Labeling nanoparticles: Dye leakage and altered cellular uptake

Abstract

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues.To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4°C and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP dye retention is confirmed when no cellular fluorescence is detected at 4°C.Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce, or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing fluorescent NP applications.3

The effect of poly(ethylene glycol) coating and monomer type on poly(alkyl cyanoacrylate) nanoparticle interactions with lipid monolayers and cells

“…15 We should note that the encapsulation of dyes into NPs can cause misinterpretation of their 16 cellular uptake in flow cytometry due to potential dye leakage [45][46][47]. We therefore confirmed 17 that no dye leakage has taken place by incubating RBE4 cells with PACA NPs at 4 ºC [48]. In 18 addition, CLSM images confirmed that the NPs were internalized.…”

A small variation in average particle size of PLGA nanoparticles prepared by nanoprecipitation leads to considerable change in nanoparticles’ characteristics and efficacy of intracellular delivery

“…In order to investigate the nanoparticles' uptake mechanisms, HEK293 cells were prepared in 6-well plates as described above and pretreated with the inhibitors (1.25 mM AMD, 45 lM CPZ, 15 lg/ml IND, and 3 lg/ml AZD) for 60 min at 37 C. For determining dye leakage or contact mediated dye transfer, uptake studies were repeated at þ4 C for PLGA 230 or PLGA 160 (Ray et al 2016, Snipstad et al 2016. Then the nanoparticles (1.25 Â 10 10 particles/ml) were added and cellular uptake was evaluated by flow cytometry following 3 h and 24 h of incubation for PLGA 230 or PLGA 160 nanoparticles, respectively.…”

BODIPY-loaded polymer nanoparticles: chemical structure of cargo defines leakage from nanocarrier in living cells

“…As release of drugs is very difficult to monitor in complex biological media and in live cells, fluorescent dyes are used as model cargos for polymer NPs. [17][18][19][20][21][22][23] Dye-loaded polymeric NPs are attractive systems to address the problem of cargo leakage. On one hand, they are powerful tools for fluorescence bioimaging in vitro and in vivo.…”

“…The encapsulated fluorescent dyes are excellent probes that can report on the cargo release from nanocarriers. [17][18][19][20][21][22][23] Dye-loaded and drug-loaded NPs share two key common requirements. The first requirement is the high loading of cargo.…”