herein.

[0333] The process of the invention often comprises selecting (i) the concentration of the effector molecule in each effector droplet and (ii) the concentration of the protein pore in each target interface outlet droplet, in each effector droplet (including in each each reservoir droplet) and in each connector droplet, and thereby achieving a desired rate of release of the effector molecule from the or each target interface outlet droplet into the target region.

[0334] Often, the process of the invention comprises selecting: the concentration of protein pore (for instance its concentration in the aqueous droplet medium of the target interface outlet droplet) to be equal to or greater than 0.1 ng mL1, and the concentration of the effector molecule (for instance its concentration in the aqueous droplet medium of the effector droplet) to be equal to or greater than 0.001 mM. Typically, for instance, the process of the invention comprises selecting the following respective concentrations for the protein pore and the effector molecule: equal to or greater than 1 ng mL'1and equal to or greater than 0.001 mM, for instance equal to or greater than 2 ng mL'1and equal to or greater than 0.01 mM, respectively. Often, the process of the invention comprises selecting the following respective concentrations for the protein pore and the effector molecule: equal to or greater than 10 ng mL'1and equal to or greater than 0.1 mM, for instance equal to or greater than 1 pg mL'1and equal to or greater than 0.5 mM. For instance, the process of the invention may comprise selecting the following respective concentrations for the protein pore and the effector molecule: equal to or greater than 2 pg mL'1and equal to or greater than 1 mM, or equal to or greater than 5 pg mL'1and equal to or greater than 2 mM, or equal to or greater than 10 pg mL'1and equal to or greater than 5 mM. Often, the respective concentrations of the protein pore and the effector molecule selected are: equal to or greater than 10 pg mL'1and equal to or greater than 10 mM, or equal to or greater than 10 pg mL'1and equal to or greater than 15 mM, or equal to or greater than 10 pg mL'1and equal to or greater than 20 mM, 25 mM, or 30 mM, respectively.

[0335] The process of the invention may comprise selecting the concentration of the protein pore (for instance its concentration in the aqueous droplet medium of the target interface outlet droplet) to be from 0.1 ng mL'1to 100 mg mL1, and selecting the concentration of the effector molecule (for instance its concentration in the aqueous droplet medium of the effector droplet) to be from 0.001 mM to 1,000 mM. Typically, for instance, the process of the invention comprises selecting the following respective concentrations for the protein pore and the effector molecule: from 1 ng mL'1to 1 mg mL'1and from 0.01 mM to 800 mM, for instance from 100 ng mL'1to 100 pg mL'1and from 0.1 mM to 600 mM, respectively. Often, the process of the invention comprises selecting the following respective concentrations for the protein pore and the effector molecule: from 1 pg mL'1to 100 ig mL1and from 0.5 RIM to 400 mM, for instance from 2 pig ml / 1to 80 pig ml / 1and from 1 RIM to 300 mM, respectively. For instance, the process of the invention may comprise selecting the following respective concentrations for the protein pore and the effector molecule: from about 1 pg ml / 1to about 60 pg ml / 1and from about 2 mM to about 200 mM, or from about 5 pg ml / 1to about 50 pg ml / 1and from about 5 mM to about 100 mM, or from about 10 pg ml / 1to about 50 pg ml / 1and from about 10 mM to about 80 mM, respectively. Often, the process of the invention comprises selecting the following respective concentrations for the protein pore and the effector molecule: from 10 pg ml / 1to 40 pg ml / 1and from 15 mM to 60 mM, or from 10 pg ml / 1to 40 pg ml / 1and from 20 mM to 50 mM, or from 25 pg ml / 1to 35 pg ml / 1and from 25 mM to 40 mM, respectively.

[0336] Concentrations of the protein pore and the effector molecule in the above ranges were found to achieve excellent desired flux into the target region and consequently the desired effect on the cells of the target region.

[0337] In terms of delivering the effector molecule from the synthetic droplet assembly to the biological cells or tissue, as discussed above, the process of the invention typically comprises allowing the effector molecule to pass from a target interface (outlet) droplet to the biological cells or tissue of the target region. Such a step relies on the target interface outlet droplet being an effector droplet itself, or being connected to an effector droplet directly, or being connected to an effector droplet indirectly via a connector droplet or a connector region. The target interface outlet droplet may for instance be connected to an effector droplet of a reservoir region directly, or it may be connected to an effector droplet of a reservoir region indirectly via a connector droplet or a connector region.

[0338] However, it can be desirable to control the onset of the flux of the effector molecule from the target interface (outlet) droplets to the biological cells or tissue of the target region. For instance, it may be desirable to delay the onset of said flux until sometime after the synthetic droplet assembly has been attached to the target region, i.e. until sometime after the step of contacting each target interface droplet with a target region which comprises the biological cells or tissue, so that each target interface droplet contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region.

[0339] This can be done by ensuring that there is no effective droplet connection between the effector droplets (which may for instance be effector droplets in a reservoir region) and the target interface droplets (for instance, target interface outlet droplets) at the time of contacting each target interface droplet with the target region, so that the effector molecule cannot enter the target interface droplets (for instance, the target interface outlet droplets) at that time and pass into the target region.

[0340] An effective droplet connection between the effector droplets and the target interface droplets (for instance, the target interface outlet droplets) may be established later, when it is desirable to start releasing the effector molecule from the target interface (outlet) droplets to thebiological cells or tissue of the target region. By “effective droplet connection”, here, is meant that the relevant droplets (i.e. the effector droplets and the target interface droplets, for instance the effector droplets and target interface outlet droplets) are connected via one or more droplet interface layers (e.g. bilayers) of amphipathic molecules which comprise a protein pore for allowing passage of the effector molecule. The relevant droplets may be connected directly, via a single droplet interface layer (e.g. bilayer) of amphipathic molecules which comprises a protein pore, or indirectly, via a plurality of droplets that are in contact with each other via such droplet interface layers (e.g. bilayers) of amphipathic molecules which comprise a protein pore.

[0341] Thus, the process of the invention may further comprise (i) ensuring that there is no effective droplet connection between any effector droplet and a target interface droplet, and subsequently (ii) providing an effective droplet connection between an effector droplet and a target interface droplet. In other words, the process of the invention may comprise “switching on” the effective droplet connection. For instance, the process may comprise (i) ensuring that there is no effective droplet connection between any effector droplet and a target interface outlet droplet, and subsequently (ii) providing an effective droplet connection between an effector droplet and a target interface outlet droplet. The “switching on” step (ii) may be performed before, during or after contacting each target interface droplet with the target region. However, it is typically performed after the contacting each target interface droplet with the target region (for instance in order to delay the onset of flux of the effector molecule to the target region, as discussed above).

[0342] Similarly, the process of the invention may comprise “switching off’ the effective droplet connection. Thus, the process of the invention may comprise (i) providing an effective droplet connection between an effector droplet and a target interface droplet, and subsequently (ii) ensuring that there is no effective droplet connection between any effector droplet and a target interface droplet. For instance, the process may comprise (i) providing an effective droplet connection between an effector droplet and a target interface outlet droplet, and subsequently (ii) ensuring that there is no effective droplet connection between any effector droplet and a target interface outlet droplet. The “switching off’ step (ii) may be performed before, during or after contacting each target interface droplet with the target region. However, it is typically performed after the contacting each target interface droplet with the target region (for instance in order to cease the flux of the effector molecule to the target region). The switching off step often, of course, follows the switching on step, and often both steps are performed after contacting each target interface droplet with the target region.

[0343] Switching on the above-defined effective droplet connection may be achieved in a number of ways, but it is often achieved by joining two regions of the droplet assembly together, for instance joining a region of the droplet assembly that comprises an effector droplet, or more typically a reservoir region, with another region of the droplet assembly that comprises the targetinterface droplets. Conversely, switching off the above-defined effective droplet connection may be achieved by breaking apart two such regions of the synthetic droplet assembly.

[0344] Thus, often in the process of the invention, the number of droplets in the synthetic droplet assembly, n, is at least 4, and the process further comprises producing the synthetic droplet assembly by contacting a first precursor droplet assembly with a second precursor droplet assembly before, during or after the step of contacting each target interface droplet with the target region. Often, this is done after the step of contacting each target interface droplet with the target region. Typically, the first precursor droplet assembly comprises at least one target interface droplet and the second precursor droplet assembly comprises at least one effector droplet, and contacting the first precursor droplet assembly with the second precursor droplet assembly enables the effector molecule to pass from the effector droplet originating from the second precursor droplet assembly to the at least one target interface droplet originating from the first precursor droplet assembly.

[0345] Typically, the first precursor droplet assembly comprises x droplets, wherein x is an integer equal to or greater than 2, wherein each of said x droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said x droplets contacts at least one other of said x droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets; and the second precursor droplet assembly comprises n-x droplets, wherein each of said n-x droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n-x droplets contacts at least one other of said n-x droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets.

[0346] Often, the first precursor droplet assembly comprises at least one target interface droplet which is a target interface outlet droplet, wherein the target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region; and the second precursor droplet assembly comprises a reservoir region which is a plurality of said effector droplets, wherein each effector droplet in the reservoir region: (i) contacts at least one other effector droplet in the reservoir region to form a layer of said amphipathic molecules as an interface between the contacting effector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the effector droplets in the reservoir region, wherein each layer of amphipathic molecules which is an interface between contacting effector droplets further comprises said protein pore,

[0347] wherein contacting the first precursor droplet assembly with the second precursor droplet assembly enables the effector molecule to pass from the reservoir region to the at least one target interface outlet droplet, optionally via a connector region as defined herein.

[0348] The synthetic droplet assembly employed in the process of the invention, may be as further defined anywhere herein. For instance, the synthetic droplet assembly employed in the process of the invention may be one in which the target interface outlet droplets together form a pattern oftarget interface outlet droplets in a target interface droplet layer, as defined above, and the process of the invention may comprise exposing the biological cells or tissue of the target region to the effector molecule in said pattern.

[0349] As discussed above, the inventors the inventors have now provided access to droplet assemblies in which droplet sizes are smaller than previously obtainable by 3D droplet printing alone. The Example hereinbelow and Figure 4(d) describe a heat-induced post-printing shrinking process, in which droplet size was reduced post-printing, by release of water from the droplets through evaporation. This advantageously allows for patterned release of an effector molecule at very high resolution. Thus, the synthetic droplet assembly employed in the process of the invention.

[0350] Therefore, the process of the invention may further comprise reducing the size of the droplets in the synthetic droplet assembly by removing water from the aqueous medium of droplets in the droplet assembly.

[0351] Often, removing water from the aqueous medium of droplets in the droplet assembly comprises evaporating said water. Usually, evaporating said water comprises heating the droplet assembly. Often, heating the droplet assembly comprises exposing the droplet assembly to a temperature of from 30 °C to 90 °C, preferably from 30 °C to 70 °C, more preferably to a temperature of from 30 °C to 50 °C, for instance to a temperature of from 35 °C to 45 °C. Heating the droplet assembly typically comprises exposing the droplet assembly to said temperature for a duration of at least 5 minutes, optionally for a duration of at least 20 minutes, or for a duration of at least 1 hour, for instance for a duration of from 1 to 4 hours.

[0352] Typically, the reducing the size of the droplets in the synthetic droplet assembly (comprising removing water from the droplets, optionally comprising evaporating said water, for instance by heating) is performed before contacting each target interface droplet with the target region. Often, when the reducing the size of the droplets is performed, the droplet assembly is disposed in a hydrophobic medium. This may be the same hydrophobic medium as the one in which the synthetic droplet assembly may be disposed prior to contacting the target interface droplets of the synthetic droplet assembly with the target region. The hydrophobic medium may be as further defined anywhere herein.

[0353] For instance, often, evaporating said water comprises heating the droplet assembly by heating said hydrophobic medium. The hydrophobic medium may be heated to a temperature as defined above, optionally for a duration as defined above.

[0354] Reducing the size of the droplets in the synthetic droplet assembly, in this way, may comprise reducing the diameter of the droplets to a diameter of less than 10 pm. For instance, each of the n droplets in the synthetic droplet assembly may be reduced to a diameter of less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm, or less than or equal to 3 pm, for example less than or equal to 2 pm. Thus, each of the n droplets in the syntheticdroplet assembly may be reduced to a diameter of from 50 nm to less than 10 pm, for instance from 50 nm to 9 pm, or from 50 nm to 8 pm. Often, each of the n droplets in the synthetic droplet assembly is reduced to a diameter of from 50 nm to 6 pm, for instance from 70 nm to 5 pm, or for example from 100 nm to 3 pm. For instance, each of the n droplets in the synthetic droplet assembly may be reduced to a diameter of from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm.

