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Photoresponsive polymer nanocarriers with multifunctional cargo a b b Subramani Swaminathan,a Jaume Garcia-Amoro ´ s, Aurore Fraix, Noufal Kandoth, b a Salvatore Sortino* and Françisco M. Raymo*

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 Received 10th September 2013 DOI: 10.1039/c3cs60324e

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

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multifunctional and photoresponsive tools for a diversity of biomedical applications.

a

Laboratory for Molecular Photonics, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida, USA 33146-0431. E-mail: [email protected] b Laboratory of Photochemistry, Department of Drug Sciences, University of Catania, Viale Andrea Doria 6, I-95125 Catania, Italy. E-mail: [email protected]

Subramani Swaminathan received a BSc in Chemistry from Ramakrishna Mission Vivekananda College (India) in 2007 and a MSc in Chemistry from the same institution in 2009. He entered the graduate program in chemistry of the University of Miami in 2010. His graduate research involves the design and synthesis of photoactivatable fluorophores and their encapsulation within the hydrophobic core Subramani Swaminathan of polymer nanoparticles. He has authored five publications in the general areas of organic synthesis and photochemistry.

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Introduction Hydrophilic and hydrophobic domains (Fig. 1) can be integrated within the very same polymer backbone to impose amphiphilic character on the overall macromolecular construct.1–7 In aqueous

Jaume Garcia-Amoro´s received a BSc in Chemistry in 2005, a MSc in Experimental Chemistry in 2006 and a PhD in Chemistry in 2011 from the University of Barcelona under the guidance of Prof. Dolores Velasco. His graduate research involved the design of new photoactive azoderivatives for their further use in realtime information transmitting systems and artificial musclelike materials. From 2011 until ´s Jaume Garcia-Amoro 2013, he was assistant professor at the same institution. In 2013, he joined the research group of Prof. Raymo at the University of Miami, as a postdoctoral associate. At the present time, his research interests are focused on the design and preparation of new molecular and supramolecular constructs for fluorescence photoactivation. He has authored 16 publications and three book chapters in the general areas of physical chemistry, materials science and photochemistry.

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Fig. 1 Amphiphilic polymers assemble noncovalently into nanosized hosts capable of encapsulating hydrophobic guests in their interior.

environments, the resulting amphiphilic polymers assemble spontaneously into particles of nanoscale dimensions. Solvophobic interactions are mostly responsible for bringing the hydrophobic domains of distinct polymer components together in order to minimize their direct exposure to water. In the process, the very same noncovalent interactions can encourage the encapsulation of hydrophobic guests in the core of the self-

Aurore Fraix received a PhD in Chemistry in 2010 from the University of Brest (France). Her graduate research involved the synthesis, characterization and formulation of new sulfurated phospholipid vectors for application in gene delivery. In 2011, she joined the research group of Prof. Sortino at the University of Catania, as a postdoctoral associate. Her current research interests are focused on the design, synthesis Aurore Fraix and characterization of molecular assemblies for multimodal therapy applications. She has authored 15 publications in the field of controlled biomedical release.

Salvatore Sortino received a PhD in Chemical Sciences in 1994 from the University of Catania (Italy) and he carried out postdoctoral research at the CNR of Bologna (Italy) until 1996. In the same year, he joined, as Assistant Professor, the University of Catania, where he is now Associate Professor of Chemistry. He was a visiting scientist at the University of Ottawa (Canada) in 1998–1999 Salvatore Sortino and at the University of Miami in 2004 and 2011. He is the author of about 160 publications. His current scientific interests are mainly focused on the development of nanoassemblies activated by light for biomedical applications.

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assembling nanoparticles. In turn, the hydrophilic domains of the polymer components protrude into the surrounding aqueous environment and ensure the effective solvation of the final nanostructured constructs. As a result, hydrophobic molecules with otherwise negligible aqueous solubility can be transported across hydrophilic environments under the assistance of these multicomponent supramolecular containers. In fact, polymer nanocarriers are promising vehicles for the delivery of drugs through the blood stream to target locations in living organisms.8–16 The possibility of making these nanoassemblies responsive to light, and as a consequence able to perform specific functions under optical control, opens fascinating prospects in nanomedicine, especially for therapeutic and imaging applications. The ease of manipulation of optical stimulations, in terms of intensity, wavelength, duration and localization, translates into a versatile approach to activate such functions, mimicking a noninvasive ‘‘optical microsyringe’’ with an exquisite spatiotemporal control.17,18

Noufal Kandoth

Noufal Kandoth received a BSc in Chemistry from the University of Calicut (India) in 2007 and a MSc in Chemistry from the Mahatma Gandhi University Kottayam (India) in 2009. He joined the group of Prof. Sortino in 2010 and obtained a PhD in Chemical Sciences in 2013. The research of his PhD program was focused on the development of light-triggered cyclodextrin-based nanoassemblies for anticancer therapy. He has authored 12 publications in the general area of photochemistry.

