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the cell. This observation demonstrates that TPE-TCF NPs
evenly distribute themselves within the cytoplasm.
ed a trend to concentrate in the nuclear region. Reasonably,
we can expect that this can be applied in more bioimaging sit-
uations when the NPs are decorated with different modifiers
besides the tat peptide.
To confirm that TPE-TCF NPs have not entered into the cell
nucleus, DAPI, a typical fluorescent dye for nuclear staining,
was used as a calibration to stain the cell nucleus. As shown
by the FL images in Figure 6A and B, bright blue (FL from
DAPI received at 450–490 nm) and red fluorescence (FL from
TPE-TCF NPs received at 600–700 nm) could be recorded in the
cell nucleus and partly observed in the cell cytoplasm when
TPE-TCF NPs and DAPI molecules were concomitantly used in
the cell staining process. Figure 6D displays the merged
images of Figure 6A and B, which indicates very good overlap-
ping. This result seems to be incompatible with that observed
in Figure 5. In fact, the treatment procedures in the two cases
are different. To let DAPI molecules penetrate into the nucleus
of the living cells, triton has to be added into the incubation
media to enlarge the size of the nuclear pore. Meanwhile, on
the surface of the NPs, each tat has six Arg residues. These
plentiful Arg residues make the TPE-TCF NPs positively charged
in aqueous media, which may be advantageous to the attrac-
tive interaction between TPE-TCF NPs and negatively charged
cell nucleus. However, without DAPI concomitantly existing in
the cell staining process, the control experiment suggested
that overlapping of the fluorescent and optical images for the
nucleus of the living cells is found only in rare cases (Fig-
ure S12, Supporting Information). Comparing the images in
Figure 6 with those demonstrated in Figure 5 and Figure S12,
in the presence of DAPI and triton, it can be seen that DAPI
plays a helpful role for the interaction between TPE-TCF NPs
and the cell nucleus. This interesting research topic is undergo-
ing further investigation.
Experimental Section
Materials
4-Bromobenzophonene and diphenylmethane were purchased
from Alfa Aesar. nBuLi, N-formylpiperdine, malononitrile, magnesi-
um ethoxide, and 3-hydroxy-3-methylbutan-2-one were purchased
from J&K. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-
[maleimide(poly- ethylene glycol)-2000] (DSPE-PEG2000-Maleimide)
was purchased from Avanti, HIV-1 tat protein(47–57)-Cys (Tyr-Gly-
Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys), DAPI and Cell-culture
products were purchased from Invitrogen Gibco. Other reagents
including p-toluene-sulfonic acid (TsOH), magnesium sulfate, am-
monium chloride, toluene, ethanol, ethyl acetate (EA), petroleum
ether (PE), and dichloromethane (DCM) were purchased from Sino-
pharm Chemical Reagent Co., Ltd. THF was distilled under normal
pressure from sodium benzophenone ketyl under nitrogen imme-
diately prior to use.
Instrumentation
1H and 13C NMR spectra were measured on a Mercury plus
400 MHz NMR spectrometer in CDCl3 with tetramethylsilane (TMS;
d=0 ppm) as the internal standard. Elemental analysis was per-
formed on a ThermoFinnigan Flash EA1112 apparatus. UV absorp-
tion spectra were taken on a Varian CARY 100 Biospectrophotome-
ter. PL spectra were recorded on a spectrofluorophotometer (RF-
5301PC, SHIMADZU, Japan). SEM images were taken on a JSM-
5510 (JEOL, Japan) scanning electron microscope. Fluorescent
images were taken with a Zeiss Axiovert 200 inverted microscope
equipped with a 100ꢁoil immersion objective with a numerical
aperture of 1.4 and an Ebq 100 Isolated electronic ballast for mer-
cury vapour compressed-arc lamps. TGA spectra were recorded on
a DSCQ 1000 (TA, USA) calorimeter. The morphology of the NPs
was studied by using a HR-TEM (JEM-2010F, JEOL, Japan). The aver-
age particle size and size distribution of the NPs were determined
by laser light scattering with particle-size analyzer (90 Plus, Broo-
khaven Instruments Co. USA) at a fixed angle of 908 at room tem-
perature. The cell images were taken on an upright Olympus laser
scanning confocal microscope (FV1000).
Conclusion
In summary, an AIE-active fluorescent dye, TPE-TCF, has been
prepared. Its emission spectrum covers the red and partial NIR
region and the emission maximum shifts from 630 to 670 nm
from when in a THF solution to the solid film. The quantum ef-
ficiency of the solid film is as high as 24.8%. In comparison
with previously reported AIE-active red-NIR emission dyes, the
synthetic route to TPE-TCF is short, containing only two simple
steps. Thus, the total yield is as high as 72%. TPE-TCF exhibits
an intramolecular charge-transfer from the TPE core to the TCF
unit, which leads to a pronounced solvatochromic effect and
a 120 nm redshifted emission from apolar to highly polar sol-
vents has been recorded. Taking the advantage of the red-NIR
emission and high quantum yield, TPE-TCF was tested in cell-
imaging studies. By loading the dye molecules into the mi-
celles built from the amphiphilic copolymer DSPE-PEG2000-tat,
TPE-TCF NPs have been fabricated. The NPs are spherical in
shape with a diameter of about 100 nm and a narrow particle-
size distribution. They are red fluorescent with an emission
peak at around 660 nm and a Stokes shift as large as 190 nm.
After incubation with Hela cells for 2 h, the confocal images
show that TPE-TCF NPs are evenly internalized in the cell cyto-
plasm. Upon co-incubation with DAPI (an organic dye that is
typically used for staining cell nuclei), TPE-TCF NPs demonstrat-
Synthesis of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)ylidene)-
malononitrile (TCF)
Malononitrile (5.9 g, 90 mmol) and magnesium ethoxide (3.8 g,
34 mmol) were added into a 100 mL two-necked round-bottomed
flask under nitrogen. 3-Hydroxy-3- methylbutan-2-one (3.2 mL,
30 mmol) and then 30 mL of ethanol were injected into the flask
and the temperature was increased to 608C. After stirring at 608C
for 8 h, the mixture was cooled to room temperature and then
evaporated. After that, 100 mL of DCM was added into the system
and the mixture was filtered. The filtrate was washed with brine
and dried over anhydrous magnesium sulfate. After filtration and
solvent evaporation, the residue was purified by silica gel column
chromatography, using DCM as the eluent and was then recrystal-
lized from ethanol. TCF (8.2 g) was obtained as a yellow crystal in
68.0% yield. 1H NMR (400 MHz, CDCl3): d=2.37 (s, 3H, CH3),
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Chem. Eur. J. 2016, 22, 1 – 9
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Wang, Yi Jia