[0355] Similarly, reducing the size of the droplets in the synthetic droplet assembly may comprise reducing the volume of the droplets to a volume of less than 0.5 pL. For instance, each of the n droplets in the synthetic droplet assembly may be reduced to a volume of less than or equal to 382 fL (femtolitres), less than or equal to 268 fL, or for instance less than or equal to 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL. Thus, each of the n droplets in the synthetic droplet assembly may be reduced to a volume of from 65 zL to less than 0.5 pL, for instance from 65 zL to 382 fL, or from 65 zL to 268 fL. Often, each of the n droplets in the synthetic droplet assembly is reduced to a volume of from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL. For instance, each of the n droplets in the synthetic droplet assembly may be reduced to a volume of from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0356] Droplet diameters and volumes can be determined by microscopy.

[0357] The invention will be described further in the following Examples.

[0358] EXAMPLES

[0359] 1. Introduction

[0360] The present inventors have appreciated that functional droplet networks (M. A. Holden et al., J. Am. Chem. Soc. 2007, 129, 8650; M. J. Booth et al., Molecular BioSystems 2017, 13, 1658; G. Villar et al., Nat Nanotechnol 2011, 6, 803; C. E. G. Hoskin et al., Nat. Chem. 2022, 1) in other contexts also referred to as synthetic tissues (H. Bayley et aL, Emerg Top Life Sci 2019, 3, 615; G. Villar et al., Science 2013, 340, 48; A. J. Lin et al., ACS Synth. Biol. 2023, 12, 1889; A. Alcinesio et al., Nat Commun 2020, 11, 2105; A. Alcinesio et al., ChemSystemsChem 2022, 4, e202100036; I. Cazimoglu et al., ACS Nano 2021, 15, 20214), exhibit potential for releasing chemical signals with high spatial and temporal resolution, as they have the ability to feature the patterning of compartments and signalling between compartments both within the droplet network and with the immediate external environment at micrometre resolution.

[0361] In this Example, 3D-printed picolitre droplet networks are provided as a universal platform that can direct cellular activity by the patterned release of chemical signals. Specifically, the controlled release of chemical signals onto populations of homogeneously-distributed Escherichia coli micro-colonies is demonstrated, eliciting patterned changes in gene expression by the precise tuning of cargo release dynamics. In addition, a method to reliably shrink our droplet networks to achieve chemical signalling with micrometre resolution (~50 pm) has been developed. The system of the invention shows improved cargo storage capacity compared to synthetic cell-based systems, while retaining the ability of releasing chemical signals from a single compartment. Further, it is shown herein that the droplet assemblies of the invention can be orientated in space through magnetism and that cargo release can be activated at defined times by the connection of reservoir networks. The versatility of the inventive system is illustrated by the spatiotemporal control of the expression of colicin E7, a proteinaceous toxin produced by specific E. coli strains (E. Cascales et al., Microbiology and Molecular Biology Reviews 2007, 71, 158), hence directing the outcome of toxin-driven bacterial competition. Taken together, the results described herein pave the way towards applications of functional droplet networks in directing cellular patterning for applications in fundamental biology and medicine that require the local control of gene expression within a target group of cells.

[0362] 2. Results and Discussion

[0363] 2.1. Interfacing Bacterial Cells with Droplet Networks

[0364] Patterned picoliter droplet networks - comprising 500 to 1000 droplets (-150 pL per droplet) connected through droplet interface bilayers (DIB) - were constructed by using a 3D droplet printer (Figure 1(a)) (G. Villar et al., Science 2013, 340, 48; WO 2014 / 087175). In brief, aqueous droplets containing L-(+)-arabinose (the chemical signal used to induce bacterial protein expression) and monomers of the pore-forming membrane protein -hemolysin (aHL) were ejected into a lipid-in-oil solution: 1,2-diphytanoyl-sn-glycero-phosphatidylcholine (DPhPC) and 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC, 2:1 molar ratio) in 35:65 (v:v) undecane: silicone oil AR20). Monomers of aHL assemble to form heptameric transmembrane pores in the DIBs, which allow the diffusion of small molecules (less than 2 kDa; L. Song et al., Science 1996, 274, 1859) between the droplets. Other protein pore types may be selected for allowing passage of larger molecules. We have previously optimized the packing of our droplets within our 3D-printed droplet networks to maximize hexagonal close-packing (A. Alcinesio et al., Nat Commun 2020, 11, 2105).

[0365] To interface these droplet networks with bacteria, droplet networks were transferred from the printing chamber (Figure 1(a), right graphic) to a lipid-in-oil solution on top of a bacterium -laden hydrogel (Escherichia coli in 30 pL of 1.5% (w / v) ultra-low gelling temperature agarose (ULGA), forming a cylindrically-shaped hydrogel with a circular area of -0.32 cm2and a thickness of -1 mm). E. coli cells were dispersed within the hydrogel at starting densities ranging from 1.6 x 107to 4.0 x 1010cells mL1. Once a droplet network (8 x 8 x 8 droplets) came into contactwith the hydrogel, bilayers formed between the external droplets of the tissue and the lipid monolayer at the hydrogel surface that were stable for weeks, with a quadratically-shaped area of ~0.3 mm2to -0.4 mm2(Figure 1(b)) (J. R. Thompson et al., Nano Lett. 2007, 7, 3875).

[0366] To observe gene expression in the E. coli population (BZB1011 Pmax:sfgfp::Tn7 pI \-PBAD.-mCherry-AMP - mCherry-inducible (Table SI)) by the release of arabinose from droplet networks, the bacteria contained the plasmid pJSl-PBAD -mCherry-AMP (Figure 7(a)) encoding a fluorescent protein, mCherry, downstream of the promoter (PBAD), which is regulated by arabinose. Once droplet networks (initially containing 33 RIM arabinose and 25 pg mL1aHL monomer) were placed on top of the bacterium-laden hydrogel (t = 0 h), aHL inserted into the DHB, establishing a flux of arabinose through the pores into the bacterium -laden hydrogel (Figure 1(c)). Over 18 h, we observed that E. coli cells were actively growing and dividing from single-cell dispersions (t = 0 h, Figure 1(d) and Figure 1(e)) to form 3D micro-colonies (R. Krishna Kumar et al., Nat Commun 2021, 12, 857), and cells directly below the droplet networks (within -50 pm to 100 pm underneath the contact area) and in close proximity to the droplet networks (within -100 pm from the edge) expressed high amounts of mCherry, quantified as the mean gene expression, IM (see Methods), of activated cells (634 a.u., Figure 1(e) and Figure 1(f)). Cells further away from the droplet network expressed only baseline levels of mCherry (179 a.u., Figure 1(e) and Figure 1(f)). An increase in fluorescence was detected at approximately t = 10 h (Figure 1(g)). This was followed by a rapid increase in mCherry expression, consistent with the all-or-nothing nature of the pBAD

[0367] system (Supplementary Note 1). After -18 h, no further increase in mCherry expression was detected. Droplet networks without aHL barely induced gene expression (Figure 1(g)), confirming the limited permeability of the lipid bilayers to arabinose (M. G. Sacerdote et al., Proceedings of the National Academy of Sciences 2005, 102, 6004) compared to other commonly used chemical signals, such as IPTG (A. Dupin et al., Nature Chem 2019, 11, 32). Therefore, localized arabinose flux from droplet networks can be controlled by the permeabilization of bilayers with aHL pores, allowing localized gene expression within a homogenous population of bacterial cells.

[0368] 2.2 Patterned Gene Expression Through Optimized Arabinose Flux

[0369] To understand and quantify the factors that control spatiotemporal release of arabinose and thus local gene expression of the bacterial population, we introduced the pattern fidelity (PF) index:

[0370] pp _ <AJ-AU)

[0371] A! ’ ’

[0372] where Ai is the intended area of expression (the x, v-planc cross-sectional area of a droplet network where aHL inserts into the DHBs), and where Au is the area of unintended expression (see Methods). We sought a F approaching a value of 1, reflecting minimal gene expression in unintended areas, Au (Figure 2(a)), while maintaining a high level of gene expression, by exploring the aHL and arabinose concentrations in the droplet networks. To evaluate control over geneexpression within intended areas of gene expression, Aj, we further introduced the normalized measure, AN, which indicated areas where no gene expression was induced within 4 / (Figure 2(a)). At a fixed concentration of arabinose (33 mM), we found that increased concentrations of aHL monomer (0 - 50 pg ml1) led to decreased PF due to increasing A u, presumably caused by a greater flux of arabinose (Figure 2(b)). Moreover, we observed an increase in total gene expression, IT (sum of all pixel values in activated pixels, see Methods), with increased aHL

[0373] concentration (Figure 2(b)), which we reasoned was due to both increased gene expression in unintended areas of gene expression, Au, and increased gene expression within Ai, which was confirmed by decreased areas of AN. At a fixed concentration of aHL monomer (50 pg mb1), arabinose concentrations higher than 33 mM also led to decreased values of PF, associated with increasing Au and h and decreasing areas of no gene expression within intended areas of gene expression, AN (Figure 2(c)).

[0374] We found that at low aHL monomer (<10 pg mL1) and low arabinose (<10 mM) concentrations we could attain PFs close to 1, however, ITwas low (<1.2 x 108a.u., Figure 2(b) and Figure 2(c)). Therefore, we settled on concentrations of aHL monomer (25 pg mL1) and arabinose (33 mM) where F was 0.77, but A was high (>1.4 x 108a.u., Figure 2(b) and Figure 2(c)) and AN was low (0.12±0.07, Figure 2(b) and Figure 2(c)). At these concentrations, we printed a range of patterned droplet networks designed to produce different release patterns of chemical signals, by using droplets that did or did not contain arabinose and aHL. These printed networks produced gene expression patterns within a homogenous population of bacteria at PFs of >0.89 (Figure 2(d)-(g), Supplementary Note 2). To further evaluate the control over induced gene expression as a consequence of arabinose release from 3D-printed synthetic tissues, we introduced AN as a means to quantify areas of no gene expression AN) within areas of intended gene expression (A / ). We noticed AN values between 0.48 and 0.85. We next investigated whether we could store larger amounts of arabinose within printed tissues while maintaining spatial control over gene expression. Therefore, we sought to vary the number of layers (4, 8, 16 in total), while keeping Ai unchanged (Figure 3(a)). The volume-to-surface-area ratio (RVSA) was defined as:

[0375] RVSA=’ (2)

[0376] where Pis the total volume of the droplet networks containing arabinose and aHL. According to this definition, droplet networks with an increasing number of network layers (4, 8, 16) are characterized by RVSA values of 0.25 mm, 0.49 mm and 1.07 mm, respectively. Increased RVSA indeed led to enhanced total gene expression, IT, and decreased areas of AN (with mean AN / AI values of 0.81±0.17, 0.57±0.18 and 0.38±0.15 for RVSA values of 0.25 mm, 0.49 mm and 1.07 mm, respectively, Figure 3(b,c)). Importantly, unintended expression, Au, barely increased (from Au / Ai values of 0.02 with 4-layered networks to 0.08 with 16-layered networks, corresponding to high PFvalues of 0.98 and 0.92, respectively, Figure 3(b)). To reduce printing time and further increase storage capacity (Supplementary Note 3), we printed droplet networks composed of 4 patterned layers at the bottom (mask) and 4 uniform layers at the top entirely composed of arabinose and aHL-containing compartments (reservoir, Figure 3(d) and Figure 3(e)). By equipping our droplet networks with magnetic handles (agarose droplets containing MagneHis™ Ni-particles) and placing a magnet placed underneath the bacterium-laden hydrogel, droplet networks were precisely guided to interface with the bacterium-laden hydrogel through the mask layers rather than the reservoir layers (Figure 3(f), Supplementary Note 3, Figures 8 and 9), retaining patterned gene induction (Figure 3(g)).

[0377] 2.3 Improved Resolution of Gene Expression with "Shrunken" Tissues

[0378] We next sought to achieve even higher spatial resolution of patterned gene expression with droplet networks. We found that chemical signal release can be achieved through a linear, single-droplet pathway (with droplet diameters of 65 pm) containing arabinose and aHL (Figure 4(a) and Figure 4(b)). In this case, gene expression was induced within a circular area of -100 pm diameter (Figure 4(c)). To further increase the resolution, we developed a heat-induced postprinting shrinking process (Figure 4(d)). At the start of this process, we observed increasing contact angles between droplets, forming more tightly packed droplet networks (droplet annealing). Then, the volume of the compartments continuously decreased, presumably as water molecules partitioned into the oil, from which water molecules eventually got released to the unsaturated atmosphere in an evaporative process (Figure 4(d)). Importantly, the general morphology of the droplet networks (Figure 4(e)) and the patterned arrangements of the chemical signal -containing droplets were barely affected (Figure 4(i)). We found that the rate at which the droplets shrank increased with temperature (Figure 4(f)), which we reasoned was due to the exponential increase in water vapor pressure (A. Wexler, J Res Natl Bur Stand A Phys Chem 1976, 80A, 775) and an increased solubility of water molecules in the oil phase with increasing temperature, causing water flow from droplet networks through the oil into the atmosphere. Additionally, we discovered that for a given droplet, the shrinking rate decreased with an increasing number of neighboring droplets. For example, we found that droplets at the periphery of droplet networks shrank more quickly than droplets in the center (Figure 4(g)), likely because water molecules from peripheral droplets partition into the oil phase first. Solute concentrations were diluted prior to printing, such that target concentrations were reached within the droplets after the evaporation process. By using shrunken droplet networks, we significantly increased spatial control compared to single -droplet pathways (Figure 4(a)) and previous patterns, such as a triangular pattern (Figure 4(h)). Shrunken networks activated gene expression within a frame-like pattern, the width of which was

[0379] <50 pm (Figure 4(i) and (j)).

