Author Correction: Dynamic actuation of glassy polymersomes through isomerization of a single azobenzene unit at the block copolymer interface.

In the version of this Article originally published, multiple changes to the "Results and discussion" section were required. In paragraph 1, "(Supplementary Fig. 1)" should have read "(Fig. 1e-j and Supplementary Fig. 1)"; in the first sentence of paragraph 3, "(R6G)" should have read "(R6G, Fig. 2i)"; in paragraph 6 in the sentence beginning "Temporal release of hydrophilic...", Supplementary Fig. 4 should have been cited after "360 nm"; in paragraph 9, in the sentence beginning "To test this...", "Fig. 4e" should have read "Fig. 4a"; in paragraph 10, in the sentence beginning "When the irradiation...", "(Fig. 4a-d)" should have read "(Fig. 4d,e)"; in paragraph 11, in the sentence beginning "Pristine PLA", "P1" should have read "P2"; and in the penultimate paragraph, in the sentence beginning "Moreover, a control PEG-PLA...", "block copolymer" should have been followed by (P5); Fig. 4g should have been Fig. 4c; "hydrophobic azobenzene small molecules" should have been followed by (12); and Fig. 4f should have been Fig. 4b. Finally, Supplementary Videos 1 and 2 were missing from the Article. All of these corrections have been made to the online versions.

Dynamic actuation of glassy polymersomes through isomerization of a single azobenzene unit at the block copolymer interface.

Nature has engineered exquisitely responsive systems where molecular-scale information is transferred across an interface and propagated over long length scales. Such systems rely on multiple interacting, signalling and adaptable molecular and supramolecular networks that are built on dynamic, non-equilibrium structures. Comparable synthetic systems are still in their infancy. Here, we demonstrate that the light-induced actuation of a molecularly thin interfacial layer, assembled from a hydrophilic- azobenzene -hydrophobic diblock copolymer, can result in a reversible, long-lived perturbation of a robust glassy membrane across a range of over 500 chemical bonds. We show that the out-of-equilibrium actuation is caused by the photochemical trans-cis isomerization of the azo group, a single chemical functionality, in the middle of the interfacial layer. The principles proposed here are implemented in water-dispersed nanocapsules, and have implications for on-demand release of embedded cargo molecules.

Amphiphilic BODIPY-Hydroporphyrin Energy Transfer Arrays with Broadly Tunable Absorption and Deep Red/Near-Infrared Emission in Aqueous Micelles.

BODIPY-hydroporphyrin energy transfer arrays allow for development of a family of fluorophores featuring a common excitation band at 500 nm, tunable excitation band in the deep red/near-infrared window, and tunable emission. Their biomedical applications are contingent upon retaining their optical properties in an aqueous environment. Amphiphilic arrays containing PEG-substituted BODIPY and chlorins or bacteriochlorins were prepared and their optical and fluorescence properties were determined in organic solvents and aqueous surfactants. The first series of arrays contains BODIPYs with PEG substituents attached to the boron, whereas in the second series, PEG substituents are attached to the aryl at the meso positions of BODIPY. For both series of arrays, excitation of BODIPY at 500 nm results in efficient energy transfer to and bright emission of hydroporphyrin in the deep-red (640-660 nm) or near-infrared (740-760 nm) spectral windows. In aqueous solution of nonionic surfactants (Triton X-100 and Tween 20) arrays from the second series exhibit significant quenching of fluorescence, whereas properties of arrays from the first series are comparable to those observed in polar organic solvents. Reported arrays possess large effective Stokes shift (115-260 nm), multiple excitation wavelengths, and narrow, tunable deep-red/near-IR fluorescence in aqueous surfactants, and are promising candidates for a variety of biomedical-related applications.

Tuning the Activation Wavelength of Photochromic Oxazines.

The activation wavelength of a photochromic oxazine can be shifted bathochromically with the introduction of a methoxy substituent on the chromophore responsible for initiating the photochemical transformation. This structural modification permits switching under mild illumination conditions, enhances the photoisomerization quantum yield and ensures outstanding fatigue resistance. Thus, these results can guide the design of new members of this family of photoresponsive molecular switches with improved photochemical and photophysical properties.

Supramolecular nanoreactors for intracellular singlet-oxygen sensitization.

An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy.

Optical writing and reading with a photoactivatable carbazole.

The fluorescence of a carbazole chromophore can be activated irreversibly under optical control with the photoinduced opening of an oxazine ring. In proximity to silver nanoparticles, the quantum efficiency of this photochemical transformation and that of the emissive process increase significantly. The plasmonic effects responsible for such enhancements, together with the photochemical and photophysical properties engineered into this particular photoactivatable fluorophore, permit the optical writing and reading of microscaled patterns at low illumination intensities.

Photoactivatable BODIPYs designed to monitor the dynamics of supramolecular nanocarriers.