Françisco M. Raymo received a Laurea in Chemistry from the University of Messina (Italy) in 1992 and a PhD in Chemistry from the University of Birmingham (UK) in 1996. He was a postdoctoral associate at the University of Birmingham (UK) in 1996–1997 and at the University of California, Los Angeles, in 1997–1999. He was appointed Assistant Professor of Chemistry at the University of Françisco M. Raymo Miami in 2000 and promoted to Associate Professor in 2004 and Full Professor in 2009. His research interests combine the design, synthesis and analysis of switchable molecular constructs for imaging applications. He has authored more than 200 publications.

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In addition, light triggering ensures fast transformations and does not affect physiological parameters, like temperature, pH and ionic strength, and, of course, such a ‘‘biofriendly’’ character is a fundamental requisite for biomedical applications. Photoresponsive properties can be engineered on supramolecular assemblies of amphiphilic polymers with the covalent introduction of appropriate chromophores in the individual macromolecular components.19–25 Photochemical transformations can then be exploited to dismember the nanostructured containers and encourage the release of their cargo. Under these conditions, external stimulations, in the form of exciting photons, offer the opportunity to expel guests from the nanosized hosts within a specific region of space at a given interval of time. In turn, such a level of spatiotemporal control permits the targeted delivery of drugs and is particularly valuable for therapeutic applications. Alternatively, photochemical reactions can be exploited to lock adjacent polymer chains together covalently. The resulting network of covalent bonds prevents the dissociation of the individual macromolecular components and ensures nanoparticle integrity. In fact, the administration of such delivery systems to living organisms can expose them to a broad range of experimental conditions. In the absence of reinforcing covalent cross links, changes in ionic strength, pH and temperature, together with the presence of interfering biomolecules and the extreme dilution possible in the blood stream, can all promote the dismembering of the nanocarriers and the premature release of their cargo. The introduction of photoswitchable groups within the covalent backbone of polymer nanoparticles offers also the opportunity to engineer fluorescent probes with unique emissive behavior.26–30 In particular, photochromic and fluorescent fragments can be integrated within the same macromolecular component and the photoinduced and reversible transformations of the former can be exploited to regulate the emission of the latter. Essentially, the same behavior can be replicated by connecting only the photochromic species covalently to the polymer network, while encapsulating the fluorescent chromophores noncovalently within the assembled nanocarriers. In both instances, energy transfer from the fluorescent groups to one of the two interconvertible states of the photochromic species can be exploited to modulate the emission intensity of the overall nanostructured assembly under optical control. In fact, the ability of these emissive polymer nanocarriers to insulate their cargo from the surrounding environment and transport it across hydrophilic media translates into the possibility of developing valuable bioimaging probes.31 As an alternative to connecting photochromic components covalently to the macromolecular backbone of polymer nanocarriers, such photoresponsive groups can be encapsulated noncovalently in the hydrophobic interior of the nanoparticles and operated successfully under these conditions.32–34 In fact, photochemical reactions, other than photochromic transformations, can also be performed inside the polymer shell of these supramolecular containers.35,36 Furthermore, the relatively constrained environment in the nanoparticle interior can have a significant influence on the outcome of these photochemical