[0380] 2.4 Switchable Induction of Gene ExpressionThe release of arabinose is initiated once aHL pores insert into the DHBs formed between the droplet network and the bacterium -laden hydrogel. We sought to activate the release by taking advantage of the modularity of droplet networks (A. Alcinesio et al., Advanced Functional Materials 2022, 32, 2107773). We printed two separate droplet networks: one contained an arabinose reservoir (the storage module), and the other contained an aHL-mediated droplet pathway (the release module). If the storage and release modules were not connected or connected incorrectly (Figure 5(a)), no gene expression was induced (Figure 5(b)). In contrast, if the modules were assembled correctly, arabinose diffused into the bacterium -laden hydrogel (Figure 5(c)) inducing patterned gene expression within the bacterial population (Figure 5(d)). By these means, we controlled the release of chemical signals by a key-lock-mechanism which couples two droplet network modules, allowing chemical signal release only once the two modules are connected correctly.

[0381] 2.5 Patterned Bacterial Competition

[0382] Finally, to demonstrate the utility of patterned chemical signal release from droplet networks, the approach was applied to control interference competition in bacterial communities (I. Brook, Critical Reviews in Microbiology 1999, 25, 155). Specific E. coli strains can inhibit the growth of closely related strains by producing and releasing (by self-lysis) proteinaceous toxins, such as DNA-damaging colicins (e.g. colicin E7 and E8, Supplementary Note 4.1-4.6, Figure 10, Figure 11, Figure 12 and Figure 13) (E. Cascales et al., Microbiology and Molecular Biology Reviews 2007, 71, 158; E. T. Granato et al., Current Biology 2020, 30, 2836).

[0383] To test the potential of droplet networks to spatially control competition between colicin-producing cells and susceptible cells, we engineered an E. coli strain to produce colicin E7 and the associated immunity and lysis proteins in the presence of arabinose (BZB1011 P max: sfgfp: :Tn7 pKCl-PBAD -ColE7-AMP - E7-inducible, Figure 6(a), Table SI, Figure 7(b)). When droplet networks containing arabinose (333 HIM) and aHL monomer (50 pg mL1) were placed on top of bacteriumladen hydrogels containing inducible E7 cells, the number of micro-colonies underneath the droplet networks was significantly reduced from 927.3±74.1 to 62.3±41.7, indicating colicin E7 release from the inducible cells through cell lysis (Supplementary Note 4.7, Figure 6(b), Figure 14(a) and Figure 14(b)).

[0384] Next, we used a reporter plasmid that produces sfGFP upon DNA damage (BZB1011 pUA66-PcolE2:sfgfp - S-GFP, Figure 6(a), Table SI, Supplementary Note 4.8) (E. T. Granato et al., Current Biology 2020, 30, 2836; D. A. I. Mavridou et al., Current Biology 2018, 28, 345) to investigate DNA damage in S-GFP cells as a consequence of colicin E7 exposure. From this, E7R-inducible (BZB1011 pYYl-PBAD -ColE7-mCherry-AMP - E7R-inducible, Table SI, Figure 7(c)) and S-GFP cells were mixed homogenously within the hydrogel at an initial ratio of

[0385] 1:1 (Figure 6(c)). In the absence of arabinose, E7R-inducible and S-GFP cells grew at similar rateswith negligible DNA damage in the S-GFP cells (as observed by the lack of sfGFP expression) over 18 h (Supplementary Note 4.8, Figure 15(a) and Figure 15 (b)). By releasing arabinose from droplet networks (50 RIM arabinose, 50 pg mb'1aHL monomer) into bacterium-laden hydrogels containing E7R-inducible and S-GFP cells at an initial ratio of 1: 1, local DNA damage in S-GFP was observed underneath the droplet networks (Figure 6(c) and Figure 15(c)), which was confirmed to correlate with cell growth inhibition (Supplementary Note 4.8, Figure 15(b)).

[0386] To maximize the mean sfGFP expression (DNA damage) and PF of DNA damage, droplet networks containing ranges of aHL and arabinose concentrations were placed on top of bacteriumladen hydrogels containing both E7R-inducible and S-GFP cells. We found that increased arabinose and aHL concentrations in droplet networks led to increased DNA damage in S-GFP cells (Figure 6(d), Figure 15(c) and Figure 16) but decreasing F values (Figure 6(e)). To achieve patterned DNA damage in S-GFP cells as a result of colicin E7 expression, droplet networks containing 66 RIM arabinose and 30 pg mL1aHL were printed, as this combination produced an intermediate level of DNA damage (mean sfGFP expression IM) of 471 a.u.), at a high PF value of 0.98. By using these conditions, patterned DNA damage was achieved with different printed patterns atPFs between 0.93 and 0.99 (Figure 6(f)).

[0387] Finally, we investigated control of the competition between E7-inducible and the natural colicin E8-expressing strain (BZB1011 Pmax:mrfpl::Tn7 pColE8 - E8, Figure 6(g), Table SI). When droplet networks induced expression of colicin E7 through arabinose release (at 333 RIM arabinose and 50 pg mL'1aHL), the number of E8 cells was significantly reduced within a localized area underneath the network. We hypothesized that the elimination after 12 h (Figure 6(h,i)) was caused by high local concentrations of colicin E7 through lysis, inducing E8 to counter-attack (as the natural E8 operon is regulated by DNA damage) through self-lysis (Supplementary Note 4.1). We found that levels of colicin E7 expression and release from E7-inducible cells necessary to eliminate colicin E8-expressing cells were only reached at an initial starting ratio, which was defined as the ratio between cells of one over the other genotype, of 9: 1 (Figure 6(j), see Methods (Definitions and Calculations)). Interestingly after 18 h, E8 cells were able to partially grow back into the cleared area (Figure 6(i)). In summary, we have shown that droplet networks can locally control which strains win or lose in well-mixed bacterial communities through the patterned-release of chemical signals that upregulate toxin production in bacterial strains.

[0388] 3. Concluding remarks in relation to the Example

[0389] In this Example, we present chemical signal-based communication between 3D-printed droplet networks and bacterial cells. The flux of arabinose, the chemical signal, can be precisely controlled, by adjusting signal and pore concentrations within the droplet networks, to produce patterned gene expression in bacterial populations. We can magnetically guide our droplet networks to land precisely onto bacterial populations, controlling where chemical signal molecules can be released.In addition, we can store chemical signals in printed droplet reservoirs, while maintaining high spatial control over gene expression. We further increased the resolution of patterned gene expression significantly to 50 pm by using evaporative shrinkage of the networks, which represents a significant improvement when compared with the millimeter resolution of previous work (T. Danino et al., Nature 2010, 463, 326; A. Tamsir et al., Nature 2011, 469, 212; T. Sohka et al., Proceedings of the National Academy of Sciences 2009, 106, 10135; J. J. Tabor et al., Cell 2009, 137, 1272; S. Basu et al, Nature 2005, 434, 1130; J. Zhang et al., J Mol Biol 2020, 432, 3137). Further, by using our key-lock mechanism the release of chemical signals can be activated at a controlled point in time.

[0390] Critically, we show the utility of our system by inducing the expression of bacteriocins in E. coli. which drives patterned DNA damage in co-cultured susceptible cells and local competition between toxin-producing strains with different potencies. Therefore, the present work demonstrates chemical signal release from 3D-printed droplet networks as a tool for the study of spatiotemporal dynamics of complex cellular behaviors within microbial communities.

[0391] Additionally, we envision that our droplet networks might encapsulate cell-free protein expression systems (M. J. Booth et al., Science Advances 2016, 2, el600056) which could mediate the release of chemical signals in response to the environment. For example, n-acyl homoserine lactones (O. D. Toparlak et al., Science Advances 2020, 6, eabb4920) released from bacterial cells could drive the expression of aHL inside droplet networks, which would then allow chemical signals, such as arabinose, to be released back onto the bacterial cells, inducing the expression of a gene of interest, thereby, establishing bidirectional communication between droplet networks and bacterial cells. To further control chemical signal release, membrane proteins that gate in response to stimuli, such as Zn2+(A. Alcinesio et al., Advanced Functional Materials 2022, 32, 2107773; O. Braha et al., Chemistry & Biology 1997, 4, 497; M. J. Booth et al., Commun Chem 2019, 2, 1) or light (C. Chang et al., Chemistry & Biology 1995, 2, 391) could be used to permeabilize membranes to achieve light-sensitive chemical signal release (C. Chang et al., Chemistry & Biology 1995, 2, 391).

[0392] We envisage that our modular system can encapsulate different types of chemical signals (such as drugs, peptide / proteins, or DNA / RNA), as well as having the ability to integrate with other effector technologies (for example lipid nanoparticles) for interactions with various cell types. Membrane pores with larger internal diameters could be used to allow larger signaling molecules to be released from droplet networks (O. D. Toparlak et al., Science Advances 2020, 6, eabb4920). For example, perfringolysin O has a diameter between 25 nm and 30 nm (T. X. Dang, Journal of Structural Biology 2005, 150, 100) as compared with the aHL pore, whose narrowest inner diameter is 1.4 nm (L. Song et al., Science 1996, 274, 1859). Moreover, chemical signals could be produced in droplet networks by cell-free protein expression or multiple types of droplet networkcould be connected to restore chemical signals once depleted (e.g. by exchanging storage modules) and to release multiple types of chemical signals (e.g. by connecting storage modules containing different chemical signals to one release module).

[0393] Further, our droplet networks could function in bulk aqueous environments by adapting previous work of forming external lipid bilayers surrounding the droplet networks to interface with cells in 3D (A. Alcinesio et al., ChemSystemsChem 2022, 4, e202100036).

[0394] Lastly, 3D-printed hybrid tissues that contain living cells might be constructed (R. Krishna Kumar et al., Nat Commun 2021, 12, 857; A. D. Graham et al., Sci Rep 2017, 7, 7004; L. Zhou et al., Advanced Materials 2020, 32, 2002183) where gradients of growth factors control the differentiation of cells at defined locations, thereby allowing the formation of complex tissues (Z. Liu et al, Progress in Materials Science 2017, 88, 467; R. Subbiah et al, Advanced Healthcare Materials 2019, 8, 1801000; L. Sardelli et al, J Appl Biomater Funct Mater 2019, 17, 2280800019829023).

[0395] In summary, the technology of the invention demonstrates patterned spatiotemporal communication between droplet networks and living cells based on chemical signals. The platform might be modified to investigate the patterning of various groups of cells through the release of various natural and synthetic signals, such as quorum sensing molecules, antibiotics and growth factors. Such an approach will prove useful in fundamental research, including the modulation of species diversity in bacterial communities or the spatiotemporal dynamics of tissue development and morphogenesis. Furthermore, the use of droplet networks in medical applications (G. Chen et al., Proceedings of the National Academy of Sciences 2022, 119, e2207525119; Z. Li et al., Science Advances 2020, 6, eaay0589; R. Chandrawati et al., Advanced Materials 2017, 29, 1604932; S. Kartha et al, Advanced Healthcare Materials 2017, 6, 1700500) is envisioned, such as in the treatment of chronic wounds, cancer, neurodegenerative diseases and spinal cord injuries.

