Implications of Quenching‐to‐Dequenching Switch in Quantitative Cell Uptake and Biodistribution of Dye‐Labeled Nanoparticles

Abstract

Ag eneral strategy to carry out cell uptake and biodistribution studies is to label nanoparticles (NPs) with afluorescent dye.However,the comparative study of different dye-loaded NPs remains difficult owingt ou ncontrolled dye quenching and de-quenching. Here we compared two types of dye-labeled NPs and demonstrated their distinct properties. NPs with dye molecules at as olid state suffer from dye quenching, so the dye release and/or NP degradation in biological environments leads to as everal-fold increase of fluorescence intensity despite the same amount of NPs,o wing to the state switch from quenchingtode-quenching. In contrast, NPs with dye molecules at asoluble state exhibit no quenching effect. To standardizet he comparative study,w ep ropose two possible solutions:u sing lower dye loading or using medium analysis for quantifying cell uptake of NPs.This work provides valuable insights into selecting valid quantification methods for bio-nano studies.

Live Cell Imaging by Förster Resonance Energy Transfer Fluorescence to Study Trafficking of PLGA Nanoparticles and the Release of a Loaded Peptide in Dendritic Cells

“…The fluorescence intensity of Cy3 or Cy5 leveled off with increasing particle concentration (Supplementary Figure S1) likely due to selfquenching. As reported in previous studies [25,30], this self-quenching phenomenon is dye-concentration-dependent. To avoid this self-quenching, NPs prepared with a fixed amount of 1 wt% Pep-Cy3 in the feed were selected for the preparation of FRET NPs containing 1.5-9 wt% PLGA-Cy5, corresponding to molar feed ratios of donor dye (Cy3) /acceptor dye (Cy5) (D/A) ranging from 5:1 to 0.8:1.…”

“…This indicates that Pep-Cy3 in the present study, in line with the previous findings, was partly released from the PLGA NPs. Recently, Yang et al [30] reported dequenching of Dil fluorescence mediated by the release of the Dil dye loaded in Cy5-labeled PLGA NP. In line with the study of Yang et al [30], the observed increase in Cy3 fluorescence of both Pep-Cy3 NP and FRET NP probably points to the dequenching of Cy3 fluorescence due to the release of Pep-Cy3 from the PLGA NP.…”

“…Recently, Yang et al [30] reported dequenching of Dil fluorescence mediated by the release of the Dil dye loaded in Cy5-labeled PLGA NP. In line with the study of Yang et al [30], the observed increase in Cy3 fluorescence of both Pep-Cy3 NP and FRET NP probably points to the dequenching of Cy3 fluorescence due to the release of Pep-Cy3 from the PLGA NP. The release of Pep-Cy3 also resulted in less donor dye present in proximity (less than 10 nm) of the acceptor dye (Cy5)-conjugated PLGA matrix and explains the lower FRET fluorescence intensity over 144 h as shown in Figure 4C (black squares).…”

FRET Ratiometric Nanoprobes for Nanoparticle Monitoring

“…However, the absolute intensity of a single fluorescence dye is often inaccurate and non-comparable because many factors could affect its intensity, including background signal, instrumental set-up, and materials screening and quenching. Furthermore, the fate of NPs in vitro or in vivo cannot be accurately monitored by using fluorescence-dye-labelled NPs, as the dye might be already separated from the NP itself due to release or degradation [3,4]. Therefore, single fluorescence probes cannot be used for the accurate monitoring of NP drug delivery.…”