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processes. Often the resulting product distributions differ significantly from those observed in bulk organic solutions. As a result, these nanoreactors can be exploited to direct photochemical processes to specific products that are otherwise difficult to generate. Moreover, their ability to transfer hydrophobic reactants into aqueous solutions offers also the opportunity to perform chemical reactions in water and avoid organic solvents.37 The covalent integration of photoresponsive fragments into polymer nanoparticles requires first structural modifications of the native chromophores aimed at the introduction of reactive functional groups.19–30 The resulting building blocks can then be polymerized, together with appropriate hydrophilic and hydrophobic monomers, into amphiphilic macromolecules able to self-assemble into the target nanoparticles. By contrast, the noncovalent introduction of photoresponsive guests within the hydrophobic interior of similar nanosized hosts requires minimal, if any, synthetic efforts. In most instances, the photoresponsive species in their native form are sufficiently hydrophobic to enter spontaneously the interior of the selfassembling host. In addition, more than one type of photoresponsive guest can be encapsulated within the same nanosized container in a single experimental step. Furthermore, the relative amounts of the co-encapsulated guests can easily be regulated with adjustments in the composition of the mixture of self-assembling components. In turn, the co-encapsulation of distinct and noninteracting photoresponsive species permits the assembly of multifunctional constructs. Alternatively, the co-encapsulated guests can actually be designed to interact with each other in order to engineer photoresponsive behavior that would otherwise be impossible to achieve with the separate components. As a result, this modular supramolecular approach permits the convenient construction of photoresponsive nanoparticles with engineered functions. This article is mostly focused on this particular class of functional nanostructured assemblies and provides a general overview of the operating principles behind the unique behavior of representative examples of these promising multicomponent and photoresponsive supramolecular constructs.

Energy transfer between co-encapsulated guests A fluorophore in the excited state can transfer energy to a complementary and proximal chromophore in the ground state.38 As a result of this process, the former species deactivates nonradiatively instead of emitting light in the form of fluorescence. Concomitantly, the excited state of the latter species is populated and, if emissive, this chromophore can subsequently return radiatively to the ground state. Thus, the excitation of the energy donor eventually produces the fluorescence of the energy acceptor under these conditions. The transfer of energy, however, requires a significant spectral overlap between the emission band of the donor and an absorption band of the acceptor. In addition, the dipoles for the two electronic transitions responsible for the overlapping bands cannot be orthogonal to each other for the transfer of energy to occur. When both requirements are

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satisfied, the excited donor can transfer energy to the acceptor, but the efficiency of the process decreases with the sixth power of the distance between the two components. In fact, a significant amount of excitation energy is transferred from a given donor to a complementary acceptor only if the two species are sufficiently close to each other. Therefore, the co-entrapment of donors and acceptors within the interior of the very same polymer nanocarrier can be exploited to keep them in close proximity and facilitate energy transfer. Indeed, the exchange of energy between co-encapsulated species offers the opportunity to probe the stability of polymer nanoparticles as well as their ability to exchange and release guests.39–50 Furthermore, similar operating principles also permit the manipulation of the fluorescence of the acceptor and the generation of luminescent nanoparticles with unique emissive behavior.51–54 The optimal spectral overlap between the emission of 1 (Fig. 2) and absorption of 2 ensures the transfer of energy from the former to the latter, if both species are in close proximity.41 Their co-entrapment within the interior of nanoparticles of 3 satisfies this condition. In fact, the emission spectrum of the resulting supramolecular constructs in water shows predominantly the fluorescence of 2 upon excitation of 1. Dilution with acetone, however, breaks the solvophobic interactions between the amphiphilic polymer components and dismembers the

Fig. 2 Nanoparticles of 3 co-encapsulate 1 and 2 in their hydrophobic interior and permit the transfer of energy from the former guest to the latter. Incubation of the resulting supramolecular constructs with KB cells causes the partial release of the energy donor in the cell membrane and the subsequent internalization of this species. The corresponding image (a, scale bar = 10 mm) reveals the fluorescence of the donor in the cell membrane and intracellular space together with the sensitized fluorescence of the acceptor in the extracellular space. Similarly, the emission spectra, recorded in the extracellular (b) and intracellular (c) space, show predominantly the emission of 2 and 1 respectively [reproduced with permission from ref. 41b].