[0396] 4. Methods

[0397] Preparing Aqueous Phases'. Lysogeny broth Miller (LB) medium (Invitrogen) was prepared by adding LB powder (25 g) to Milli-Q® water (I L) and autoclaving. 5 x M9 minimal salts solution was prepared by adding M9 minimal salts (2.82 g, Sigma- Aldrich) to Milli-Q® water (100 mL) and autoclaving. Casamino acid solution was prepared by adding Calbiochem® OmniPur® Casamino acid (5 g , Merck) to Milli-Q® water (50 mL) and autoclaving. The final M9 minimal medium consisted of 1 x M9 minimal salts, MgSO4 (2 mM), CaCL (0.1 mM, filter-sterilized using a 0.22-pm polyethersulfone membrane (Millex-GP Syringe Filter Unit)), casamino acids (0.2% (w / v), Sigma) and D-glucose (24 mM) or glycerol (24 mM). Bacterium -laden hydrogels were prepared by adding ultra-low gelling agarose (Sigma-Aldrich) to the M9 minimal medium (at a final concentration of 1.5% (w / v)) and autoclaving. The hydrogel solution was kept molten in a water bath at 37°C andprepared freshly before every experiment. Antibiotics were dissolved in Milli-Q® water, filter-sterilised (0.22-pm polyethersulfone membrane) and frozen (-20°C) as stock solutions (ampicillin: 100 mg mL1, kanamycin: 50 mg nil . Sigma- Aldrich). The final concentrations of antibiotics in media were 100 pg mL'1and 50 pg mL'1for ampicillin and kanamycin, respectively.

[0398] Construction of Recombinant DNA'. The E. coli strain BZB1011 was used for all Golden Gate cloning experiments in this study. E. coli cells were cultured in LB medium at 37°C with shaking at 250 rpm and plated on LB agar, incubated at 37°C. The LB medium and agar were supplemented with the appropriate antibiotics, either ampicillin (100 pg mL ) or kanamycin (50 pg mL ' ). For blue-white screening, agar plates were additionally supplemented with IPTG (0.1 mM, Sigma Aldrich) and Xgal (40 pg mL1, Thermo Fisher). Constructs (pKCl- T D^Co / E -AMP and fY \-PBAD>ColE7-mCherry-MAf see Table SI) were made using the MoClo kit and cloning method89. The following components were added to a 0.2 mL PCRtube: DNA components (10 ftnol each), Bsal (10 U) or BbsI (10 U) restriction enzyme (NEB), T4 Ligase (20 U, NEB), and lx T4 DNA ligase buffer (NEB). Milli-Q® water was then added to bring the total volume to 10 pL. Reaction mixtures were incubated in a thermocycler for 40 cycles of digestion and ligation (37°C for digestion for 2 min, 16°C for ligation for 5 min), followed by 5 min at 50°C and a heat kill step at 80°C for 10 min. The mixtures were then held at 4°C, and 4 pL was used to transform cells. Transformants were selected using lacZa blue-white screening.

[0399] Competent Cell Preparation and Cell Transformation'. An overnight culture of E. coli BZB1011 cells was set up by inoculating the cells into LB medium, followed by shaking (225 rpm) at 37°C for no longer than 12 h. The overnight culture was inoculated into LB (50 mL) at an ODeoo of 0.05 and grown at 37°C with shaking (225 rpm) until the ODeoo reached 0.4-0.6. The culture was centrifuged at 4481 x g at 4°C for 10 min. The supernatant was removed and the cell pellet was resuspended in a solution (25 mL) containing CaCL (100 mM) and glycerol (15% (v / v)) at 4°C. After 45 min, the cells were pelleted again by centrifugation at 4481 x g at 4°C for 10 min. The supernatant was removed and the cell pellet was re-suspended in a solution (5 mL) containing CaCL (100 mM) and glycerol (15% (v / v)) at 4°C before aliquoting and storing at -80°C.

[0400] Chemically competent E. coli cells (BZB1011, 750 pL) were thawed on ice and of the plasmid of interest (100 ng) was added. After 30 min on ice, the cells were heat-shocked for 45 sec at 42°C. After 2 min on ice, the cells were added to pre-warmed super optimal broth (SOC, 1 mL) at 37°C and incubated for 1 h. The cells were then inoculated onto LB-agar plates supplemented with antibiotics (100 pg mL1ampicillin, 50 pg mL1kanamycin). After overnight culture at 37°C, a single colony was picked and inoculated into LB (4 mL), supplemented with antibiotics

[0401] (100 pg mL1ampicillin, 50 pg mL1kanamycin), and shaken for no longer than 12 h at 37°C (225 rpm). The culture was then mixed with an equal volume of glycerol (50% (v / v)) solution. Glycerol stocks were stored at -80°C.Preparation of Bacterium-laden Hydrogels'. E. coli cells (BZB1011) were pipeted from glycerol stocks into a round botom tube containing LB (4 mL) supplemented with antibiotics ( 100 pg ml1ampicillin, 50 pg mL1kanamycin). Cells were grown for no longer than 12 h at 37°C with shaking (225 rpm). 40 pL of overnight culture were then transferred to a round botom tube containing LB (4 mL) supplemented with antibiotics (100 pg mL1ampicillin, 50 pg mL1kanamycin) and grown for 3 h at 37°C with shaking (225 rpm). Based on the ODeoo, which was measured using a spectrophotometer (BioRad SmartSpec™ Plus, with a conversion factor of 1.0 = 108cells mL1), appropriate amounts of culture were added to a 1.5 mL tube and centrifuged for 8 min at 8000 x g. The supernatant was removed and the cells were re-suspended in appropriate amounts of pre-heated (37°C) M9 medium containing ultra-low gelling agarose (final concentration: 1.5% (w / v)) to form a molten bacterium-laden hydrogel solution with the desired cell density (i.e. ODeoo 0.02, 2 or 4.5). In case of experiments involving two bacterial strains (i.e. bacterial interference competition) the amount of cells were adjusted according to the starting ratio to reach the desired total cell concentration. The resulting cell suspension (30 pL) was added to a number of wells (<30) of a 96-well plate according to the number of experimental conditions and solidified at 4°C for 35 min.

[0402] Expression and Purification of aHI . The aHL monomers were prepared by transforming E. coli BL21(DE3) pLysS cells (Agilent) with the pT7-aHL-DsH6 plasmid90and inoculated onto LB-agar plates containing antibiotics (carbenicillin, 50 pg mL1. chloramphenicol, 34 pg mL1). A single colony from the plate was picked and inoculated in LB (10 mL) for the pre -culture. An LB culture (400 mL) containing the same antibiotics was inoculated with overnight pre-culture (4 mL). This expression culture was shaken at 37°C at 250 rpm for approximately 3 h until the ODeoo reached 0.6, when it was cooled to 18°C before the addition of IPTG (2 mL, 0.1 M, Fluorochem) to induce protein expression. The culture was further shaken at 18°C at 200 rpm overnight. The cells were then harvested by centrifugation in a Beckman J25 centrifuge at 5000 rpm for 20 min at 4°C and resuspended in lysis buffer (10 mL, 50 RIM Tris-HCl, pH 8.0, 150 RIM NaCl, 10 RIM imidazole, 0.1% Triton X-100, 5% glycerol, 2 RIM TCEP with an EDTA-free protease -inhibitor tablet (ThermoFisher)). Lysis was then performed by the addition of lysozyme (250 pL, 40 mg niL1. ThermoFisher), universal nuclease (2 pL, 250 U L ThermoFisher) and MgCL-containing solution (25 pL, 2 M), and incubation on ice for 1 h. The lysate was sonicated at 40% amplitude for 3 min in a 30 s-ON-30 s-OFF pulse train on ice (VCX 500, Sonics). The supernatant was cleared by centrifugation at 29000 x g for 45 min at 4°C and transferred to a gravity column containing Ni-NTA resin (1 mL, bed volume, ThermoFisher). The lysate supernatant and resin mixture were mixed at 4°C on a platform rotator for 1 h. The column was washed with washing buffer (2 x 15 mL, 50 RIM Tris-HCl, pH 8.0, 500 RIM NaCl, 20 RIM imidazole, 2 RIM TCEP, 0.1% Triton X-100 and 5% glycerol) and eluted with elution buffer (50 RIM Tris-HCl, pH 8.0, 500 RIM NaCl, 250 RIMimidazole, 2 RIM TCEP, 0.1% Triton X-100 and 5% glycerol). The fractions (~10 mL) containing aHL were combined and loaded onto a HiLoad 26 / 600 Superdex 200pg (Cytiva) SEC column equilibrated with SEC buffer (10 RIM Tris-HCl, pH 8.0, 150 RIM NaCl, 2 RIM TCEP and 5% glycerol) at 4°C. Fractions containing monomers of aHL were concentrated (1 mg mL1) and stored at -80°C as aliquots. The mass of the monomer was verified by LC-MS. On average, 9 mg pure aHL monomer after SEC purification was obtained from culture (400 mL).

[0403] Preparation of Lipid-in-oil Solutions'. Lipids (l,2-diphytanoyl-sn-glycero-3 -phosphocholine (DPhPC, 4ME 16:0-18:1 PC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC, 16:0-18:1 PC); Avanti Polar Lipids) were dissolved in anhydrous chloroform (2.5 mL, 10 mg niL1. Sigma-Aldrich). The final lipid composition DPhPC:POPC (2: 1 molar ratio) was prepared in chloroformcleaned, Teflon capped glass vials (Supelco®). The chloroform was evaporated under nitrogen and the remaining solvent removed by placing the vials under vacuum for 24 h. The vials were stored at -80°C under argon. Before use, the vials were brought to room temperature for 15 minutes and a pre-mixed oil solution consisting of undecane (Sigma-Aldrich) and silicone oil (AR20, Wacker) in a ratio of 35:65 (v / v) was added. The lipid-in-oil solution was vortexed and then

[0404] sonicated (Branson 2800 ultrasonic bath 230 V) for 1 h at 25 to 35°C and vortexed again before use. The total concentration of lipids was 2 RIM.

[0405] 3D-Printing of Droplet Networks'. The droplet networks used in this work were formed by using a 3D-printing device as described elsewhere (I. Cazimoglu, M. J. Booth, H. Bayley, ACS Nano 2021, 15, 20214). Briefly, an aqueous solution (M9 minimal medium supplemented with various concentrations of L-(+)-arabinose (0 - 333 RIM, Sigma-Aldrich), aHL monomer (0 - 50 pg mL1) and cascade blue dextran (250 pvi. Invitrogen, Cat. D1976) was ejected from a glass nozzle into the lipid-in-oil solution in a printing cuvette. The printing cuvettes (composed of special optical glass (SOG), Starma Scientific) were mounted on a micromanipulator stage (Patch Star 7000, Scientifica), which moved in xyz-dircction so to position the static glass nozzle according to a printing map. During droplet ejection, monolayers of lipid assemble spontaneously at the interface between the aqueous droplets and the lipid-in-oil solution. Lipid bilayers form between neighboring droplets when lipid monolayers contact one another. The placement of individual droplets with various contents can be controlled by using a multiple nozzle setup, where patterned droplet networks are formed by initializing the printing software according to the relative position of the glass nozzles. The droplet size (60 - 120 pm in diameter) was controlled by adjusting pulse voltage and / or pulse duration of the piezo driver.

[0406] Placement of Droplet Networks on Bacterium-laden Hydrogels'. Lipid-in-oil solution (50 pL) was pipetted on top of bacterium -laden hydrogels in a 96-well plate and incubated at room temperature for 15 min. Then, droplet networks were transferred to the lipid-in-oil solution using a pipette, when they sank forming robust DHBs at the interface with the hydrogel. After network placementthe 96-well plates were incubated at 37°C for 18 h until further analysis (see pattern fidelity quantification by image analysis).

[0407] Pattern Fidelity Quantification by Image Analysis'. Bacterium -laden hydrogels and droplet networks were imaged with an epi-fluorescence microscope (Leica DMI8, camera: DFC7000T) and a laser scanning confocal microscope (Zeiss LSM780). Epi-fluorescence images were recorded after placement of the droplet networks (at t = 0 h and t = 18 h, unless stated otherwise). The same settings were used throughout this work with 5x magnification (objective: N PLAN 5x / 0.12 DRY): cascade blue dextran: Zex: 327 - 383 nm, Xem: 435 - 485 nm, exposure time: 600 ms, gain: 1; sfGFP: Zex: 450 - 490 nm, Zem: 500 - 550 nm, exposure time: 200 ms (for constitutive expression of sfGFP) or 1 s (for bacterial interference competition), gain: 1; mCherry: Zex: 540 - 552 nm, Zem: 567 - 643 nm, exposure time: 2 s, gain: 1. Images were composed of 1920 pixels in x- direction (xmax) and 1440 pixels in v-di recti on (ymax), spanning a field of view of 2.4887 mm (x-direction) by 1.8662 mm (y-di recti on) and were analyzed by using MATLAB R2021b. The pixel value range spanned 0 to 4095. Baseline gene expression of mCherry, lB. arising from bacteriumladen hydrogels without arabinose, was computed based on the mean of all pixel intensity values ^x,ymcherry

[0408]

[0409] fluorescenceimages, where x is the xthpixel in x-direction and y is they* pixel in v-direction of an image):

[0410] y ymax r lB=X=1 y=1 xymcherry =179 a.u. (3)

[0411]

[0412] xmaxymax Accordingly, pixel intensity values >179 a.u. were considered to reflect induced gene expression of mCherry (termed activated pixels). The mean gene expression, IM, of all activated pixels in mCherry fluorescence images was computed as follows:

[0413] ^xmaxMymax , v nri _ / _ Lx=1Ly=1‘X, y mCherryXmCherry

[0414]

[0415] 'M - r , I4) where, H(z) is the Heaviside step function, defined as:

[0416] (0 if z < 0

[0417]

[0418] (5)

[0419] and where Nais the total number of activated pixels.

