In Situ, Noncovalent Labeling and Stimulated Emission Depletion-Based Super-Resolution Imaging of Supramolecular Peptide Nanostructures.

Supramolecular materials have gained substantial interest for a number biological and nonbiological applications. However, for optimum utilization of these dynamic self-assembled materials, it is important to visualize and understand their structures at the nanoscale, in solution and in real time. Previous approaches for imaging these structures have utilized super-resolution optical imaging methods such as STORM, which has provided important insights, but suffers from drawbacks of complex sample preparation and slow acquisition times, thus limiting real-time in situ imaging of dynamic processes. We demonstrate a noncovalent fluorescent labeling design for STED-based super-resolution imaging of self-assembling peptides. This is achieved by in situ, electrostatic binding of anionic sulfonates of Alexa-488 dye to the cationic sites of lysine (or arginine) residues exposed on the peptide nanostructure surface. A direct, multiscale visualization of static structures reveals hierarchical organization of supramolecular fibers with sub-60 nm resolution. In addition, the degradation of nanofibers upon enzymatic hydrolysis of peptide could be directly imaged in real time, and although resolution was compromised in this dynamic process, it provided mechanistic insights into the enzymatic degradation process. Noncovalent Alexa-488 labeling and subsequent imaging of a range of cationic self-assembling peptides and peptide-functionalized gold nanoparticles demonstrated the versatility of the methodology for the imaging of cationic supramolecular structures. Overall, our approach presents a general and simple method for the electrostatic fluorescent labeling of cationic peptide nanostructures for nanoscale imaging under physiological conditions and probe dynamic processes in real time and in situ.

Focused ultrasound blood brain barrier opening mediated delivery of MRI-visible albumin nanoclusters to the rat brain for localized drug delivery with temporal control.

There is an ongoing need for noninvasive tools to manipulate brain activity with molecular, spatial and temporal specificity. Here we have investigated the use of MRI-visible, albumin-based nanoclusters for noninvasive, localized and temporally specific drug delivery to the rat brain. We demonstrated that IV injected nanoclusters could be deposited into target brain regions via focused ultrasound facilitated blood brain barrier opening. We showed that nanocluster location could be confirmed in vivo with MRI. Additionally, following confirmation of nanocluster delivery, release of the nanocluster payload into brain tissue can be triggered by a second focused ultrasound treatment performed without circulating microbubbles. Release of glutamate from nanoclusters in vivo caused enhanced c-Fos expression, indicating that the loading capacity of the nanoclusters is sufficient to induce neuronal activation. This novel technique for noninvasive stereotactic drug delivery to the brain with temporal specificity could provide a new way to study brain circuits in vivo preclinically with high relevance for clinical translation.