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nanoscaled hosts. In turn, the physical separation of the two guests prevents energy transfer and results in the appearance of the fluorescence of 1 in the corresponding emission spectrum. Thus, the spectral complementarity of the two guests offers the opportunity to assess the integrity of such supramolecular constructs using relatively simple fluorescence measurements. The transfer of energy between co-encapsulated guests can be exploited to probe the ability of polymer nanocarriers to deliver hydrophobic molecules intracellularly.41b For example, a fluorescence image (a of Fig. 2) of KB cells incubated with nanoparticles of 3, containing equimolar amounts of 1 and 2, reveals the partial separation of donors and acceptors. The red color in the extracellular space corresponds to the sensitized fluorescence of the energy acceptor. The green color on the cell membrane and in the intracellular space is, instead, the fluorescence of the energy donor. In fact, the emission spectra (b and c in Fig. 2), recorded in the extracellular and intracellular spaces upon excitation of the donor, show predominantly the emission bands of 2 and 1 respectively. These observations demonstrate that the polymer nanocarriers retain their cargo in the extracellular space and permit energy transfer, but release the energy donor in the lipophilic cell membrane. Interestingly, the very same experiment performed at a temperature of 4 1C, instead of 37 1C, did not result in the accumulation of the energy donor in the membrane. This relatively low temperature strengthens the solvophobic interactions holding the hydrophobic domains of the macromolecular components together and inhibits the escape of the entrapped guests. The ability of 1 to transfer energy to 2 can also be exploited to monitor the exchange of guests between independent polymer nanocarriers.43 In particular, nanoparticles of 4 (Fig. 3) can be loaded with either the energy donor or the energy acceptor and then they can be mixed in the same solution. The initial emission spectrum of the mixture, recorded upon excitation of the donor, reveals predominantly the fluorescence of 1. The subsequent acquisition of spectra (a in Fig. 3) over the course of 48 hours, however, shows the gradual decrease of the emission band of 1 with the concomitant increase of that of 2. These observations indicate that an energy-transfer pathway is established after mixing and that the efficiency of the process increases up to a stationary value in a few hours. Thus, independent nanoparticles can partially exchange their guests to produce supramolecular assemblies with mixtures of donors and acceptors in their interiors. The disulfide bridge in one of the two side-chains of 4 can be exploited to cross link independent polymer components after their assembly into a nanoparticle. Under the influence of dithiothreitol, a fraction of the disulfide groups is reduced to the corresponding thiols. The resulting functional groups can then react with disulfide linkages in adjacent polymer chains to lock covalently the many macromolecular components of each nanoparticle. The intercomponent disulfide cross links, however, can be cleaved with the addition of an appropriate reducing agent. As a result, the stability of these particular nanocarriers, and hence their ability to retain entrapped guests, can be controlled with redox stimulations. For example, glutathione can encourage the reductive cleavage of the disulfide cross links, promote the

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Fig. 3 Cross-linked nanoparticles of 4, doped independently with 1 and 2, exchange their guests upon mixing to produce supramolecular assemblies with both donors and acceptors in their interior. The emission spectra (a), recorded upon excitation of the donor after mixing and over the course of 48 hours, reveal the establishment of energy transfer and confirm guest exchange. Incubation of the cross-linked nanoparticles with MCF-7 cells results in the partial intracellular release of the donor because of the reductive cleavage of the disulfide bonds bridging the macromolecular components of each nanoparticle. The corresponding images reveal the fluorescence of the released donor in one channel (b) and the sensitized emission of the acceptor in the other (c) [reproduced with permission from ref. 43a and b].

partial release of co-encapsulated donors and acceptors and suppress the associated energy-transfer process. Furthermore, the concentration of glutathione is relatively high in the intracellular environment, while it is generally negligible in the extracellular space. Thus, the cellular internalization of the cross-linked nanoparticles can be accompanied by the cleavage of their disulfide links and the intracellular release of their cargo. Indeed, dual-channel fluorescence images (b and c in Fig. 3) of MCF-7 cells incubated with cross-linked nanoparticles of 4, containing equimolar amounts of 1 and 2, indicate the intracellular release of the energy donor. The green color in one detection channel is the emission of the released donor and the red color in the other corresponds to the sensitized fluorescence of the acceptor. The opportunity to constrain energy pairs within the interior of polymer nanoparticles also permits the realization of luminescent probes with photophysical properties specifically engineered for bioimaging applications.54 In particular, the transfer of energy from a donor to a complementary acceptor ensures a large separation between the excitation wavelength of the former and the emission wavelength of the latter. Such a large shift, in combination with the selection of an acceptor able to emit in the near-infrared region, translates into the possibility of recording fluorescence images with minimal background signal. Furthermore, the modularity of these supramolecular constructs enables the optimization of the absorption coefficient and energy-transfer efficiency with simple

