A high precision approach allowing light-triggered, programmed cell-sized vesicle rupture is described, with particular emphasis on self-assembled polymersome capsules. The mechanism involves a hypotonic osmotic imbalance created by accumulation of new photogenerated species inside the lumen, which cannot be compensated due to the low water permeability of the membrane. This simple and versatile mechanism can be adapted to a wealth of hydrosoluble molecules, which are either able to generate reactive oxygen species or undergo photocleavage. Ultimately, in a multi-compartmentalized and cell-like system, the possibility to selectively burst polymersomes with high specificity and temporal precision, and consequently deliver small encapsulated vesicles (both polymersomes and liposomes) is demonstrated. Polymersomes are mechanically robust self-assembled vesicular structures that are widely studied and are proving central in increasing research and application areas ranging from nanomedicine to artificial cell design. Control over their membrane diffusion properties and structural integrity is crucial for the development of new complex systems, such as artificial cells. Compartmentalization is central in biological cells. Indeed, physical separation of biochemical species allows metabolic reactions to take place independently and simultaneously in a confined and crowded space. For decades, different methods have been proposed to construct elaborate structures that have been developed in the field of lipid and polymer chemistry. Amongst others, double emulsion techniques , phase transfer of emulsion droplets over an interface, layer-by-layer assembly or microfluidics have proved efficient in affording micron-sized vesicles, allowing the encapsulation of distinct biochemical species in different compartments and the ability to control simple biomimetic enzymatic reactions in a confined space. One additional major characteristic of natural cells is their ability to initiate metabolic reactions at a specific time and at a desired location, independently and repeatedly. In this regard, temporal control is crucial in artificial cell systems. However, most of the designed synthetic systems to date lack some control over the initiation of the reactions, which are generally induced by passive diffusion of species across semi-permeable membranes, either as a result of intrinsic membrane permeability or by the incorporation of channels or pores into the membrane, resulting in a slow release of reactants. As a result, a remaining major challenge concerns the ability to trigger specific reactions by selectively and rapidly inducing the release of species from independent compartments, while controlling their concentration. Herein, we introduce a tunable protocol for light-driven specific polymersome rupture in time and space, which combines the advantages of utilizing light as a trigger and the fast release of components from bursting vesicles. Our system is based on laser excitation of hydrophilic dyes encapsulated in the lumen of distinct giant poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) polymersomes, across the whole visible spectrum gamut. Upon excitation the dye is degraded, either through photofragmentation or reactive oxygen species (ROS)-mediated degradation, leading to an increase of the internal osmotic pressure until subsequent polymersome rupture. This process allows for a precise and fast release of entrapped species from different compartments. Additionally, such a selective mechanism allows discrimination between two types of polymersomes within a group of many and to successively trigger the release of their content without altering the remaining vesicles. This system offers great potential for the development of cell mimics where different species encapsulated in distinct organelle-like compartments have to be released independently, in a controlled manner, but also for the release of other (bio)active compounds. [a] A. Peyret, E. Ibarboure, L. Beauté, Dr. O. Sandre, Prof. Dr. S. Lecommandoux Laboratoire de Chimie des Polymères Organiques, LCPO Université de Bordeaux CNRS, Bordeaux INP, UMR 5629, F-33600 Pessac, France. E-mail: lecommandoux@enscbp.fr [b] Dr. A. Tron, R. Rust, Dr. N. D. McClenaghan Institut des Sciences Moléculaires Université de Bordeaux, CNRS UMR 5255, 33405 Talence, France. E-mail : nathan.mcclenaghan@u-bordeaux.fr Supporting information for this article is given via a link at the end of the document. Scheme 1. a) Schematic representation of osmotic pressure increase in polymersomes. The impermeable membrane prevents water from entering the vesicle or the internal solution to leak out and the osmotic pressure remains imbalanced until the vesicle ruptures. b) Schematic representation of osmotic pressure increase in liposomes. The internal osmotic pressure increases transiently but it is rapidly compensated by water diffusion through the tenfold more permeable membrane of the liposome or