[0420] The total gene expression, IT, was calculated as the sum of pixel values of all activated pixels in mCherry fluorescence images:

[0421]

[0422] IT = SSr r <T.,mch,„yX - IO. (6) using Equation (5).The area of intended gene expression, Aj, was determined by multiplying the total number of activated pixels in cascade blue dextran (CBD) fluorescence images with the area of a single pixel, APiX(1.68 x IO-6mm2):

[0423] A

[0424]

[0425] , = & H(Jx,ycBD- ICBD) X Apix, (7) where Ix,ycBDare the pixel values of cascade blue dextran fluorescence images and ICBD = 500 a.u. was set as an appropriate threshold to capture the outline of the area comprising of droplets containing cascade blue dextran and, hence, arabinose and aHL. The same step function was used (see Equation (5)).

[0426] The area of unintended gene expression, Au, was calculated by multiplying the total number of pixels outside of Ai with Apix

[0427]

[0428] = SSr - / «) X X (S) The area of no gene expression, A-,-. was calculated by multiplying the total number of pixels within Ai with Apix

[0429] AN= 1IXX-^ 11^X\H(IX V- IB) X H(lx v^n- lCBD} \ -x. A„ix. (9)

[0430]

[0431] was computed by using Equation (1).

[0432] For bacterial interference competition, PF was determined as described above, with sfGFP as the read-out for induced gene expression, as opposed to mCherry. Here, a threshold value of >200 was used, which was determined from the baseline expression of sfGFP as a consequence of colicin E7 baseline expression levels in bacteria without the presence of arabinose.

[0433] Volume -to-surf ace -area Ratio (RVSA)'. First, the area of intended gene expression, Ai, of a droplet network was determined by multiplying the number of cascade blue dextran pixels reaching the threshold of 500 with Apix. Based on Ai, the pixel size (mm2) and the number of droplets per layer, the cross-sectional area of a single droplet was determined (mm2). This information was used to determine the volume of a single droplet (mm3) and the volume of a droplet network based on the total number of droplets (mm3). The volume-to-surface-area ratio RVSA was then determined as shown in Equation (2), yielding a value (mm) describing the relation between volume and area of intended gene expression, Ai.

[0434] Magnetic Handles for Guided Landing of Droplet Networks'. Magnetic handles were prepared by adding a pre-heated (60°C) solution (composition not stated by manufacturer) containing Ni- particles (MagneHis™ Ni-Particles, Promega) to pre-heated (60°C) M9 medium containing 1.5% (w / v) of ULGA at a volume ratio of 2:3. Droplets (50 - 250 pm diameter) were then ejected (Femto Jet 4x, Eppendorf) into lipid-in-oil (same composition used for printing of droplet networks) and cooled at 4°C for 35 min. The gelled droplets were transferred next to the comers of a dropletnetwork by pipetting and pushed onto the droplet network to which they adhered through DIB formation. The droplet network was then placed on top of a bacterium-laden hydrogel, under which a magnet had been placed to direct the orientation of the droplet network.

[0435] Heat-induced Dehydration of Droplet Networks'. Droplet networks were printed in SOG cuvettes, which contained lipid-in-oil solution (500 pL). The cuvettes were then placed in the center of a transparent heating plate (Leica Thermo Plate), which was set to 36 °C or 43 °C. The temperature in the lipid-in-oil solution was allowed to reach equilibrium as determined with a sensor (Thorlabs, TSP01). The printing solution was diluted with Milli-Q® water so that the concentrations of the components in the droplet networks would be as desired after shrinkage (36 °C or 43 °C). Once the desired decrease in volume was attained as determined by microscopy, a shrunken network was brought to room temperature for 30 min before transfer on top of the bacterium-laden hydrogel. Droplet Networks as Switchable Modules'. Two kinds of droplet networks were printed separately; one acted as a storage module and the other as a release module. Functional droplets in storage modules (containing 33 RIM arabinose, 50 pg mL'1aHL and 250 M cascade blue dextran; cyan compartments, Figure 5) were connected with functional droplets in release modules (containing 50 pg mL'1aHL and 250 M cascade blue dextran; yellow compartments, Figure 5). Storage and release modules were connected by gently pushing them together in lipid-in-oil solution by using a pipet. Once the droplet networks were connected correctly, such that arabinose could diffuse from the storage modules through aHL pores to release modules, gene expression was induced in bacterial cells according to the pattern formed by the functional droplets of the release module at the xv-planc (e.g. a stripe-like pattern, Figure 5(d)).

[0436] Definitions and Calculations'.

[0437] Mean Gene Expression IM'. Mean gene expression (mCherry or sfGFP) refers to the sum of all pixel values in activated pixels divided by the total number of activated pixels, where pixels are considered activated when baseline expression levels are reached (see Pattern fidelity quantification by image analysis).

[0438] Total Gene Expression If Total gene expression (mCherry or sfGFP) refers to the sum of all pixel values in activated pixels, where pixels are considered activated when baseline expression levels are reached (see pattern fidelity quantification by image analysis).

[0439] Starting Ratio: Starting ratio refers to the ratio between cells of one over the other genotype in a bacterial community at the start of an experiment when the two genotypes were mixed homogeneously in an ULGA hydrogel solution (t = 0 h).

[0440] Bacterial competition: Bacterial competition refers to the process by which one individual decreases the survival or reproduction of others.

[0085]

[0441] Statistical Analysis'.Epi-fluorescent microscopy images were processed according to “Pattern Fidelity Quantification by Image Analysis”, which was visualized in Figure 2(a). The data is presented as mean ± standard deviation (SD) and the number of replicates is stated in each figure caption. For analysis of statistical differences between groups it was first tested whether the data was normally distributed using the Shapiro-Wilk test (p < 0.05). Given groups were normally distributed, significance between groups was tested performing a two-sample t-test. If data of at least one group was not normally distributed, significance between groups was tested by performing a Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0442] Supplementary Note 1: Arabinose-induced Gene Expression: pBAD Rationale

[0443] We chose pBAD as an inducer system for multiple reasons: 1) The permeability of arabinose (chemical signal) was minimal (Figure 1(h)) through lipid bilayers (comprising of a lipid composition 2: 1 (molar ratio) DPhPC:POPC) formed between compartments within droplet networks (droplet interface bilayers = DIBs) and between droplet networks and the

[0444] hydrogel (droplet hydrogel bilayers = DHBs). This allowed us to control the arabinose flux from the droplet networks into the bacterium-laden hydrogels by changes in both arabinose and aHL concentration, and prevented diffusion of arabinose between compartments within the droplet networks unless aHL was present in the DIBs. 2) pBAD systems have been reported to behave as an all-or-nothing system (D. A. Siegele et al., Proc Natl Acad Sci U S A 1997, 94, 8168), which we reasoned would be ideal to achieve patterned gene expression based on arabinose flux from droplet networks. We hypothesized that below or above a critical arabinose concentration, the gene expression in cells would be low and high, respectively, rather than a gradient going from low to high. Hence, a tight population gene expression pattern could be achieved depending on the arabinose gradient released overtime. This all-or-nothing system works once a critical concentration of arabinose is reached, resulting in high expression levels of genes downstream of the PBAD promoter, such as mCherry or cxE7. At the same time, increased activity of the transcriptional regulator AraC induces the expression of araE. which encodes for the arabinose transporter AraE. This increases the uptake of arabinose, leading to a positive feedback loop that rapidly increases protein expression in the cells until a maximum is reached.

[0445] Supplementary Note 2: Pattern Fidelity as a Measure of Gene Expression Controllability As described, our F measure describes the controllability of gene expression in E. coli populations by comparing the area of unintended gene expression, Au, to the area of intended gene expression, Ai. Another measure we considered was factoring AN, which refers to the area within Ai, where no gene expression was induced. However, this measure was not chosen to optimize arabinose release from 3D-printed droplet networks, as we did not encounter areas within Ai wheregene expression was not induced (unless droplets did not form DHBs with the hydrogel (see Figure 3(g))). Therefore, our measure was chosen to quantify gene expression in areas outside of the intended gene expression area as a measure over controlled release of arabinose. However, in addition we reported values of A, (Figure 2(g)), which takes into account areas within A where insufficient DHB formation as a consequence of printing defects and irregularities of the surface of bacterium -laden hydrogels prevented arabinose release.

[0446] By using cascade blue dextran to reveal where arabinose was released from droplet networks, we could determine the area of unintended gene expression, AuandAjv (Figure 2(a)). However, in some cases, the definitive location of arabinose release was imperfect due to imaging limitations (A. Alcinesio et al, Nat Commun 2020, 11, 2105). This is because the layers of droplets in our droplet networks are stacked by shifting every other layer in both the x- and v-dircctions such that hexagonally-packed structures can form. So, droplets of the same position in x and y within the droplet networks were offset depending on the layer. Therefore, accurate determination of the droplets from which arabinose was being released into the bacterium-laden hydrogel was difficult in some cases. In particular, this may cause inaccuracies in locating fine patterns, such as singledroplet diffusive pathways (Figure 4(b)) or patterns in mask layers, where the fluorescence signal from cascade blue dextran in droplets comprising reservoir layers may interfere with the fluorescence signal from cascade blue dextran droplets in the mask layers (Figure 10 and 11).

[0447] Supplementary Note 3: Magnetic Beads for Guiding Tissue Placement

[0448] Our 3D droplet printing technology can be time-consuming when printing intricate patterns. This is because, 1) droplets are positioned one after another, 2) when printing droplets of different compositions (e.g. with or without arabinose and ocHL), two printing nozzles are used of which the printing stage has to be moved between the nozzle positions. For example, printing droplet networks composed of 10 x 10 x 8 droplets (in xvz-dircction) takes 67 minutes for a cross-like pattern and 87 minutes for an arrow-like pattern. To overcome this, we reasoned that only the bottom layers (masks) in the droplet networks are necessary to be patterned, as long as this ‘mask’ layer was connected to a reservoir of arabinose and ocHL-containing droplets to supply arabinose to the mask layers. Therefore, we printed cubic, arabinose-containing ‘reservoirs’ (4-8 layers) on top of the mask layers (4 layers) connecting reservoirs to the mask via aHL-mediated droplet diffusive pathways. Using this method, we reduced our printing time significantly, for example, the printing time for droplet networks composed of twelve cross-like patterned layers (9 x 9 x 12 droplets) was 56 minutes, while the printing time for droplet networks composed of 4 mask layers (9 x 9 x 4 droplets) of the same pattern and 8 reservoir layers (7 x 7 x 8) on top was 41 minutes. Moreover, this method stored arabinose more efficiently within the droplet network in terms of occupiedvolume, as 8 reservoir layers were composed of a total of 392 aHL and cascade blue dextran-containing droplets as opposed to 136 in 8 patterned layers.

[0449] To avoid reservoir droplets from accidentally rolling to the patterned bottom layer of droplet networks during printing and, hence, disrupting intended patterns, reservoirs were designed to be slightly smaller than bottom layers (e.g. 6 x 6 droplets (reservoir) instead of 8 x 8 droplets (mask)). As a result, we found that droplet networks predominantly flipped before they landed on top of the bacterium -laden hydrogel (i.e. the reservoir interfaced with the hydrogel instead of the mask layer, Figure 8(a)). Out of 45 droplet networks composed of patterned masks and a reservoir, 88.9% flipped during the transfer and landed with the reservoir facing the hydrogel. In this case, arabinose would diffuse directly from the reservoir to the cells, resulting in gene expression patterns reflecting the shape of the reservoir, rather than the intended pattern (Figure 8(b)). This occurred likely due to hydrodynamic forces in the viscous lipid-in-oil solution, aligning the smaller reservoir towards the hydrogel. To overcome the flipping, we developed a mechanism to control the landing of droplet networks on top of the hydrogel. This comprised of attaching droplets containing magnetic beads (1.5% w / v ultra-low gelling agarose and nickel magnetic beads) to the comers of the mask layers before the transfer and then placing a magnet underneath the bacterium-laden hydrogel to guide the mask layer towards the hydrogel. Using this method, the correct landing was achieved in 76.5% of 34 transferred droplet networks, allowing for patterned gene expression in bacterial cells by the mask layers rather than the reservoir layers (Figure 8(c)).