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adjustments in the ratio of their self-assembling components. For example, a single nanoparticle of 7 (Fig. 4) can accommodate more than 6000 guests, in the shape of 5 and 6, to produce a supramolecular construct with an absorption coefficient in excess of 107 M 1 cm 1 and an almost unitary energy-transfer efficiency. In fact, the resulting luminescent assemblies are brighter than semiconductor quantum dots and permit the acquisition of nearinfrared images of animal models. Specifically, the insertion of a capillary tube, filled with an aqueous dispersion of the doped nanoparticles, in a mouse phantom (a in Fig. 4) enables the detection of the sensitized near-infrared emission of 6, after the excitation of 5, in the region of interest (b in Fig. 4) with negligible background signal in the rest of the sample. The introduction of an energy donor in the hydrophobic interior of a polymer nanocarrier, together with a photoswitchable acceptor, permits the modulation of the emission of the former under optical control.34a In particular, 8 (Fig. 5) and 9 can be co-encapsulated within nanoparticles of 11. The two guests, however, lack the spectral complementarity required for the transfer of energy between them to occur. In fact, the excitation of 8 results in the characteristic borondipyrromethene fluorescence, in spite of the presence of 9 within the same supramolecular container. Nonetheless, ultraviolet illumination encourages the isomerization of 9 to 10. The photogenerated and zwitterionic isomer has an extended chromophoric fragment able to absorb light in the same spectral region where 8 emits. As a result, 10 can accept the excitation

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Fig. 4 Nanoparticles of 7 co-encapsulate 5 and 6 in their hydrophobic interior and encourage the transfer of energy from the former guest to the latter. Insertion of a capillary tube, filled with an aqueous dispersion of the doped nanoparticles, in a mouse phantom (a) permits the detection of the sensitized near-infrared emission of 6, after the excitation of 5, with minimal background fluorescence (b) [reproduced with permission from ref. 54].

energy of 8 and suppress its fluorescence. In addition, the photoisomerization of 9 to 10 is reversible. Under visible irradiation, 10 reverts to 9 and loses its ability to accept energy from 8. Thus, alternating ultraviolet and visible irradiation steps translates into the modulation of the emission intensity (a in Fig. 5) of the energy donor, because of the activation and deactivation of the energy-transfer pathway. Furthermore, these polymer nanocarriers can cross the membrane of living cells and transport their cargo in the cytosol. Indeed, a fluorescence image (b in Fig. 5) of CHO cells, incubated with the doped nanoparticles, clearly reveals the characteristic fluorescence of the donor in the intracellular space. Under these conditions, the photoinduced generation of the acceptor and its reisomerization can be exploited, once again, to turn off and on fluorescence intracellularly under the influence of optical stimulations.

Electron transfer between co-encapsulated guests The photoinduced transition of a chromophore from its ground state to one of the accessible excited states imposes a significant change in electronic configuration.38 In particular, the absorption of one photon with an appropriate wavelength is accompanied by the transition of one electron from an occupied molecular orbital to an unoccupied molecular orbital. The resulting singly-occupied orbitals can accept an electron from or donate an electron to a complementary species respectively.

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Fig. 5 Nanoparticles of 11 can co-encapsulate 8 and 9 in their hydrophobic interior. However, 9 cannot accept the excitation energy of 8 and these supramolecular systems display the characteristic fluorescence of the borondipyrromethene component. Under ultraviolet illumination, 9 switches to 10 and activates an energy-transfer pathway with the concomitant quenching of the fluorescence of 8. Visible irradiation regenerates 9 and restores the initial fluorescent state. As a result, the emission intensity (a) of 8 can be switched off and on by alternating ultraviolet and visible illumination steps. Furthermore, these switchable supramolecular constructs can cross the membrane of CHO cells (b, scale bar = 50 mm) and permit the intracellular photomodulation of fluorescence [reproduced with permission from ref. 34a].

