Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

Macromolecular Crowding as an Intracellular Stimulus for Responsive Nanomaterials.

Stimuli-responsive materials are exploited in biological, materials, and sensing applications. We introduce a new endogenous stimulus, biomacromolecule crowding, which we achieve by leveraging changes in thermoresponsive properties of polymers upon high concentrations of crowding agents. We prepare poly(2-oxazoline) amphiphiles that exhibit lower critical solution temperatures (LCST) in serum above physiological temperature. These amphiphiles stabilize oil-in-water nanoemulsions at temperatures below the LCST but are ineffective surfactants above the LCST, resulting in emulsion fusion. We find that the transformations observed upon heating nanoemulsions above their surfactant's LCST can instead be induced at physiological temperatures through the addition of polymers and protein, rendering thermoresponsive materials "crowding responsive." We demonstrate that the cytosol is a stimulus for nanoemulsions, with droplet fusion occurring upon injection into cells of living zebrafish embryos. This report sets the stage for classes of thermoresponsive materials to respond to macromolecule concentration rather than temperature changes.

Stimuli Induced Uptake of Protein-Like Peptide Brush Polymers.

Recently, we presented a strategy for packaging peptides as side-chains in high-density brush polymers. For this globular protein-like polymer (PLP) formulation, therapeutic peptides were shown to resist proteolytic degradation, enter cells efficiently and maintain biological function. In this paper, we establish the role charge plays in dictating the cellular uptake of these peptide formulations, finding that peptides with a net positive charge will enter cells when polymerized, while those formed from anionic or neutral peptides remain outside of cells. Given these findings, we explored whether cellular uptake could be selectively induced by a stimulus. In our design, a cationic peptide is appended to a sequence of charge-neutralizing anionic amino acids through stimuli-responsive cleavable linkers. As a proof-of-concept study, we tested this strategy with two different classes of stimuli, exogenous UV light and an enzyme (a matrix metalloproteinase) associated with the inflammatory response. The key finding is that these materials enter cells only when acted upon by the stimulus. This approach makes it possible to achieve delivery of the polymers, therapeutic peptides or an appended cargo into cells in response to an appropriate stimulus.