Photodynamic therapy (PDT) has been explored as a therapeutic strategy to clear toxic amyloid aggregates involved in neurodegenerative disorders such as Alzheimer's disease. A major limitation of PDT is off-target oxidation, which can be lethal for the surrounding cells. We have shown that a novel class of oligo-pphenylene ethynylenes (OPEs) exhibit selective binding and fluorescence turn-on in the presence of prefibrillar and fibrillar aggregates of disease-relevant proteins such as amyloid-β (Aβ) and αsynuclein. Concomitant with fluorescence turn-on, OPE also photosensitizes singlet oxygen under illumination through the generation of a triplet state, pointing to the potential application of OPEs as photosensitizers in PDT. Herein, we investigated the photosensitizing activity of an anionic OPE for the photo-oxidation of Aβ fibrils and compared its efficacy to the well-known but nonselective photosensitizer methylene blue (MB). Our results show that, while MB photo-oxidized both monomeric and fibrillar conformers of Aβ40, OPE oxidized only Aβ40 fibrils, targeting two histidine residues on the fibril surface and a methionine residue located in the fibril core. Oxidized fibrils were shorter and more dispersed but retained the characteristic β-sheet rich fibrillar structure and the ability to seed further fibril growth. Importantly, the oxidized fibrils displayed low toxicity. We have thus discovered a class of novel theranostics for the simultaneous detection and oxidization of amyloid aggregates. Importantly, the selectivity of OPE's photosensitizing activity overcomes the limitation of off-target oxidation of traditional photosensitizers and represents an advancement of PDT as a viable strategy to treat neurodegenerative disorders.

activation. Despite extensive work over the last 15 years, the biophysical mechanisms of INS are still debated. Multiple downstream molecular effectors have been implicated, however the ability of IR to evoke cellular responses in range of cell phenotypes suggests that the mechanism has a universal basis. While cell membrane dynamics and nanoporation have been explored, limitations in imaging technology make it difficult to study such fast photothermal processes such as INS. Electrophysiology and electrodynamic modeling suggests that pulsed IR light nonspecifically depolarizes irradiated cells by thermallymediated transient alterations in cell membrane structure (e.g. trans-cis isomerization of fatty acid chains). While the electrodynamic model fits experimental data well, the hypothesis lacks direct experimental evidence of lipid structural changes. Nonlinear Raman imaging (NRI), (e.g. coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)) is a rapidly evolving technique that is particularly good for observing lipid dynamics based on vibrational spectra changes. We have developed a platform to observe vibrational spectral changes during INS to study lipid dynamics in cellular model systems. We will present our methods on validating NRI to identify vibrational spectral changes to pulsed IR light in microbeads, application towards several live neural cell models, and the biophysical implications of the results. A deeper understanding of biophysical mechanisms underlying INS will provide the basis for optimizing delivery of infrared light for neuromodulation and shed light on novel neuromodulation technologies. Extracellular plaques composed of b-sheet rich amyloid-beta (Ab) fibrils are believed to cause neurodegeneration and cognitive decline in Alzheimer's disease. Fibrils can form through a primary nucleation-dependent aggregation pathway or secondary nucleation mechanism in which preformed fibrils cause Ab monomers to misfold and aggregate into amyloid fibrils. As Ab aggregation is believed to occur during the prolonged asymptomatic stage before neurodegeneration, it is important to gain insights into the aggregation process. Photodynamic therapy utilizes photosensitizing molecules to produce reactive oxygen species, which in turn can oxidize an array of biomolecules including proteins. We have recently shown that a novel Ab fibril sensor, the anionic phenylene ethynylene oligomer OPE1-, exhibits controllable photosensitizing activity. When bound to Ab fibrils, OPE1-becomes highly fluorescent, sensitizes the production of singlet oxygen, and induces fibril oxidation upon irradiation. As the singlet oxygen species is short-lived, the controlled photosensitization thus lowers off-target oxidation. In this study we evaluated the effects of OPE1-induced Ab40 fibril oxidation on its seeding potency. Ab40 aggregation seeded by non-oxidized and oxidized fibrils were characterized by Thioflavin-T fluorescence assay and compared. Morphologies of the fibrils produced by both seeding condition...

Photodynamic therapy (PDT) has been explored as a therapeutic strategy to clear toxic amyloid aggregates involved in neurodegenerative disorders such as Alzheimer’s disease. A major limitation of PDT is off-target oxidation, which can be lethal for the surrounding cells. We have shown that a novel class of oligo-p-phenylene ethynylene-based compounds (OPEs) exhibit selective binding and fluorescence turn-on in the presence of pre-fibrillar and fibrillar aggregates of disease-relevant proteins such as amyloid-β (Aβ) and α-synuclein. Concomitant with fluorescence turn-on, OPE also photosensitizes singlet oxygen under illumination through the generation of a triplet state, pointing to the potential application of OPEs as photosensitizers in PDT. Herein, we investigated the photosensitizing activity of an anionic OPE for the photo-oxidation of toxic Aβ aggregates and compared its efficacy to the well-known but non-selective photosensitizer methylene blue (MB). Our results show that while MB photo-oxidized both monomeric and fibrillar conformers of Aβ40, OPE oxidized only Aβ40 fibrils, targeting two histidine residues on the fibril surface and a methionine residue located in the fibril core. Oxidized fibrils were shorter and more dispersed, but retained the characteristic β-sheet rich fibrillar structure and the ability to seed further fibril growth. Importantly, the oxidized fibrils displayed low toxicity. We have thus discovered a class of novel theranostics for the simultaneous detection and oxidization of amyloid aggregates. Importantly, the selectivity of OPE’s photosensitizing activity overcomes the limitation of off-target oxidation of currently available photosensitizers, and represents a significant advancement of PDT as a viable strategy to treat neurodegenerative disorders.