In turn, the photoinduced transfer of an electron converts the excited chromophore into a radical anion or cation respectively and prevents its radiative deactivation to the ground state. Thus, photoinduced electron transfer ultimately results in fluorescence quenching. This process, however, requires the quenching species to have an appropriate oxidation or reduction potential for it to donate or accept an electron to or from the excited chromophore respectively. In addition, the rate of electron transfer decreases exponentially with the physical separation between donor and acceptor and, therefore, the two species exchanging an electron must be in close proximity for the transfer to occur. This stringent condition can be satisfied by entrapping complementary donors and acceptors within the hydrophobic interior of polymer nanocarriers.55–58 Furthermore, the environment around the species exchanging an electron can be manipulated by adjusting the composition of the

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Fig. 6 Nanoparticles of 12 entrap 13 and 14 in their hydrophobic interior and facilitate the transfer of an electron from the latter to the former upon excitation. As a result, the fluorescence (a) of 13 decreases with the concentration of 14. The rate constant for electron transfer can be determined from the concentration dependence of the emission intensity (b). Its value increases with the incorporation of 15 in the supramolecular envelope surrounding donors and acceptors [reproduced with permission from ref. 57c].

supramolecular container in order to regulate the rate of the photoinduced process. For example, nanoparticles of 12 (Fig. 6) can co-encapsulate 13 and 14 in their interior.57 Under these conditions, 13 accepts an electron from 14 upon excitation and its fluorescence is quenched. Consistently, the characteristic emission band (a in Fig. 6) of 13 decreases in intensity with the concentration of 14. The actual rate constant for the electron transfer process can be determined from the analysis of the concentration dependence of the emission intensity. Interestingly, the very same analysis performed in the presence of 15 reveals a significant increase in the rate of electron transfer with the concentration of this component (b in Fig. 6). The hydrophobic domain of this particular surfactant encourages its noncovalent integration within the polymer nanocarriers. In turn, the polar heads of the incorporated molecules alter the ionic strength in the immediate environment surrounding donors and acceptors and facilitate electron transfer. Thus, the composition of the supramolecular host enveloping the photoresponsive guests can be engineered to regulate their ability to exchange electrons upon excitation.

Proton transfer between co-encapsulated guests The ability of polymer nanoparticles to entrap distinct guests within their hydrophobic interior can be exploited to encourage the photoinduced transfer of protons between encapsulated species. In particular, the pairing of a photoacid generator with a halochromic compound inside a nanocarrier permits the control of the spectroscopic signature of the latter by addressing the former with optical stimulations. These operating principles can be implemented with nanoparticles of 11 loaded with equimolar amounts of 16 (Fig. 7) and 17.59 The absorption

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spectrum of 16 shows a band centered in the ultraviolet region and extending into the visible region. Upon illumination at a wavelength positioned in the visible tail of this band, 16 releases hydrobromic acid to produce 18. The released acid can encourage the opening of the oxazine ring of 17 with the formation of 19. This structural transformation brings the coumarin fragment in conjugation with the adjacent 3H-indolium cation and shifts bathochromically its absorption band. Indeed, the absorption spectrum (a in Fig. 7), recorded before irradiation, shows a band at 412 nm for the coumarin appendage of 17 and that (b in Fig. 7), acquired after illumination reveals a band at 595 nm for the extended 3H-indolium chromophore of 19. Illumination at a wavelength positioned within the photogenerated absorption band excites selectively 19 and results in the appearance of an intense emission band in the corresponding spectrum (c in Fig. 7). Thus, the photoacid generator can mediate the interconversion of the two states of the halochromic component to permit the photoinduced activation of fluorescence. In fact, a fluorescence image (d in Fig. 7) of the doped nanoparticles, deposited on glass, shows emissive objects after the photoinduced activation of fluorescence inside the polymer nanocarriers. The analysis of the emission intensity across the fluorescent objects indicates an average feature size of 270 nm, while the actual average diameter of each polymer nanoparticle is only 14 nm. The apparent mismatch in physical dimensions is a result of diffraction. This phenomenon is inherent to the optics of conventional fluorescence microscopes and imposes wavelength dependence on the spatial resolution of the final image. As a result, fluorescent objects with dimensions that are significantly smaller than their emission wavelength appear to be significantly larger than their actual size. The ability to photoactivate fluorescence, however, offers the opportunity to overcome diffraction and acquire images with resolution at the

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Fig. 7 Illumination of 16 with visible light produces 18 with the concomitant transfer of a proton to 17 and the formation of 19. The absorption spectra recorded before (a) and after (b) irradiation show a significant bathochromic shift associated with the photoinduced transformation. Illumination at a wavelength positioned within the photogenerated absorption band excites 19 selectively and results in fluorescence (c). Nanoparticles of 11 with 16 and 17 in their interior can be imaged, after fluorescence activation, and can be identified in the corresponding diffraction-limited (d) and sub-diffraction (e, scale bar = 500 nm) images. The latter was reconstructed on the basis of multiple fluorescence activation and bleaching steps to yield a sixfold improvement in spatial resolution [reproduced with permission from ref. 59a].

