Redox-Triggered Activation of Heavy-Atom-Free Photosensitizer and Implications in Targeted Photodynamic Therapy.

A strategy for a redox-activatable heavy-atom-free photosensitizer (PS) based on thiolated naphthalimide has been demonstrated. The PS exhibits excellent reactive oxygen species (ROS) generation in the monomeric state. However, when encapsulated in a disulfide containing bioreducible amphiphilic triblock copolymer aggregate (polymersome), the PS exhibits aggregation in the confined hydrophobic environment, which results in a smaller exciton exchange rate between the singlet and triplet excited states (TDDFT studies), and consequently, the ROS generation ability of the PS was almost fully diminished. Such a PS (in the dormant state)-loaded redox-responsive polymersome showed excellent cellular uptake and intracellular release of the PS in its active form, which enabled cell killing upon light irradiation due to ROS generation. In a control experiment involving aggregates of a similar block copolymer, but lacking the bioreducible disulfide linkage, no intracellular reactivation of the PS was noticed, highlighting the importance of stimuli-responsive polymer assemblies in the area of targeted photodynamic therapy.

DNA nanodevices map enzymatic activity in organelles.

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.

A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome.

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.

Hydrogen-bonding-induced chain folding and vesicular assembly of an amphiphilic polyurethane.

We have reported synthesis and vesicular assembly of a novel amphiphilic polyurethane with hydrophobic backbone and hydrophilic pendant carboxylic acid groups which were periodically grafted to the backbone via a tertiary amine group. In aqueous medium the polymer chain adopted a folded conformation which was stabilized by intrachain H-bonding among the urethane groups. Such a model was supported by concentration and solvent-dependent FT-IR, powder XRD, and urea-mediated "denaturation" experiments. Folded polymer chains further formed vesicular assembly which was probed by dynamic light scattering, TEM, AFM, SEM, and fluorescence microscopic studies, and dye encapsulation experiments. pH-dependent DLS and fluorescence microscopic studies revealed stable polymersome in entire tested pH window of 3.5-11.0. Zeta potential measurements showed a negatively charged surface in basic pH while a charge-neutral surface in neutral and acidic pH. MTT assay with CHO cell line indicated good cell viability.

Amphiphilic random copolymer vesicle induces differentiation of mouse C2C12 myoblasts.

Herein we report that vesicular assembly from a simple non-ionic amphiphilic random copolymer initiates extremely efficient myotube formation from C2C12 myoblast cells in standard growth media lacking horse serum. Control experiments with structurally related polymers and a small molecule suggest the absolute necessity of the vesicular assembly as well as the particular hydrophilic functionality in mediating such high yielding muscle cell generation. The LDH assay indicates that the membrane integrity is retained during cell-cell fusion. Expression of various myogenic factors such as MyoD, myogenin and P-21 has been examined in the presence of the polymersome and control molecules to rationalize this serendipitous discovery.

pH-responsive aggregation of amphiphilic glyco-homopolymer.

The first example of amphiphilic glyco-homopolymers is reported and their aggregation properties as a function of solution pH are studied. Two structurally similar polymers with different hydrophobicity (C8 and C6 alkyl chains) are examined. Both polymers form micelle-type aggregates in aqueous solution. The size and micro-environment of the aggregates are found to be strongly dependent on solution pH because of the state of protonation of the tertiary amine group. At acidic pH, swollen multi-micellar aggregates are formed, presumably because of the electrostatic repulsion among the ammonium ions. At basic pH more compact particles are found, which further co-assemble to generate either garland type (C8) or fractal-aggregates (C6).

Vesicular assembly and thermo-responsive vesicle-to-micelle transition from an amphiphilic random copolymer.

Vesicular assembly from a thermo-responsive amphiphilic random copolymer is reported. Vesicle-to-micelle transition above a critical morphology transition temperature (CMTT) resulted in selective triggered release of encapsulated hydrophilic guests over hydrophobic ones. The aggregation pattern of a control polymer indicated a defined role of the methacrylamide groups in the polymer backbone for such unprecedented self-assembly from a simple polymer.

Aggregation and pH responsive disassembly of a new acid-labile surfactant synthesized by thiol-acrylate Michael addition reaction.

Nucleophilic thiol-acrylate Michael reaction between a hydrophobic thiol and hydrophilic acrylate derivative generated a nonionic surfactant with acid-labile ß-thiopropionate linker. Micellization of the surfactant, its ability to encapsulate hydrophobic dye, acid-induced disruption of the aggregate and pH-selective dye release profile have been revealed in this report. The micellar aggregates were found to be stable under neutral conditions, but they could be disrupted in acidic pH (5.3), and thus the encapsulated hydrophobic dye molecules could be selectively released. Appropriate control experiments revealed that the sulfur atom in the ß-position is essential for acidic hydrolysis of the ester functionality of the surfactant.