Dissecting extracellular and intracellular distribution of nanoparticles and their contribution to therapeutic response by monochromatic ratiometric imaging

Efficient delivery of payload to intracellular targets has been identified as the central principle for nanomedicine development, while the extracellular targets are equally important for cancer treatment. Notably, the contribution of extracellularly distributed nanoparticles to therapeutic outcome is far from being understood. Herein, we develop a pH/light dual-responsive monochromatic ratiometric imaging nanoparticle (MRIN), which functions through sequentially lighting up the intracellular and extracellular fluorescence signals by acidic endocytic pH and near-infrared light. Enabled by MRIN nanotechnology, we accurately quantify the extracellular and intracellular distribution of nanoparticles in several tumor models, which account for 65-80% and 20-35% of total tumor exposure, respectively. Given that the majority of nanoparticles are trapped in extracellular regions, we successfully dissect the contribution of extracellularly distributed nanophotosensitizer to therapeutic efficacy, thereby maximize the treatment outcome. Our study provides key strategies to precisely quantify nanocarrier microdistribtion and engineer multifunctional nanomedicines for efficient theranostics.

A precise and general FRET-based method for monitoring structural transitions in protein self-organization

Proteins assemble into a tremendous variety of dynamic and functional structures. Sensitive measurements directly in cells with a high spatiotemporal resolution are needed to distinguish these different assemblies. Here, we demonstrate precise and continuous monitoring of cytoplasmic protein self-assemblies and their structural transitions. Intermolecular FRET with both the donor and acceptor protein at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We measure different assembly structures, transient intermediate states’ kinetics, and assembly formation resolved in space and time. Specifically, the method recapitulates that i) the mutant Huntingtin exon1 (mHttex1) protein first forms low-FRET and presumably less ordered assemblies in yeast and human cells, which develop into high-FRET aggregates, ii) the chaperone DNAJB6b prevents low-FRET mHttex1 assemblies, yet coassembles with mHttex1 aggregates, and iii) FUS’ condensates have mutation-dependent nanoscopic structures. FACS measurements allow assembly measurement in a high-throughput manner crucial for screening efforts, while fluorescence microscopy provides spatiotemporally-resolved measurements on the single-condensate level during a cell’s lifetime to assess the biological consequences. Implementation in other native or non-native proteins could provide insight into many studies involving protein condensation or aggregation.

Quantification of bone marrow interstitial pH and calcium concentration by intravital ratiometric imaging

The fate of hematopoietic stem cells (HSCs) in the bone marrow can be directed by microenvironmental factors including extracellular calcium ion concentration (T h can vary significantly with bone remodeling, but the local e around individual HSCs in vivo remains unknown. Here we developed an intravital ratiometric imaging approach to quantify the absolute pH and in the mouse calvarial bone marrow, taking into account the pH sensitivity of the calcium probe and the wavelength-dependent optical loss through bone to ensure unbiased ratiometric analyses. We uncovered substantial heterogeneity in f (1.0 - 3.6 mM) surrounding steady-state HSCs. While the lowest a (0.3 mM) was found in regions dominated by bone formation, HSCs were not found in those locations. This work thus established a tool to further investigate t and pH in the HSC niche under malignant or stressed conditions and can be broadly applied to other tissue types.