nanometer level. Indeed, an image (e in Fig. 7) of the very same sample, reconstructed sequentially on the basis of fluorescence activation, shows fluorescent objects with an average size of only 42 nm. This particular image was acquired by illuminating the sample continuously using a laser, operating at 405 nm, to excite the photoacid generator and produce the fluorescent species. The intensity of the laser was maintained at a value sufficiently low to ensure the presence of only a sparse population of fluorescent species at a given time. The concomitant illumination of the sample with a second laser, operating at 532 nm, was exploited to excite the fluorescent species, produced after proton transfer, localize them at the single-molecule level and, eventually, bleach them. Under these conditions, sequential activation and bleaching steps permit the localization of evolving populations of fluorophores over the course of hundreds of frames. The coordinates of the localized species can then be compiled into a single map to generate an image with spatial resolution that is solely dependent on the number of photons detected per molecule and the background noise. Therefore, this approach overcomes the wavelength dependence imposed by diffraction on conventional images and translates into a significant improvement in spatial resolution.

Co-encapsulation of noninteracting guests The exchange of either electrons or energy between co-encapsulated guests requires either redox or spectral complementarity, respectively. Similarly, the transfer of protons between species entrapped within the same polymer nanocarrier requires

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functional groups capable of releasing and capturing protons. These interguest interactions can be avoided intentionally with a careful choice of chromophoric groups. Under these conditions, distinct photoresponsive guests can be operated in parallel within the same nanosized host to impose multifunctional character of the resulting supramolecular assembly. In particular, the possibility of exploiting the fluorescence of one guest to image the nanoparticles together with the ability of another to release bioactive species permits diagnosis and therapy in tandem and, therefore, is especially valuable for theranostic applications.60 Under ultraviolet illumination, the oxazine ring of 17 opens to bring the coumarin appendage in conjugation with the 3H-indolium cation of 20 (Fig. 8).61 The cationic chromophore of the resulting zwitterionic isomer is essentially identical to that of the protonated counterpart 19. In fact, this structural transformation also shifts bathochromically the main absorption band of the coumarin fluorophore. Irradiation at a wavelength positioned within the photogenerated absorption band can, therefore, be exploited to excite selectively 20 with concomitant fluorescence. This species, however, reverts spontaneously back to 17 on a microsecond timescale. As a result, the fluorescence of this system can be switched reversibly on the basis of the photoinduced opening and thermal closing of the oxazine ring. By contrast, the 4-nitroaniline chromophore of 21 can be excited with visible light to encourage the formation of 22 with the concomitant release of nitric oxide. This bioactive species has a fundamental role in cancer therapy, if its generation is accurately controlled,62,63 and can be monitored conveniently using electrochemical measurements. Indeed, the generation of nitric oxide translates into an amperometric signal and,

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Fig. 8 Nanoparticles of 23 co-encapsulate 17 and 21 in their hydrophobic interior and permit the photoinduced control of both guests in parallel. The photochemical and reversible interconversion of 17 and 20 translates into the modulation of the emission intensity (a) of one component. The photoinduced and irreversible transformation of 21 into 22 produces nitric oxide and an amperometric signal (b) under illumination [reproduced with permission from ref. 61].

therefore, its formation can be probed electrochemically during irradiation. These particular switchable fluorophore and nitricoxide generator can be co-encapsulated within nanoparticles of 23 and operated together under optical control without interference. In particular, the emission intensity (a in Fig. 8) can be switched on and off relying on the reversible interconversion of 17 and 20, while nitric-oxide bursts (b in Fig. 8) can be produced on the basis of the irreversible transformation of 21 into 22. Furthermore, these polymer nanocarriers can cross the membrane of A375 melanoma cells and transport their photoresponsive and multifunctional cargo to the cytosol. Under these conditions, the fluorescence of one guest can be employed to visualize the internalized nanoparticles and the release of nitric oxide from the other can be exploited to photoinduce cell mortality. Further complexity can be engineered into similar nanoparticles with an appropriate choice of noninteracting guests. Specifically, pairs of molecules able to emit in distinct spectral regions without mutual interference permit the acquisition of fluorescence images with two detection channels in parallel.

