Sicheng Tang

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.

Energy-Transfer Schemes To Probe Fluorescent Nanocarriers and Their Emissive Cargo

A strategy to probe supramolecular nanocarriers and their cargo in the intracellular space was developed on the basis of fluorescence measurements and energy transfer. It relies on the covalent attachment of an energy donor, or acceptor, to the macromolecular backbone of amphiphilic polymers and the noncovalent encapsulation of a complementary acceptor, or donor, in the resulting micelles. In aqueous environments, these macromolecules self-assemble into nanostructured constructs and bring the complementary chromophores in close proximity to enable efficient energy transfer. These supramolecular assemblies travel from the extracellular to the intracellular space and retain their integrity in the process. Indeed, donors and acceptors remain close to each other after internalization, and excitation of the former chromophores translates into significant intracellular emission from the latter. Furthermore, these supramolecular assemblies exchange their components with fast kinetics in aqueous dispersions because of the reversible character of the noncovalent contacts holding them together. As a result, micelles incorporating exclusively the donors and nanocarriers containing only the acceptors scramble their chromophoric building blocks, upon mixing, to allow the transfer of energy. These dynamic processes can be reproduced in the intracellular environment with the sequential incubation of cells with the two sets of complementary nanostructured assemblies. Thus, these operating principles and choice of supramolecular synthons are particularly valuable to monitor self-assembling nanocarriers and their cargo inside living cells and can facilitate the elucidation of the behavior of these promising delivery vehicles in a diversity of biological specimens.

Synthesis in living cells with the assistance of supramolecular nanocarriers

Independent supramolecular nanocarriers can transport pairs of complementary reactants inside living cells in two consecutive incubation steps. After the second internalization step, the nonemissive reactants produce a fluorescent product with the concomitant appearance of intense fluorescence exclusively in the intracellular space. These results demonstrate that supramolecular delivery can be exploited to perform chemical reactions inside target cells and can lead to valuable strategies for the intracellular synthesis of drugs.

Highlighting Cancer Cells with Halochromic Switches

Halochromic coumarin-oxazine prefluorophores and targeting folate ligands can be connected covalently to the side chains of amphiphilic polymers. The resulting macromolecular constructs assemble into nanoparticles in aqueous environments. The prefluorophores do not produce any detectable fluorescence at neutral pH, but are converted into fluorophores with intense visible emission at acidic pH. Protonation opens the oxazine heterocycle to shift bathochromically the coumarin absorption and activate fluorescence with a brightness per nanoparticle approaching 5 × 105 M-1 cm-1. This value translates into a 170-fold enhancement relative to the isolated fluorophores dissolved in organic solvent. The folate ligands direct these multicomponent constructs into acidic intracellular compartments of folate-positive cells, where the prefluorophores switch to the corresponding fluorophores and produce fluorescence. The pH-induced activation of the signaling units ensures negligible background fluorescence from the extracellular matrix, which instead limits considerably the contrast accessible with model systems incorporating conventional nonactivatable fluorophores. Furthermore, no intracellular fluorescence can be detected when the very same measurements are performed with folate-negative cells. Nonetheless, control experiments demonstrate that the covalent connection of the prefluorophores to the polymer backbone of the amphiphilic constructs is essential to ensure selectivity. Model systems with prefluorophores noncovalently encapsulated cannot discriminate folate-positive from -negative cells. Thus, our structural design for the covalent integration of activatable signaling units and targeting ligands within the same nanostructured assembly together with the photophysical properties engineered into the emissive components offer the opportunity to highlight cancer cells selectively with high brightness and optimal contrast.

Supramolecular delivery of fluorescent probes in developing embryos

Self-assembling nanocarriers of amphiphilic polymers encapsulate hydrophobic fluorophores in their hydrophobic interior and, upon injection in Drosophila melanogaster embryos, release their cargo into the cellular blastoderm. The fluorescence of the delivered probes can be detected immediately upon injection to allow the visualization of the very early stages of embryogenesis.

A Photoswitchable Fluorophore for the Real-Time Monitoring of Dynamic Events in Living Organisms

This study reports the synthesis of a photoactivatable fluorophore with optimal photochemical and photophysical properties for the real-time tracking of motion in vivo. The photoactivation mechanism designed into this particular compound permits the conversion of an emissive reactant into an emissive product with resolved fluorescence, under mild illumination conditions that are impossible to replicate with conventional switching schemes based on bleaching. Indeed, the supramolecular delivery of these photoswitchable probes into the cellular blastoderm of Drosophila melanogaster embryos allows the real-time visualization of translocating molecules with no detrimental effects on the developing organisms. Thus, this innovative mechanism for fluorescence photoactivation can evolve into a general chemical tool to monitor dynamic processes in living biological specimens.