Self-assembling nanoparticles of amphiphilic polymers can transport hydrophobic molecules across hydrophilic media and, as a result, can be valuable delivery vehicles for a diversity of biomedical applications. Strategies to monitor their dynamics noninvasively and in real time are, therefore, essential to investigate their translocation within soft matrices and, possibly, rationalize the mechanisms responsible for their diffusion in biological media. In this context, we designed molecular guests with photoactivatable fluorescence for these supramolecular hosts and demonstrated that the activation of the fluorescent cargo, under optical control, permits the tracking of the nanocarrier translocation across hydrogel matrices with the sequential acquisition of fluorescence images. In addition, the mild illumination conditions sufficient to implement these operating principles permit fluorescence activation within developing Drosophila melanogaster embryos and enable the monitoring of the loaded nanocarriers for long periods of time with no cytotoxic effects and no noticeable influence on embryogenesis. These photoresponsive compounds combine a borondipyrromethene (BODIPY) chromophore and a photocleavable oxazine within their covalent skeleton. Under illumination at an appropriate activation wavelength, the oxazine ring cleaves irreversibly to bring the adjacent BODIPY fragment in conjugation with an indole heterocycle. This structural transformation shifts bathochromically the BODIPY absorption and permits the selective excitation of the photochemical product with concomitant fluorescence. In fact, these operating principles allow the photoactivation of BODIPY fluorescence with large brightness and infinite contrast. Thus, our innovative structural design translates into activatable fluorophores with excellent photochemical and photophysical properties as well as provides access to a general mechanism for the real-time tracking of supramolecular nanocarriers in hydrophilic matrices.

Autocatalytic fluorescence photoactivation.

We designed an autocatalytic photochemical reaction based on the photoinduced cleavage of an α-diketone bridge from the central phenylene ring of a fluorescent anthracene derivative. The product of this photochemical transformation sensitizes its own formation from the reactant, under illumination at a wavelength capable of exciting both species. Specifically, the initial and direct excitation of the reactant generates the product in the ground state. The subsequent excitation of the latter species results in the transfer of energy to another molecule of the former to establish an autocatalytic loop. Comparison of the behavior of this photoactivatable fluorophore with that of a model system and the influence of dilution on the reaction progress demonstrates that the spectral overlap between the emission of the product and the absorption of the reactant together with their physical separation govern autocatalysis. Indeed, both parameters control the efficiency of the resonant transfer of energy that is responsible for establishing the autocatalytic loop. Furthermore, the proximity of silver nanoparticles to reactant and product increases the energy-transfer efficiency with a concomitant acceleration of the autocatalytic process. Thus, this particular mechanism to establish sensitization offers the opportunity to exploit the plasmonic effects associated with metallic nanostructures to boost photochemical autocatalysis.

Plasmonic activation of a fluorescent carbazole-oxazine switch.

The covalent attachment of a carbazole fluorophore to an oxazine photochrome permits the reversible activation of fluorescence under optical control. Ultraviolet irradiation with a pulsed laser opens the oxazine ring to shift bathochromically the absorption of the carbazole component. Concomitant visible illumination excites selectively the carbazole fluorophore of the photochemical product to produce fluorescence. The photogenerated and fluorescent species reverts spontaneously on a submicrosecond timescale to the initial nonemissive state of the carbazole-oxazine dyad. The photochemical and photophysical properties engineered into this particular molecular switch allow the convenient monitoring of plasmonic effects on photochemical reactions with fluorescence measurements. In close proximity to silver nanoparticles, visible illumination with a continuous-wave laser also results in fluorescence activation. The metallic nanostructures enable the two-photon excitation of the oxazine component to induce the photochromic transformation and then facilitate the one-photon excitation of the photochemical product to generate fluorescence. Thus, these operating principles offer the opportunity to avoid altogether the need of pulsed ultraviolet irradiation to trigger the photochromic transformation and, instead, allow fluorescence activation with a single visible source operating at low illumination power.

Intracellular guest exchange between dynamic supramolecular hosts.

Decyl and oligo(ethylene glycol) chains were appended to the same poly(methacrylate) backbone to generate an amphiphilic polymer with a ratio between hydrophobic and hydrophilic segments of 2.5. At concentrations greater than 10 µg mL(-1) in neutral buffer, multiple copies of this particular macromolecule assemble into nanoparticles with a hydrodynamic diameter of 15 nm. In the process of assembling, these nanoparticles can capture anthracene donors and borondipyrromethene acceptors within their hydrophobic interior and permit the transfer of excitation energy with an efficiency of 95%. Energy transfer is observed also if nanocarriers containing exclusively the donors are mixed with nanoparticles preloaded separately with the acceptors in aqueous media. The two sets of supramolecular assemblies exchange their guests with fast kinetics upon mixing to co-localize complementary chromophores within the same nanostructured container and enable energy transfer. After guest exchange, the nanoparticles can cross the membrane of cervical cancer cells and bring the co-entrapped donors and acceptors within the intracellular environment. Alternatively, intracellular energy transfer is also established after sequential cell incubation with nanoparticles containing the donors first and then with nanocarriers preloaded with the acceptors or vice versa. Under these conditions, the nanoparticles exchange their cargo only after internalization and allow energy transfer exclusively within the cell interior. Thus, the dynamic character of such supramolecular containers offers the opportunity to transport independently complementary species inside cells and permit their interaction only within the intracellular space.