[0450] Next, we investigated the number of mask layers that were necessary to consistently induce patterned gene expression. We printed droplet networks composed of 1, 2, 3 and 4-layered masks comprising a stripe-like pattern and an 8-layered reservoir on top (33 RIM arabinose and 50 pg mb'1aHL). Accordingly, 4 connected mask layers were necessary to induce stripe-like gene expression in bacterial cells (Figure 3(d)-Figure 3(g) and Figure 8(d)).

[0451] Finally, we compared gene expression patterns induced by arabinose release from droplet networks with and without reservoirs. Droplet networks with reservoirs consisted of 4-layered masks encoding for a single-droplet pathway and 4-layered reservoirs on top (Figure 9(a)), whereas droplet networks without reservoirs consisted of 8-layered masks encoding for a single -droplet pathway. Using both types of droplet networks (with and without reservoirs), single-droplet gene expression patterns could be induced. Further, we found that there was no significant difference between droplet network type regarding PF and mean mCherry expression (Figure 9(b)), confirming that droplet networks composed of masks and reservoirs are not only more efficient in terms of printing time and occupied space but also induced gene expression at similar spatial resolution compared to droplet networks comprised completely of mask layers.Supplementary Note 4: E. coli Interference Competition

[0452] Supplementary Note 4,1: Colicin biology

[0453] E. coli can produce colicins, protein toxins that target susceptible E. coli strains, which is crucial for interference competition between strains[3,4]. These colicins, specifically group A colicins (E7, E8), are encoded on a plasmid (pCol, type I plasmids: 6-10 kb) with about 20 copies per cell. Type I plasmids carry group A colicins, which parasitize the Ton system in E. coli for entry into the periplasm. Group A colicins include nuclease colicins (E7, E8) causing DNA damage to susceptible cells which mostly cause cell death[5].

[0454] On the pCol plasmid, the colicin operon is controlled by the LexA protein, which represses the SOS promoter. The SOS response, triggered by DNA damage, upregulates RecA, enabling LexA self-cleavage. This allows RNA polymerase binding to the SOS promoter for colicin operon transcription, leading to colicin expression. Nuclease colicins are associated with two genes: one encoding the colicin and the other the cognate immunity protein. The immunity protein is constitutively expressed, preventing self-intoxication. Last, the operon also contains a gene that encodes the lysis protein needed for colicin release1'1.

[0455] Supplementary Note 4,2: Differences Between Competing Strains

[0456] We utilized six distinct strains in our inducible competition assays:

[0457] 1) S (BZB1011): a susceptible strain to DNA damage (and as a consequence, likely death) by colicins E7 and E8. It does not carry a colicin-producing plasmid (Table SI, 2)

[0458] 2) S-GFP (BZB1011 pUA66-PcolE2::gfp): a susceptible strain to DNA damage (and as consequence, likely death) by colicins E7 and E8. It also does not carry a colicin-producing plasmid but, but harbours a DNA damage -reporting plasmid (Table SI, 5)

[0459] 3) E7-inducible (BZB1011 pKC \-PBAD>ColE7-AA y. a strain that upregulates the colicin E7 operon (toxin, immunity, and lysis proteins) in the presence of arabinose, and is susceptible to colicin E8 (Table SI, 3)

[0460] 4) E7R-inducible (BZB1011 pYYl-PBAD -ColE7-mCherry-AMP) a strain that upregulates the colicin E7 operon (toxin, immunity, and lysis proteins) and the mCherry protein in the presence of arabinose, and is susceptible to colicin E8 (Table SI, 4)

[0461] 5) E7 (BZB1011 pColE7): the natural colicin E7-producing strain that upregulates the colicin E7 operon when experiencing DNA damage, and is susceptible to colicin E8 (Table SI , 6) 6) E8 (BZB1011 pColE8): the natural colicin E8-producing strain that upregulates the colicin E8 operon when experiencing DNA damage, and is susceptible to colicin E7 (Table SI, 7).Notably, colicin-producing strains (E7-inducible, E7R-inducible, E7 and E8) possess additional mechanisms fortoxin release. These strains exhibit a basal toxin production rate. For E8, approximately 1 in 200 cells stochastically upregulate toxin and lysis protein production[5]; the basal rate is assumed to be lower for the E7-inducible strain. Specifically for the natural colicin E8 producer, E8 can amplify toxin production through autoinduction. This density-dependent mechanism involves clonemates being more likely to upregulate their colicin E8 operon when in the proximity of an E8 cell that releases toxins. This occurs because E8 produces lower amounts of cognate immunity protein so that the probability of DNA damage is increased upon import of a clonemates’ released colicin E8[5].

[0462] Supplementary Note 4,3: Testing Growth Inhibition Using Agar Overlay Assay

[0463] To test whether our E7-inducible (BZB1011 pKC -PBAD -ColE7-MAP) strain expressed colicin E7 upon induction with arabinose, we performed overlay assays (Figure 10(a)-(d), see Supplementary Methods), where E7-inducible cells were spotted on top of cells that natively produce colicin E7 (BZB1011 pColE7 - E7), (Table SI, 6) or colicin E8 (BZB1011 pColE8 -E8), (Table SI, 7) and susceptible cells (BZB1011 - S), (Table SI, 2). Using an agar overlay assay (K. E. S. Avelar, Letters in Applied Microbiology 1999, 29, 264) when E7-inducible was spotted on top of an E7 top agar, E7 -inducible continued to grow when no arabinose was added to the plates. Adding arabinose concentrations of 0.5% (w / v), 1% (w / v) and 5% (w / v) to the agar caused complete lysis of E7-inducible. In contrast, E7 continued to grow at all arabinose concentrations used (0% (w / v) to 5% (w / v)), due to it expressing the E7 immunity protein (Figure 10(a)).

[0464] When E7-inducible was spotted on E8 top agar, no growth was observed of E7-inducbile after 18 h (Figure 10(b)). We reasoned this was because of the higher basal expression of E8 compared to E7-inducible, and E8’s ability to respond to an attack from E7 as the natural operon is upregulated by DNA damage. Taken together E8 is dominant because it can produce a lot more toxin than the uninduced E7 -inducible strain (see colicin biology section).

[0465] With arabinose concentrations of >0.5% (w / v) in the agar, E7-inducible was activated, leading to not only mass lysis of E7-inducible (as the entire colicin operon, including the lysis protein is under control of the PBAD promoter), but also growth inhibition in native colicin E8-expressing cells, because of the high concentration of colicin E7 released from E7-inducible cells (Figure 10(b)).

[0466] In the case where E7-inducible was spotted on top of LB agar containing susceptible

[0467] cells (BZB1011 - S), E7-inducible continued to grow when no arabinose was present. However, a halo around the location where E7-inducible was spotted, presumably because of low basal levels of E7 expression in LB medium, affecting susceptible cells at the boundary of the E7-induciblespot (Figure 10(c)). In contrast, when E7-inducible was spotted on top of M9 agar containing susceptible cells (BZB 1011 - S), both E7-inducible and S cells grew next to each other when no arabinose was present (Figure 10(d)). As soon as arabinose was added to the plates (> 0.5%(w / v)) E7-inducible was activated, leading to growth inhibition of susceptible cells both in LB agar plates (Figure 10(c)) and in M9 medium agar plates (Figure 10(d)).

[0468] Supplementary Note 4,4: Actively-Dividing Cells Are Required for Colicin Competition Next, we investigated whether growth of susceptible cells can be inhibited by induced expression of colicin E7 at a range of arabinose concentrations (0 RIM to 333 HIM) when both cells were mixed homogeneously at an equal starting ratio (1: 1) in M9 ultra-low gelling agarose (ULGA, 1.5% w / v). At atotal starting cell density of 3.6 x 109cells mb'1the mean GFP expression of E7-inducible, arising from constitutive expression of GFP, decreased only slightly with increasing arabinose concentrations (Figure 11(a)), indicating a low number of lysis events and, hence, minimal expression of colicin E7. This is supported by the fact that the mean RFP expression of susceptible cells, arising from constitutive expression of RFP, did not decrease significantly with increasing arabinose concentrations (Figure 11(a)). We hypothesized that colicin E7 expression was low because not many cell divisions of the E7-inducible strain occurred in the hydrogel at these high cell densities.

[0469] To confirm whether lower cell densities, and hence whether sustained cell divisions during interference competition is required for E7-inducible to express colicin E7 in M9 ULGA, we decreased the starting cell density of E7-indicuble to 1.6 x 107cells mL1within the hydrogel. Indeed, the number of micro-colonies arising from E7-inducible decreases with increasing arabinose concentrations within the M9 ULGA gels, suggesting induced expression of colicin E7 lysis protein at lower cell densities (Figure 11(b)). Similarly, when both E7-inducible and susceptible cells were mixed homogeneously at equal starting ratio and a combined starting cell density of 1.6 x 107cells mL1the number of E7 -inducible micro-colonies decreased with increasing arabinose concentrations. No micro-colonies were observed of susceptible cells after 18 hours of co-culture, which suggests that baseline expression levels of colicin E7 in the absence of arabinose was sufficient to inhibit the growth of susceptible cells (Figure 11(c)). Hence, the homogeneous distribution of E7-inducible and susceptible cells in our bacterium -laden hydrogel enhanced the effect of colicin E7 baseline expression compared to the agar overlaying assay.

[0470] Therefore, we decreased the starting ratio from initially 1: 1 to 1:9 and 1:99 (number of E7-inducible cells to number of susceptible cells). At a starting ratio of 1:9 and total starting cell density of 1.6 x 107cells mL'1both E7-inducible and susceptible cells grew to homogeneously-distributed micro-colonies within M9 ULGA gels when no arabinose was present (Figure 11(d)).With increasing arabinose concentrations both the number of E7-inducible and susceptible microcolonies decreased, suggesting both lysis of E7-inducible and DNA-damaging effects on S cells. At a starting ratio of 1:99 of E7 -inducible to susceptible cells, less susceptible cells were inhibited presumably because of the much lower starting densities of E7 -inducible and hence lower release concentrations colicin E7 (Figure 11(e)).

[0471] Supplementary Note 4,5: Droplet Networks and Colicin Competition

[0472] From our above results, we hypothesized that arabinose released from droplet networks into bacterium-laden M9 ULGA gels (supplemented with 24 RIM glucose) could induce localized lysis of E7-inducible cells that would, in turn, clear a localized area of susceptible cells when the two strains were incubated at a starting ratio of 1:9. Therefore, we placed droplet networks containing 33 RIM arabinose and 50 pg mb'1aHL on top of bacterium -laden hydrogels containing E7-inducible and S cells. Droplet networks were placed either immediately after the gels were formed or 24 h after the gels were formed. However, when droplet networks were place after the gels were formed, the number of micro-colonies of both E7-inducible and susceptible cells did not change significantly, indicating that arabinose release from droplet networks did not induce levels of colicin E7 required for clearing of susceptible cells. The time point of tissue placement in regards to formation of the gel (just after or 24 hours after formation) did not affect the final number of E7-inducible and S cells (Figure 12(a) and (b)).

[0473] We reasoned higher concentrations of arabinose within droplet networks were required to induce sufficient expression of colicin E7 in E7-inducible. However, a ten-fold increase in arabinose concentration (333 HIM) did not lead to lysis of E7-inducible within 42 h of the competition assay against S cells, as confirmed by the number of micro-colonies arising from E7-inducible cells (Figure 12(c)). Therefore, the number of micro-colonies arising from susceptible cells did not change significantly when comparing droplet networks with or without arabinose (Figure 12(d)).

[0474] Supplementary Note 4,6: Importance of Culture Medium for Colicin Competition

[0475] We wondered whether the culture medium in the gels had a significant impact on induced gene expression of E7-inducible. In particular, we hypothesized that glucose, which was supplemented to M9 as a carbon source throughout previous experiments, could act as catabolic repressor, limiting gene expression levels (L. M. Guzman et al., J Bacteriol 1995, 177, 4121; C. G. Miyada et al., Proceedings of the National Academy of Sciences 1984, 81, 4120). Our hypothesis was based on the differences in basal expression of E7-inducible from overlay assays (Figure 10(c) and 10(d)), where without addition of arabinose E7 -inducible visibly inhibited and did not visibly inhibit S cells when LB and M9 medium were used, respectively. To test these effects of mediacomposition on colicin expression in E7-inducible, we monitored cell lysis in E7-inducible populations in ULGA gels (at a starting cell density of 3.6 x 109cells mL1) composed of M9 supplemented with 24 RIM glucose, M9 supplemented with 24 RIM glycerol or LB at arabinose concentrations of 0 mM, 6 mM, 33 RIM and 66 RIM. We monitored cell lysis by tracking constitutive sfGFP expression (on the chromosome of E7-inducible), as and indication of cell viability, and propidium iodide staining in E7-inducible, as indication of cell death, which has previously been shown to confirm cell lysis (E. T. Granato et al., Current Biology 2020, 30, 2836). A decrease in sfGFP expression and increase in propidium iodide fluorescence would indicate cell lysis (as propidium iodide can only penetrate cells and bind to DNA when the membrane is compromised). While the mean gene expression of sfGFP did not change significantly when comparing glucose and glycerol as supplemented carbon source in M9 medium, a significant decrease in sfGFP was observed for gels composed of LB with increasing arabinose concentrations (Figure 13(a)).