Structural Implications on the Properties of Self-Assembling Supramolecular Hosts for Fluorescent Guests

Nine amphiphilic macromolecules with decyl and oligo(ethylene glycol) side chains, randomly distributed along a common poly(methacrylate) backbone, were synthesized from the radical copolymerization of appropriate methacrylate monomers. The resulting amphiphilic constructs differ in (1) the ratio between their hydrophobic and hydrophilic components, (2) the length of their oligo(ethylene glycol) chains, and/or (3) the molecular weight. When the ratio between hydrophobic and hydrophilic segments is comprised between 6:1 and 1:2, the macromolecules assemble spontaneously into particles with nanoscaled dimensions in neutral buffer and capture hydrophobic borondipyrromethene chromophores in their interior. However, the critical concentration required for the assembly of these supramolecular hosts as well as their hydrodynamic diameter, supramolecular weight, and number of constituent macromolecular building blocks all vary monotonically with the ratio between hydrophobic and hydrophilic components. Specifically, the critical concentration decreases and the other three parameters increase as the relative hydrophobic content raises. Furthermore, an increase in the relative hydrophobic content also discourages interchromophoric interactions between entrapped guests in both ground and excited states as well as delays access of potential quenchers. In fact, these observations demonstrate that the hydrophobic components must be in excess over their hydrophilic counterparts for optimal supramolecular hosts to assemble. Indeed, a ratio of 6:1 between the numbers of decyl and oligo(ethylene glycol) side chains appears to be ideal for this particular structural design. Under these conditions, supramolecular hosts assemble spontaneously even at relatively low polymer concentrations and their fluorescent guests do not escape into the bulk aqueous solution, despite the reversibility of the noncovalent interactions holding the supramolecular container together. Thus, these systematic investigations provide invaluable structural guidelines to design self-assembling supramolecular hosts with optimal composition for the effective encapsulation of fluorescent guests and can lead to ideal delivery vehicles for the transport of imaging probes to target locations in biological samples.

Self-Assembling Nanoparticles of Amphiphilic Polymers for In Vitro and In Vivo FRET Imaging

Self-assembling nanoparticles of amphiphilic polymers are viable delivery vehicles for transporting hydrophobic molecules across hydrophilic media. Noncovalent contacts between the hydrophobic domains of their macromolecular components are responsible for their formation and for providing a nonpolar environment for the encapsulated guests. However, such interactions are reversible and, as a result, these supramolecular hosts can dissociate into their constituents amphiphiles to release the encapsulated cargo. Operating principles to probe the integrity of the nanocarriers and the dynamic exchange of their components are, therefore, essential to monitor the fate of these supramolecular assemblies in biological media. The co-encapsulation of complementary chromophores within their nonpolar interior offers the opportunity to assess their stability on the basis of energy transfer and fluorescence measurements. Indeed, the exchange of excitation energy between the entrapped chromophores can only occur if the nanoparticles retain their integrity to maintain donors and acceptors in close proximity. In fact, energy-transfer schemes are becoming invaluable protocols to elucidate the transport properties of these fascinating supramolecular constructs in a diversity of biological preparations and can facilitate the identification of strategies to deliver contrast agents and/or drugs to target locations in living organisms for potential diagnostic and/or therapeutic applications.

Semiconductor Quantum Dots with Photoresponsive Ligands

Photochromic or photocaged ligands can be anchored to the outer shell of semiconductor quantum dots in order to control the photophysical properties of these inorganic nanocrystals with optical stimulations. One of the two interconvertible states of the photoresponsive ligands can be designed to accept either an electron or energy from the excited quantum dots and quench their luminescence. Under these conditions, the reversible transformations of photochromic ligands or the irreversible cleavage of photocaged counterparts translates into the possibility to switch luminescence with external control. As an alternative to regulating the photophysics of a quantum dot via the photochemistry of its ligands, the photochemistry of the latter can be controlled by relying on the photophysics of the former. The transfer of excitation energy from a quantum dot to a photocaged ligand populates the excited state of the species adsorbed on the nanocrystal to induce a photochemical reaction. This mechanism, in conjunction with the large two-photon absorption cross section of quantum dots, can be exploited to release nitric oxide or to generate singlet oxygen under near-infrared irradiation. Thus, the combination of semiconductor quantum dots and photoresponsive ligands offers the opportunity to assemble nanostructured constructs with specific functions on the basis of electron or energy transfer processes. The photoswitchable luminescence and ability to photoinduce the release of reactive chemicals, associated with the resulting systems, can be particularly valuable in biomedical research and can, ultimately, lead to the realization of imaging probes for diagnostic applications as well as to therapeutic agents for the treatment of cancer.

Far-Red Photoactivatable BODIPYs for the Super-Resolution Imaging of Live Cells

The photoinduced disconnection of an oxazine heterocycle from a borondipyrromethene (BODIPY) chromophore activates bright far-red fluorescence. The high brightness of the product and the lack of autofluorescence in this spectral region allow its detection at the single-molecule level within the organelles of live cells. Indeed, these photoactivatable fluorophores localize in lysosomal compartments and remain covalently immobilized within these organelles. The suppression of diffusion allows the reiterative reconstruction of subdiffraction images and the visualization of the labeled organelles with excellent localization precision. Thus, the combination of photochemical, photophysical and structural properties designed into our fluorophores enable the visualization of live cells with a spatial resolution that is inaccessible to conventional fluorescence imaging.