10.1021/acs.biomac.9b00823.s001
Marc P. Wolf
Marc P.
Wolf
Kegang Liu
Kegang
Liu
Thomas F. W. Horn
Thomas F. W.
Horn
Patrick Hunziker
Patrick
Hunziker
FRET in a Polymeric Nanocarrier: IR-780 and IR-780-PDMS
American Chemical Society
2019
FRET dye partners IR -780-PDMS
FLIM
NIR
IR -780-loaded micellar nanocarriers
fluorescence-lifetime imaging microscopy
IR -780-PDMS
PDT
herewith-described FRET method
donor
IR -780
Confocal laser scanning microscopy
micellar nanocarriers
2019-10-18 15:36:05
Journal contribution
https://acs.figshare.com/articles/journal_contribution/FRET_in_a_Polymeric_Nanocarrier_IR-780_and_IR-780-PDMS/10003229
We
introduce a method to monitor the integrity of micellar nanocarriers
using a novel fluorescent dye, IR-780-PDMS and Förster resonance
energy transfer (FRET) as a readout. In addition, these dye-loaded
nanocarriers can be used as a phototoxic agent in vitro. Mainly, a
nanocarrier was designed, based on a previously described amphiphilic
ABA-copolymer (Pip-PMOXA-PDMS-PMOXA-Pip) scaffold that incorporates
the fluorescent FRET dye partners IR-780-PDMS (donor) and IR-780 (acceptor).
The confirmation of FRET (that only occurs when donor and acceptor
are in the required close proximity of less than ∼10 nm) in
the nanocarriers is used to prove that the acceptor dye (IR-780) is
still contained in its hydrophobic core. We measured such FRET signals
of the nanocarriers also upon cellular uptake into HeLa cells using
fluorescence-lifetime imaging microscopy (FLIM). Confocal laser scanning
microscopy after incubation with nanocarriers demonstrated the intracellular
uptake of the particles and their localization in an intracellular
granular pattern. To demonstrate the intactness of the nanocarriers
by detection of FRET we measured the fluorescence lifetime (FLIM)
of the donor dye. FLIM showed that both types of lifetimes, that of
the quenched donor, and that of the unquenched donor were present,
in a granular pattern and homogeneously in the cytosol, respectively,
indicating the presence of intracellular intact and disintegrated
micellar nanocarriers. These data show that the herewith-described
FRET method allows monitoring the intactness of nanocarriers while
en route to the target, and also that the cargo is delivered and released
within a potential target cell. In addition, near-infrared (NIR) irradiation
of IR-780-loaded micellar nanocarriers leads to photocytotoxicity,
which we demonstrated in in vitro experiments. Our findings open potential
avenues in photodynamic therapy (PDT) of cancer.