[0476] Moreover, propidium iodide fluorescence increased when cells were in LB medium supplemented with arabinose as compared to M9 medium, indicating increased cell lysis (Figure 13(b)). The combination of decreased sfGFP expression and increased propidium iodide intensity indicated a significant reduction in number of micro-colonies formed with increasing arabinose concentrations. Interestingly, the propidum ioide fluorescence was significantly increased for gels composed of M9 supplemented with glycerol at arabinose concentrations of 33 mM and 66 mM compared to gels supplemented with glucose, indicating increased cell lysis in the presence of glycerol as compared to glucose (Figure 13(b)).

[0477] Supplementary Note 4,7: Local Lysis

[0478] Next, we investigated whether E7-inducible cells could be lysed when droplet networks containing 333 mM of arabinose and 50 pg mL'1of aHL monomer were placed on top of bacterium -laden hydrogels composed of LB ULGA. For this we formed gels containing E7-inducible at a starting cell density of 1.6 x 107cells mL'1and measured the number of micro-colonies in the periphery and center when droplet networks were or were not placed on top of the bacterium-laden hydrogels (Figure 14(a) and Figure 14(b)). Center refers to the area directly underneath droplet networks and the same areas when no droplet networks were placed, whereas periphery refers to the area outside of where droplet networks were placed (Figure 14(a)). So, droplet networks were used as reference region of interest both when droplet networks were placed on top of the bacterium -laden hydrogel (+ST) or when droplet networks were not placed on top(-ST). Critically, the cell density of E7-inducible cells was significantly reduced both in the periphery and center when droplet networks were placed on top of the bacterium-laden hydrogel compared to when no droplet networks were present (Figure 14(b)), suggesting that local lysis of E7-inducible is indeed possible when arabinose is released from droplet networks in LB.As the localized expression of colicin E7 was achieved in LB, we investigated the effect of toxin release on susceptible cells at different starting ratios. Here, the total starting cell density was kept constant at 1.6 x 107cells mL1, while altering the starting ratio of E7-inducible to susceptible cells from 1:9 to 9:1. We found that the relative abundance of E7-inducible decreased when droplet networks containing 333 RIM of arabinose and 50 pg mL'1of aHL monomer were placed on top of the bacterium-laden hydrogel (Figure 14(c)). However, the cell density of susceptible cells was not affected significantly when droplet networks were placed on top of the bacterium-laden hydrogel as compared to when they were not. Taken together, these results indicated that the initial assumption of distinctive cell clearing of susceptible cells as a consequence of induced E7 expression may be limited within the time course of our experiments.

[0479] Supplementary Note 4,8: Colicin E2 Promoter as Indication of DNA Damage

[0480] From our experiments, we thought a susceptible strain was needed to report on DNA damage as an indication of cell inhibition, rather than focusing on cell clearing. We reasoned that the natural promoter of colicin-expressing strains, which is regulated by DNA damage (see colicin biology section), could act as reporter of DNA damage in susceptible cells. Therefore, we transformed susceptible cells with a reporter plasmid (pUA66-Pc» / / i2:.s g / / j) (E. T. Granato et al., Current Biology 2020, 30, 2836; D. A. I. Mavridou et al., Current Biology 2018, 28, 345) which allowed us to monitor the activity of PCOIE2 based on GFP expression levels and, hence, levels of DNA-damage that susceptible cells may experience as a consequence of induced colicin E7 expression. In summary, susceptible cells (S-GFP) would express GFP in response to DNA-damage. Additionally, to track colicin operon activity in the E7-inducible strain, we created a reporter strain with mCherry downstream of the colicin E7 lysis gene, i.e. when the operon was active, fluorescent protein would be produced (BZB1011 ^ N\-PBAD.-ColE7-mCherry-PMP, E7R-inducible).

[0481] First, we mixed E7R-inducible cells and S-GFP cells at equal starting ratio and a total starting cell density of 1.6 x 107cells mL1, or S-GFP cells only at a starting cell density of 0.8 x 107cells mL1, in M9 ULGA gels supplemented with 24 RIM of glycerol and a range of arabinose concentrations (0 RIM to 8 RIM, Figure 15(a)). After 18 hours of culture at 37°C we found that mCherry expression increased with increasing arabinose concentrations, indicating increasing levels of colicin E7 expression with increasing concentrations of arabinose (Figure 15(b)). The mean GFP expression in S-GFP cells increased significantly at arabinose concentrations of 0.25 RIM and 0.5 RIM in the gels as compared to when no arabinose was present in the gels. However, a further increase of arabinose (>0.5 RIM) concentration caused a significant drop in GFP expression (Figure 15(b)), of which we assumed cells were killed too quickly (with high concentrations of expressed colicin E7) to report on DNA-damage. To correlate GFP expression with the ability of S-GFP cells to replicate, we re-suspended cells from the ULGA gels and plated them on selective LB agar plates (withkanamycin selecting for S-GFP cells) and measured the relative number of colony forming units (RCFU) of S-GFP cells after 12 hours at 37°C:

[0482] RCFU =N

[0483] Iv CN

[0484] FlCFU100%, (SI)

[0485] uJ si ■ngl ,e

[0486] where NCFUrefers to the number of colony forming units at a certain concentration of arabinose in the gels before plating (0 RIM to 8 mM) and NCFusinglerefers to the number of colony forming units when only S-GFP cells were seeded in ULGA gels without E7R-inducible cells and without arabinose (single). We found a significant drop in RCFU at an arabinose concentration of 0.5 mM in the ULGA gels, confirming that the observed increase in GFP indeed was an indication of cell inhibition of S-GFP cells.

[0487] From this, we printed droplet networks containing 50 pg mL1of aHL monomer and a range of arabinose concentrations (0 mM to 33 mM) and placed them on top of homogeneously-distributed cells of E7R-inducible and S-GFP cells at equal starting ratio and a total cell density of

[0488] 1.6 x 107cells mL1. We did not observe any DNA damage in S-GFP cells when droplet networks contained <3 mM of arabinose. However, at 16 mM of arabinose the number of micro-colonies of S- GFP cells that experienced DNA damage increased significantly, both underneath the droplet network and in the periphery. Further increase in arabinose (33 mM) led to a decrease in the number of micro-colonies expressing sfGFP, suggesting decreased viability, i.e. cells were inhibited too quickly to report on DNA-damage (Figure 15(c)).

[0489] In order to achieve patterned DNA damage in S-GFP cells, we adjusted both arabinose (8 mM, 12 mM and 16 mM) and aHL monomer concentrations (0 pg mL1, 10 pg mL1, 25 pg mL'1and 50 pg mL1) so to control the release of arabinose from droplet networks. With increasing arabinose and aHL concentrations the number of S-GFP cells experiencing DNA-damage increased both underneath droplet networks (Figure 16(a)), and outside of where droplet networks were placed (Figure 16(b)). Moreover, the mean sfGFP expression increased with increasing arabinose and aHL concentrations (Figure 16(c)), while the mean micro-colony 2D cross-sectional area of S- GFP cells increased up to 198.5 pm2before it decreased to 63.7 pm2(Figure 16(d)).

[0490] Table SI. Summary of strains and plasmids.

[0491] Strain and Recombinant Abbreviation Promoter Description Source of genotype DNA recombinant _ DNA _ 1 BZB1011 pJS -PBAD - mCherry- pBAD Constitutive This study Pmax:sfgfp::Tn7 mCherry- inducible expression of

[0492] AMP sfGFP,

[0493] inducedexpression of

[0494] mCherry

[0495] BZB1011 - S - Constitutive Pmax:mrfpl::Tn7 expression of

[0496] RFP BZB1011 pKCl- E7-inducible pBAD Constitutive This study Pmax:sfgfp::Tn7 PBAD.- expression of

[0497] Co / / -.7-AMP sfGFP,

[0498] induced

[0499] expression of

[0500] colicin E7

[0501] BZB1011 pYYl- E7R- pBAD Constitutive This study Pmax:sfgfp::Tn7 PBAD.- inducible expression of

[0502] ColE7- sfGFP, mCherry- induced

[0503] AMP expression of

[0504] colicin E7 and

[0505] mCherry

[0506] BZB1011 pUA66- S-GFP pColE2 Unlabeled, E. T. Granato PcolE2::sfgfp GFP et al., Current expression Biology 2020, upon sensed30’2836’D' DNA-damage ^avridou

[0507]

[0508] et al., Current Biology 2018, 28, 345 BZB1011 pColE7 pcolE7 SOS Constitutive L. Ghazaryan Pmax:mrfpl::Tn7 expression of et al, Arch RFP, natural Microbiol colicin E7 2014, 196, 753 plasmid

[0509] BZB1011 pColE8 pcolE8 SOS Constitutive L. Ghazaryan Pmax:mrfpl::Tn7 expression of et al, Arch RFP, natural Microbiol colicin E8 2014, 196, 753 plasmid

[0510] Supplementary Methods

[0511] Growth overlay assay

[0512] Plates for growth inhibition assay consisted of two layers of solidified agar. The bottom layer did not contain cells, while the top layer contained bacterial cells. LB / M9 agar plates (1.5% w / v) were prepared by pouring 20 mL of liquid LB / M9 per petri dish before drying in a laminar flow hood. Overnight cultures of both the susceptible strain and toxin-expressing strain were prepared by inoculation from glycerol stocks one day prior to the experiment. Strains were grown in a tube containing 4 mL of LB on a shaker (225 rpm) at 37°C for no longer than 12h, before the overnight culture of susceptible cells was inoculated at an OD of 0.05 in a tube containing 4 mL of LB and grown at 37°C with shaking (225 rpm). When susceptible cells reached an OD of 0.6, 200 pL of the cell suspension was added to 6 mL of melted LB / M9 agar (0.75% w / v) before pouring 6 mL ofthe resulting cell suspension per petri dish on the dried 20 mL of LB / M9 agar. Both the bottom agar and top agar (containing susceptible cells) of the LB / M9 agar plates contained arabinose concentrations of 0% w / v, 0.5% w / v, 1% w / v or 5% w / v. The plates were dried for 1 h. Once dried, 0.5 pL of the toxin-expressing stain was pipetted on top of the LB / M9 agar plate in serial dilutions of 10° (undiluted), 10-1, IO-2, IO-3and 10-4. The plates were dried for 15 min before placed in a static incubator at 37°C for 12 h. Plates were imaged using a gel imager and epi-fluorescent microscope.

[0513] Propidium iodide staining

[0514] Cell death staining was performed using propidium iodide by preparing a stock solution (1.5 mM) of propidium iodide in DMSO. Then, a working solution was prepared by diluting the stock solution with H2O to 150 pvi. The working solution was added to the bacterial cell suspension in M9 ULGA prior to gel solidification to reach a final concentration of 5 pvi.

[0515] Determination of Relative Colony Forming Units (CFU)

[0516] M9 ULGA gels (supplemented with 24 mM of glycerol) were formed containing E7-inducible (“Single”) or E7-inducible and S-GFP cells at a starting ratio of 1 : 1 and a total starting cell density of 1.6 x 107cells mL1. The bacterial cells were cultured for 18 h at 37 °C in the M9 ULGA gels supplemented by varying concentrations of arabinose (0 mM - 8 mM), before resuspending the bacterium -laden hydrogels by pipetting to form 100-fold dilutions. Then, the bacterium-containing solutions were plated onto LB agar plates containing 50 pg mL 'of kanamycin, which selected for S-GFP cells. The LB agar plates were than incubated for another 12 h at 37 °C, before the relative number of colony forming units arising from S-GFP cells were determined according to equation S 1.

[0517] 5. Testing Further Effector Molecules

[0518] 5.1 HC1 applied to astrocytes

[0519] A single aqueous droplet containing aHL and hydrochloric acid (HC1) was deposited onto a layer of astrocytes. Cellular responses were monitored using a Fluo-4 calcium assay. Transient fluctuations in intracellular calcium levels indicated that local pH changes induced by the droplet triggered a localized signaling response in the cells. Panel (a) of Figure 17 provides a schematic representation of the droplet-cell interface (not to scale), while panel (b) of Figure 17 shows the droplet positioned over the cells (left) and the corresponding calcium response (right).

