Labelling primary immune cells using bright blue fluorescent nanoparticles

Abstract

Tracking cell movements is an important aspect of many biological studies. Reagents for cell tracking must not alter the biological state of the cell and must be bright enough to be visualized above background autofluorescence, a particular concern when imaging in tissue. Currently there are few reagents compatible with standard UV excitation filter sets (e.g. DAPI) that fulfill those requirements, despite the development of many dyes optimized for violet excitation (405 nm). A family of boron-based fluorescent dyes, difluoroboron β-diketonates, has previously served as bio-imaging reagents with UV excitation, offering high quantum yields and wide excitation peaks. In this study, we investigated the use of one such dye as a potential cell tracking reagent. A library of difluoroboron dibenzoylmethane (BF₂dbm) conjugates were synthesized with biocompatible polymers including: poly(L-lactic acid) (PLLA), poly(ε-caprolactone) (PCL), and block copolymers with poly(ethylene glycol) (PEG). Dye–polymer conjugates were fabricated into nanoparticles, which were stable for a week at 37 °C in water and cell culture media, but quickly aggregated in saline. Nanoparticles were used to label primary splenocytes; phagocytic cell types were more effectively labelled. Labelling with nanoparticles did not affect cellular viability, nor basic immune responses. Labelled cells were more easily distinguished when imaged on a live tissue background than those labelled with a commercially available UV-excitable cytoplasmic labelling reagent. The high efficiency in terms of both fluorescence and cellular labelling may allow these nanoparticles to act as a short-term cell labelling strategy while wide excitation peaks offer utility across imaging and analysis platforms.