through sub-critical resealing pores, resulting in vesicle swelling without irreversible rupture. COMMUNICATION Author manuscript of Angew. Chem. Int. Ed. 2017, 56 1566-1570 Figure 2. a) Photocleavage of N-diethyl, O-({7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl carbamate (coumarin derivative) under irradiation. b) Electronic absorption spectrum of a 80 μM coumarin derivative in aqueous solution before and after (dashed line) 30 min irradiation at 365 nm with a 200 W Hg-Xe lamp. c) Confocal observation of a 10 mM coumarin-loaded GUV (green channel, emission range of coumarin, 485 nm). The vesicle undergo fast (few milliseconds) rupture upon irradiation at 405 nm (50 mW, 25%). Scale bar = 10 μm. Poly(butadiene)-b-poly(ethylene oxide) (PBut2.5-b-PEO1.3) giant unilamellar vesicles (GUVs) were prepared by a previously reported emulsion-centrifugation method. As suggested in Scheme 1. a), due to the limited water permeability of the polymersome membrane, compared to liposomes, we initially hypothesized that a sudden increase in the internal osmotic pressure of the vesicles would lead to efficient rupture of the membrane. Indeed, water would be unable to diffuse into the cavity fast enough to compensate for the pressure difference between the lumen and the external medium. The outcome is that the membrane is exposed to a large lateral tension and ruptures irreversibly to release pressure. On the other hand, liposomes exhibit a tenfold larger permeability towards water compared to polymersomes (Supplementary Information (SI) p. 14,15), and whenever a pore opens up, the lateral stress on the membrane can be relaxed by hydrodynamic flow from inner to outer solutions through transient pores reported by many groups on large or giant liposomes irrespective of the means used to stress their bilayer: osmotic pressure, applied electric field, lipid photo-oxidation or membrane dye illumination (Scheme 1. b). Osmotic pressure was also shown to induce shell rupture of layer-by-layer coated gel beads releasing microcapsules. To test our hypothesis, a photodegradation experiment was performed to confirm that fast in situ molecule fragmentation and subsequent osmotic pressure increase in the lumen of giant polymersomes could indeed cause vesicle rupture. In this context, N-diethyl, O-({7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl carbamate (coumarin derivative (11), SI, p. 5-7) was synthesized, inspired by a previously described procedure. It has been established that coumarin derivatives undergo heterolytic C-O bond cleavage under UV irradiation. This cleavage results in the formation of a carbamate ion. After decarboxylation of the carbamate, carbon dioxide and diethylamine are released (Φreaction = 0.003 on irradiating at 405 nm) (figure 1. a). Cleavage of the molecule was confirmed by a decrease and a shift of the absorption band after 30 min UV irradiation (365 nm, 200 W Hg-Xe lamp) (figure 1. b). This photoinduced coumarin cleavage feature was used as a way to increase the osmotic pressure inside the polymersomes. The molecule was encapsulated inside the PBut2.5-b-PEO1.3 GUVs. The vesicles were then irradiated under confocal observation (405 nm, 50 mW, 25%) resulting in a fast (few milliseconds) explosion (figure 1. c). As a control, dye-free (sucrose-loaded) polymersomes were irradiated at 405 nm and coumarin-loaded polymersomes were irradiated at 488 nm and 561 nm. In all cases, no rupture was observed, confirming that the explosion results from coumarin selective irradiation. Figure 1. a) Chemical structure of calcein and confocal images of a 15 mM calcein-loaded polymersome irradiated at 488 nm, with laser intensity 40 mW, 5% (green channel, emission range of calcein, 520 nm). b) Chemical structure of methylene blue (MB) and confocal images of a 10 mM MBloaded polymersome irradiated at 633 nm with laser intensity 10 mW, 90% (red channel, emission range of MB, 660 nm). c) Electronic absorption spectrum of a 30 μM calcein photosensitizer in aqueous solution before and after (dashed line) 30 min irradiation in the 400 – 550 nm range with a 200 W Hg-Xe lamp equipped with a bypass filter, showing photoinduced degradation. d) Electronic absorption spectrum of a 30 μM methylene blue in water solution before and after (dashed line) 30 min irradiation in the 240 – 550 nm range with a 200 W Hg-Xe lamp COMMUNICATION Author manuscript of Angew. Chem. Int. Ed. 2017, 56 1566-1570 In order to broaden the scope and versatility of the release process, we reasoned that as increased osmolarity is a colligative process, any molecule able to degrade/cleave following illumination would potentially provide a complementary alternative release pathway. In this context, we chose two hydrophilic fluorescent dyes, calcein and methylene blue (MB), that are known to be effective photosensitizers. Upon irradiation in the visible region, they generate reactive oxygen species (ROS) including singlet oxygen (O2) via energy transfer from th