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Furthermore, the excitation events ultimately leading to emission can be coupled with photochemical reactions designed to yield different products. Under these conditions, the simultaneous photoinduced generation of two distinct species in a precise region of space can be accurately imaged with fluorescence measurements in two distinct channels. Compounds 24 (Fig. 9) and 25 satisfy all these design requirements and can be both encapsulated within nanoparticles of 23.64 The phthalocyanine and benzofurazan chromophores of the two guests produce red and green fluorescence, respectively, upon excitation. In addition, part of the excited phthalocyanine guests undergo intersystem crossing and sensitize the generation of singlet oxygen, which is, of course, the main cytotoxic species in the photodynamic therapy of cancer. Furthermore, the 4-nitroaniline fragment of 25 is essentially identical to that incorporated in 21 and, once again, releases nitric oxide under illumination without any interference from the covalently-bound green-fluorescent tag. It follows that the excitation of 24 produces red fluorescence and singlet oxygen, while excitation of 25 generates green fluorescence and nitric oxide. Indeed, these multifunctional

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Fig. 9 Nanoparticles of 23 co-encapsulate 24 and 25 in their hydrophobic interior. Under illumination, the former guest sensitizes the generation of singlet oxygen and produces fluorescence. Similarly, the latter guest releases nitric oxide and produces fluorescence as well. The emission bands of the two guests, however, are resolved across the visible region. Therefore, the intracellular release of singlet oxygen (a) and nitric oxide (b) can be monitored in parallel by recording fluorescence images with dual detection [reproduced with permission from ref. 61].

supramolecular assemblies can be operated intracellularly and probed with dual detection within the intracellular space. Images recorded after the incubation of the doped polymer nanocarriers with A375 melanoma cells reveal green (a in Fig. 9) and red (b in Fig. 9) fluorescence in the intracellular space upon excitation of 25 and 24 respectively. Moreover, the simultaneous photogeneration of singlet oxygen and nitric oxide in the cytosol induces amplified cell mortality, as a result of additive/synergistic effects associated with these two cytotoxic species.

Conclusions Amphiphilic polymers assemble into nanoscale particles, capable of enveloping hydrophobic molecules in an aqueous environment. Solvophobic interactions between the hydrophobic domains of the macromolecular components and their guests are responsible for these supramolecular events. This relatively simple strategy permits the encapsulation of multiple photoresponsive species with distinct functions within the very same polymer nanocarrier to produce multifunctional assemblies with unique properties. Specifically, the co-encapsulation of complementary donors and acceptors within these nanostructured hosts enforces them in close proximity and facilitates the exchange of electrons, energy or protons upon light excitation. In turn, such interguest interactions can be exploited to assess the stability of the nanoparticles, monitor their ability to release their cargo, engineer luminescent probes with outstanding photophysical properties and switch fluorescence under optical control. Alternatively, subsets of guests with specific functions can be designed to retain their individual properties without mutual interference, after co-encapsulation in the nanocarriers. This modular approach for the construction of supramolecular assemblies offers the opportunity to engineer multifunctional

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character into a single nanoparticle. In particular, luminescent constructs able to release distinct bioactive molecules intracellularly can be developed on the basis of these general operating principles. Indeed, these fascinating studies demonstrate that polymer nanocarriers are versatile scaffolds for the modular integration of multiple components into a single supramolecular construct with functions that would otherwise be impossible to replicate with the separate constituents. Thus, further investigations aimed at the elucidation of the basic factors governing their assembly and at the identification of viable mechanisms for the design of further complexity into their functions will presumably lead to invaluable nanostructured materials for a diversity of biomedical applications.

Acknowledgements `s postdoctoral grant from the JGA acknowledges a Beatriu de Pino Generalitat de Catalunya (2011 BP-A-00270). SS acknowledges the Marie Curie Program (237962 CYCLON, FP7-PEOPLE-ITN-2008), the MIUR (Projects PRIN 2008, PRIN 2011) and the AIRC (Project IG-12834) for financial support. FMR thanks the National Science Foundation (CAREER Award CHE-0237578, CHE-0749840 and CHE-1049860) for supporting his research program.

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