Development of Ln(III) Derivatives as 19F Parashift Probes.

19F parashift probes with paramagnetically shifted reporter nuclei provide attractive platforms to develop molecular imaging probes. These probes enable ratiometric detection of molecular disease markers using a direct detection technique. Here, we describe a series of trivalent lanthanide (Ln(III)) complexes that are structural analogues of the clinically approved MR contrast agent (CA) ProHance to obtain LnL 19F parashift probes. We evaluated trans-gadolinium paramagnetic lanthanides compared to diamagnetic YL for 19F chemical shift and relaxation rate enhancement. The paramagnetic contribution to chemical shift (δPCS) for paramagnetic LnL exhibited either shifts to lower frequency (δPCS < 0 for TbL, DyL, and HoL) or shifts to higher frequency (δPCS > 0 for ErL, TmL, and YbL) compared to YL 19F spectroscopic signal. Zero-echo time pulse sequences achieved 56-fold sensitivity enhancement for DyL over YL, while developing probe-specific pulse sequences with fast delay times and acquisition times achieved 0.6-fold enhancement in limit of detection for DyL. DyL provides an attractive platform to develop 19F parashift probes for ratiometric detection of enzymatic activity.

Musculoskeletal imaging of senescence.

Senescent cells play a vital role in the pathogenesis of musculoskeletal (MSK) diseases, such as chronic inflammatory joint disorders, rheumatoid arthritis (RA), and osteoarthritis (OA). Cellular senescence in articular joints represents a response of local cells to persistent stress that leads to cell-cycle arrest and enhanced production of inflammatory cytokines, which in turn perpetuates joint damage and leads to significant morbidities in afflicted patients. It has been recently discovered that clearance of senescent cells by novel "senolytic" therapies can attenuate the chronic inflammatory microenvironment of RA and OA, preventing further disease progression and supporting healing processes. To identify patients who might benefit from these new senolytic therapies and monitor therapy response, there is an unmet need to identify and map senescent cells in articular joints and related musculoskeletal tissues. To fill this gap, new imaging biomarkers are being developed to detect and characterize senescent cells in human joints and musculoskeletal tissues. This review article will provide an overview of these efforts. New imaging biomarkers for senescence cells are expected to significantly improve the specificity of state-of-the-art imaging technologies for diagnosing musculoskeletal disorders.

HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.