Photoresponsive polymer nanocarriers with multifunctional cargo.

Nanoparticles with photoresponsive character can be assembled from amphiphilic macromolecular components and hydrophobic chromophores. In aqueous solutions, the hydrophobic domains of these species associate to produce spontaneously nanosized hosts with multiple photoresponsive guests in their interior. The modularity of this supramolecular approach to nanostructured assemblies permits the co-encapsulation of distinct subsets of guests within the very same host. In turn, the entrapped guests can be designed to interact upon light excitation and exchange electrons, energy or protons. Such photoinduced processes permit the engineering of properties into these supramolecular constructs that would otherwise be impossible to replicate with the separate components. Alternatively, noninteracting guests with distinct functions can be entrapped in these supramolecular containers to ensure multifunctional character. In fact, biocompatible luminescent probes with unique photochemical and photophysical signatures have already emerged from these fascinating investigations. Thus, polymer nanocarriers can become invaluable supramolecular scaffolds for the realization of multifunctional and photoresponsive tools for a diversity of biomedical applications.

Fluorescence photoactivation by ligand exchange around the boron center of a BODIPY chromophore.

Chelation of the boron center of the borondipyrromethene (BODIPY) platform by a catecholate ligand results in effective fluorescence suppression. Electron transfer from the chelating unit to the adjacent chromophore upon excitation is responsible for fluorescence quenching. Under the influence of a photoacid generator, the catecholate chelator can be exchanged with a pair of methoxide ligands. This photoinduced transformation prevents electron transfer and efficiently activates the fluorescence of the BODIPY chromophore.

Activation of BODIPY fluorescence by the photoinduced dealkylation of a pyridinium quencher.

The photoinduced cleavage of a 2-nitrobenzyl group from a pyridinium quencher covalently attached to the meso position of a BODIPY fluorophore activates the emission of the latter. This photochemical transformation prevents the transfer of one electron from the BODIPY platform to its heterocyclic appendage upon excitation and, as a result, permits the radiative deactivation of the excited fluorophore. This versatile mechanism for fluorescence switching can translate into the realization of an entire family of photoactivatable fluorophores based on the outstanding photophysical properties of BODIPY chromophores.

Superresolution imaging with switchable fluorophores based on oxazine auxochromes.

The spatial resolution of fluorescence microscopes is limited by diffraction to about half of the light wavelength, hampering the observation of many important intracellular processes. Recent emerging techniques have overcome that diffraction barrier using the temporal discrimination of close objects that are otherwise unresolved or blurred within the spatial resolution of the microscope. The key of these techniques is to switch the signal of fluorescence markers on and off exploiting their distinct molecular states, and detect and localize these markers at the single-molecule level. This underlying principle highlights the critical role of the photophysical properties of the probes, and the importance of finding adequate switching mechanisms. Here, we present strategies to achieve fluorescence modulation based on novel molecular assemblies containing a [1,3]oxazine as the two states, building block responsible for the transformation. Two different triggering events, based on the photochromic and halochromic properties of the oxazine, induce a large absorption and emission bathochromic shift of a pendant fluorophore, as the ultimate fluorescence switching event. The implementation of these approaches to achieve spatial resolution beyond the diffraction limit is also discussed.

Fluorescence photoactivation by intermolecular proton transfer.

We designed a strategy to activate fluorescence under the influence of optical stimulations based on the intermolecular transfer of protons. Specifically, the illumination of a 2-nitrobenzyl derivative at an activating wavelength is accompanied by the release of hydrogen bromide. In turn, the photogenerated acid encourages the opening of an oxazine ring embedded within a halochromic compound. This structural transformation extends the conjugation of an adjacent coumarin fluorophore and enables its absorption at an appropriate excitation wavelength. Indeed, this bimolecular system offers the opportunity to activate fluorescence in liquid solutions, within rigid matrixes and inside micellar assemblies, relying on the interplay of activating and exciting beams. Furthermore, this strategy permits the permanent imprinting of fluorescent patterns on polymer films, the monitoring of proton diffusion within such materials in real time on a millisecond time scale, and the acquisition of images with spatial resolution at the nanometer level. Thus, our operating principles for fluorescence activation can eventually lead to the development of valuable photoswitchable probes for imaging applications and versatile mechanisms for the investigation of proton transport.