TADF Dye-Loaded Nanoparticles for Fluorescence Live-Cell Imaging

Abstract

Thermally activated delayed fluorescence (TADF) molecules offer nowadays a powerful tool in the development of novel organic light emitting diodes due to their capability of harvesting energy from non-emissive triplet states without using heavy-metal complexes. TADF emitters have very small energy difference between the singlet and triplet excited states, which makes thermally activated reverse intersystem crossing from the triplet states back to the singlet manifold viable. This mechanism generates a long-lived delayed fluorescence component which can be explored in the sensing of oxygen concentration, local temperature, or used in time-gated optical cell-imaging, to suppress interference from autofluorescence and scattering. Despite this strong potential, until recently the application of TADF outside lighting devices has been hindered due to the low biocompatibility, low aqueous solubility and poor performance in polar media shown by the vast majority of TADF emitters. To achieve TADF luminescence in biological media, careful selection or design of emitters is required. Unfortunately, most TADF molecules are not emissive in polar media, thus complexation with biomolecules or the formation of emissive aggregate states is required, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein, we demonstrate a facile method with great generalization potential that maintains the photophysical properties of solvated dyes by combining luminescent molecules with polymeric nanoparticles. Using an established swelling procedure, two known TADF emitters are loaded onto polystyrene nanoparticles to prepare TADF emitting nanomaterials able to be used in live-cell imaging. The obtained particles were characterized by optical spectroscopy and exhibited the desired TADF emission in aqueous media, due to the polymeric matrix shielding the dye from solvent polarity effects. The prepared nanoparticles were incubated with live human cancer cells and showed very low cytotoxicity and good cellular uptake, thus making fluorescence microscopy imaging possible at low dye concentrations.

Applying TADF Emitters in Bioimaging and Sensing—A Novel Approach Using Liposomes for Encapsulation and Cellular Uptake

“…This is in line with what has been reported previously (Crucho et al, 2020;Okkelman et al, 2020). The third, and most long-lived decay phase, is attributed to PF from DPTZ-DBO2 nanoparticles, in line with the observation by Crucho et al, 2020, who reported a very similar PF lifetime for the same type of DPTZ-DBO2 nanoparticles (Crucho et al, 2020). Consequently, we cannot claim there is a DF component in the emission from DPTZ-DBO2 nanoparticles internalised in NIH 3T3 cells, based on the fit of the data in Figure 5.…”

“…In this part of the work, we also investigated polystyrene nanoparticles containing the TADF emitter DPTZ-DBTO2, internalised in NIH 3T3 cells. The application of these nanoparticles in bioimaging was also described by Crucho et al, 2020 , but for MCF-7 cells. Confocal images of DPTZ-DBTO2 nanoparticles in NIH 3T3 cells can be found in the SI ( Supplementary Figure S5 ).…”

“…The longer DF lifetimes observed by Crucho et al, 2020 is outside the measurement range of our TCSPC detection system. Furthermore, based on observations by Crucho et al, 2020, from the same DPTZ-DBO2 nanoparticles, the intensity of the DF component would very likely be 2-3 orders of magnitude weaker than the PF component (Crucho et al, 2020). This would make the detection of a DF component very challenging in a fluorescence microscopy experiment.…”

“…This TADF system is unique as it can function as an intracellular sulfite sensor ( Yin et al, 2020 ). The relatively small body of previous works shows intracellular localisation in lysosomes ( Xiong et al, 2014 ; Qi et al, 2020 ), in most cases, but also in the cytosol ( Crucho et al, 2020 ) and on the cell membrane ( Li et al, 2017 ). The unique and unusual time domains of the TADF emission remain the centerpiece, driving the development of TADF systems for bioimaging.…”

“…Accordingly, there is a need for further functionalization of TADF emitters to improve their biocompatibility. Thus far, the approaches taken have included various encapsulation methods, intending to shield the TADF emitter from the cellular environment whilst enhancing cellular uptake and retention ( Xiong et al, 2014 , Li et al, 2017 , Qi et al, 2020 Luo et al, 2020 , Crucho et al, 2020 ). In this work, we developed a novel method for TADF emitter confinement and cellular delivery/uptake based on liposomes.…”