[0520] 5.2 HC1 applied to OVCAR cellsA network (droplet assembly) of aqueous droplets, each containing aHL and hydrochloric acid (HC1) was deposited onto a layer of OVCAR cells (an ovarian cancer cell line). Cellular responses were monitored using a Fluo-4 calcium assay. Transient fluctuations in intracellular calcium levels indicated that local pH changes induced by the droplets triggered a localized signaling response in the cancer cells. Panel (a) of Figure 18 provides a schematic representation of the droplet-cell interface (not to scale), while panel (b) is a graph showing the corresponding calcium response of the OVCAR cells.

[0521] 5.3 Application of isoprenaline and doxorubicin to cardiomyocytes

[0522] A single aqueous droplet containing aHL and a small molecule drug (either isoprenaline or doxorubicin) was deposited onto a layer of cardiomyocytes. Cellular responses were monitored by measuring the frequency of cardiomyocyte contractions in response to the drug released from the droplet via aHL. Panel (a) presents a schematic representation of the droplet-cell interface (not to scale). Panel (b) shows the percentage change in beat frequency 5 minutes after drug release. Panel (bl) demonstrates that isoprenaline increases the frequency of contraction over a concentration range of 10 nM to lOOnM, with beat frequency rising proportionally across this range. Panel (b2) shows that doxorubicin decreases the frequency of contraction over a concentration range of 500 pM to 5 mM, with beat frequency decreasing as concentration increases.

[0523] Further embodiments of the invention are described in the following numbered clauses.

[0524] 1. A system for delivering an effector molecule to biological cells or tissue, which system comprises:

[0525] (a) a target region which comprises biological cells or tissue; and

[0526] (b) a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0527] wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet additionally contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, and

[0528] at least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.. A system according to clause 1 wherein the means for producing the effector mole...

Claims

1. CLAIMS1. A system for delivering an effector molecule to biological cells or tissue, which system comprises:(a) a target region which comprises biological cells or tissue; and(b) a synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous droplet medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous droplet medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet additionally contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, andat least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.

2. A system according to claim 1 wherein at least one target interface droplet is a target interface outlet droplet, wherein the target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface outlet droplet and (ii) the target region, further comprises said protein pore.

3. A system according to claim 1 or claim 2 wherein the number of target interface droplets, m, is equal to or greater than two and:at least one of the target interface droplets is a target interface outlet droplet, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface outlet droplet and (ii) the target region, further comprises said protein pore; andat least one other of the target interface droplets is a mask droplet, wherein each mask droplet does not comprise said protein pore and does not comprise the effector molecule.

4. A system according to claim 2 or claim 3 wherein:the number of target interface droplets, m, is at least 4, preferably at least 16, and more preferably at least 64; andat least two of the target interface droplets are target interface outlet droplets, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface outlet droplet and (ii) the target region, further comprises said protein pore; andat least two other of the target interface droplets are mask droplets, wherein each mask droplet does not comprise said protein pore and does not comprise the effector molecule.

5. A system according to claim 4 wherein the target interface droplets together form a flat or curved layer of droplets which is a target interface droplet layer, wherein each target interface droplet in said target interface droplet layer contacts at least one other target interface droplet in the target interface droplet layer to form a layer of said amphipathic molecules as an interface between the contacting target interface droplets.

6. A system according to claim 5 wherein the at least two target interface outlet droplets together form a pattern of target interface outlet droplets in the target interface droplet layer, optionally wherein the target interface droplets which are not target interface outlet droplets are mask droplets.

7. A system according to claim 6 wherein the pattern of target interface outlet droplets in the target interface droplet layer corresponds to the shape of an area of the target region to be exposed to the effector molecule,optionally wherein the pattern of target interface outlet droplets is a ring, triangle, heart, cross, arrow or smiley face,optionally wherein the or each target interface outlet droplet is an effector droplet.

8. A system according to any one of claims 2 to 7 wherein the synthetic droplet assembly comprises a reservoir region which is a plurality of said effector droplets, wherein each effector droplet in the reservoir region: (i) contacts at least one other effector droplet in the reservoir region to form a layer of said amphipathic molecules as an interface between the contacting effector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the effector droplets in the reservoir region, wherein each layer of amphipathic molecules which is an interface between contacting effector droplets further comprises said protein pore,optionally wherein the number of the effector droplets in the reservoir region is at least 20, preferably at least 50, and more preferably at least 100.

9. A system according to claim 8 wherein at least one of the effector droplets in the reservoir region contacts a target interface outlet droplet, to form a layer of said amphipathic molecules as an interfacebetween the contacting effector droplet and target interface outlet droplet, wherein each layer of said amphipathic molecules between an effector droplet and a target interface outlet droplet further comprises said protein pore.

10. A system according to claim 8 wherein at least one of the effector droplets in the reservoir region is a target interface outlet droplet.

11. A system according to claim 8 wherein at least one of the n droplets in the synthetic droplet assembly is a connector droplet, wherein each connector droplet further comprises a protein pore for allowing passage of the effector molecule into and out of the connector droplet,optionally wherein each connector droplet contacts at least two other of the n droplets, to form a layer of said amphipathic molecules as an interface between the connector droplet and each of the at least two other droplets, wherein each layer of said amphipathic molecules further comprises said protein pore.

12. A system according to claim 11 wherein the connector droplet contacts an effector droplet in the reservoir region and a target interface outlet droplet.

13. A system according to claim 11 wherein the synthetic droplet assembly comprises a connector region which is a plurality of said connector droplets, wherein each connector droplet in the connector region: (i) contacts at least one other connector droplet in the connector region to form a layer of said amphipathic molecules as an interface between the contacting connector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the connector droplets in the connector region, wherein each layer of amphipathic molecules which is an interface between contacting connector droplets further comprises said protein pore.

14. A system according to claim 13 wherein at least one of the connector droplets in the connector region contacts an effector droplet in the reservoir region, and at least one other of the connector droplets in the connector region contacts a target interface outlet droplet.

15. A system according to claim 13 or claim 14 wherein the synthetic droplet assembly comprises at least one said connector region, wherein each target interface outlet droplet in the synthetic droplet assembly contacts a connector droplet in a connector region, and wherein at least one of the connector droplets in each connector region contacts an effector droplet in the reservoir region.

16. A system according to any one of claims 4 to 15 wherein a plurality of the n droplets in the synthetic droplet assembly are mask droplets, wherein each mask droplet does not comprise a protein pore and does not comprise the effector molecule.

17. A system according to claim 16 wherein the synthetic droplet assembly comprises at least one mask region, wherein a mask region is a plurality of said mask droplets, wherein each mask droplet in the mask region contacts at least one other mask droplet in the mask region to form a layer of said amphipathic molecules as an interface between the contacting mask droplets.

18. A system according to claim 17 wherein:• the target interface droplets together form a flat or curved layer of droplets which is a target interface droplet layer, wherein each target interface droplet in said target interface droplet layer contacts at least one other target interface droplet in the target interface droplet layer to form a layer of said amphipathic molecules as an interface between the contacting target interface droplets;• at least two of the target interface droplets in the target interface droplet layer are target interface outlet droplets, wherein each target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region, wherein the layer of amphipathic molecules which is an interface between (i) the target interface (outlet) droplet and (ii) the target region, further comprises said protein pore;• the target interface droplets in the target interface droplet layer which are not target interface outlet droplets are mask droplets;• the synthetic droplet assembly comprises a reservoir region which is a plurality of said effector droplets, wherein each effector droplet in the reservoir region: (i) contacts at least one other effector droplet in the reservoir region to form a layer of said amphipathic molecules as an interface between the contacting effector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the effector droplets in the reservoir region, wherein each layer of amphipathic molecules which is an interface between contacting effector droplets further comprises said protein pore;• the synthetic droplet assembly comprises at least one connector region wherein the or each connector region is a plurality of said connector droplets, wherein each connector droplet in the connector region: (i) contacts at least one other connector droplet in the connector region to form a layer of said amphipathic molecules as an interface between the contacting connector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the connector droplets in the connector region, wherein each layer of amphipathic molecules which is an interface between contacting connector droplets further comprises said protein pore;• each target interface outlet droplet in the synthetic droplet assembly contacts a connector droplet in a connector region, and wherein at least one of the connector droplets in the or each connector region contacts an effector droplet in the reservoir region; and• the synthetic droplet assembly comprises at least one mask region, wherein the or each mask region is disposed between mask droplets in the target interface droplet layer and effector droplets in the reservoir region,optionally wherein:the or each connector region is disposed between target interface outlet droplets in the target interface droplet layer and effector droplets in the reservoir region, and the target interface outlet droplets together form a pattern of target interface outlet droplets in the target interface droplet layer, optionally wherein the same pattern is formed by the connector region or regions disposed between the target interface outlet droplets in the target interface droplet layer and the effector droplets in the reservoir region, and / oroptionally wherein said pattern corresponds to the shape of an area of the target region to be exposed to the effector molecule, optionally wherein the pattern is a cross, ring, triangle, heart, arrow or smiley face.

19. A system according to any one of claims 2 to 18 wherein the (i) concentration of the effector molecule in each effector droplet and the (ii) concentration of the protein pore in each target interface outlet droplet (and optionally in each reservoir droplet, connector droplet and / or effector droplet), are selected to achieve a desired rate of release of the effector molecule from the or each target interface outlet droplet into the target region,optionally wherein said desired rate of release of the effector molecule is selected to achieve a desired effect in said biological cells or tissue in the target region.

20. A system according to any one of the preceding claims which further comprises magnetic particles attached to the synthetic droplet assembly, suitable for moving the synthetic droplet assembly relative to the target region using a magnet,optionally wherein the system further comprises a magnet for moving the synthetic droplet assembly relative to the target region.

21. A synthetic droplet assembly which comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, andat least one of the n droplets is an effector droplet, wherein each effector droplet further comprises an effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n.

22. A process for delivering an effector molecule from a synthetic droplet assembly to biological cells or tissue,wherein the synthetic droplet assembly comprises n droplets, wherein n is an integer equal to or greater than 2, wherein each of said n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said n droplets contacts at least one other of said n droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,wherein at least one of the n droplets is a target interface droplet, wherein each target interface droplet is suitable for contacting a target region comprising biological cells or tissue, to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region, wherein the number of said target interface droplets, m, is from 1 to n, andat least one of the n droplets is an effector droplet, wherein each effector droplet further comprises the effector molecule or means for producing the effector molecule, wherein the number of said effector droplets, p, is from 1 to n,and wherein the process comprises:contacting each target interface droplet with a target region which comprises the biological cells or tissue, so that each target interface droplet contacts the target region to form a layer of the amphipathic molecules as an interface between the target interface droplet and the target region.

23. A process according to claim 22 wherein the synthetic droplet assembly is as further defined in any one of claims 2 to 19 and the process further comprises allowing the effector molecule to pass from a target interface outlet droplet to the target region, by passing through said protein pore of the layer of amphipathic molecules which is an interface between (i) the target interface outlet droplet and (ii) the target region.

24. A process according to claim 23 which comprises selecting (i) concentration of the effector molecule in each effector droplet and the (ii) concentration of the protein pore in each target interface outlet droplet (and optionally in each reservoir droplet, connector droplet and / or effector droplet), and thereby achieving a desired rate of release of the effector molecule from the or each target interface outlet droplet into the target region,wherein achieving said desired rate of release of the effector molecule achieves a desired effect in said biological cells or tissue in the target region.

25. A process according to any one of claims 22 to 24 wherein n is at least 4 and the process further comprises producing the synthetic droplet assembly by contacting a first precursor droplet assembly with a second precursor droplet assembly before, during or after the step of contacting each target interface droplet with the target region,wherein the first precursor droplet assembly comprises at least one target interface droplet and the second precursor droplet assembly comprises at least one effector droplet, and contacting the first precursor droplet assembly with the second precursor droplet assembly enables the effector molecule to pass from the effector droplet originating from the second precursor droplet assembly to the at least one target interface droplet originating from the first precursor droplet assembly,optionally wherein:the first precursor droplet assembly comprises at least one target interface droplet which is a target interface outlet droplet, wherein the target interface outlet droplet further comprises a protein pore for allowing passage of the effector molecule from the target interface outlet droplet to the target region; andthe second precursor droplet assembly comprises a reservoir region which is a plurality of said effector droplets, wherein each effector droplet in the reservoir region: (i) contacts at least one other effector droplet in the reservoir region to form a layer of said amphipathic molecules as an interface between the contacting effector droplets, and (ii) further comprises a protein pore for allowing passage of the effector molecule between the effector droplets in the reservoir region, wherein each layer of amphipathic molecules which is an interface between contacting effector droplets further comprises said protein pore,wherein contacting the first precursor droplet assembly with the second precursor droplet assembly enables the effector molecule to pass from the reservoir region to the at least one target interface outlet droplet, optionally via a connector region as defined in any one of claims 13 to 15 and 18.I l l

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