Live-cell-permeable poly(p-phenylene ethynylene)

Article abstract of DOI:10.1002/anie.200701991

Light up my life: Stable, bright, conjugated-polymer nanoparticles (CPNs) show promise for fluorescence imaging of live cells. The cell-permeable CPNs are synthesized by a simple solvent exchange, and accumulate exclusively in the cytosol (see picture) wi

Full text of DOI:10.1002/anie.200701991

Angewandte
Chemie
DOI: 10.1002/anie.200701991
Imaging Agents
Live-Cell-Permeable Poly(p-phenylene ethynylene)**
Joong Ho Moon,* William McDaniel, Paul MacLean, and Lawrence F. Hancock
Fluorescent labeling and detection of target biological
molecules in live cells is an essential way of studying complex
and dynamic cellular processes.[1] Many fluorescent dyes[2]
and engineered fluorescent proteins[3] are widely used for
these applications because of their small size and biocompat-
ibility. However, poor photostability of these probes limits
their broad applicability in long-term monitoring of live cells
with high sensitivity. Quantum dots (QDs) are considered an
alternative probe owing to their excellent optical properties,
such as high photostability, narrow emission, and high bright-
ness.[4] However, the inherent toxicity of QDs (mainly from a
heavy-metal core, such as divalent cadmium ions) causes
concern in long-term monitoring of cellular events.[5,6] In
addition, difficulties associated with surface modification of
QDs also retard their applications in live-cell systems. There-
fore, novel materials that over-
solution. An amine containing poly(p-phenylene ethynylene)
(PPE) was designed and fabricated into CPN in water to
demonstrate live-cell imaging. The CPNs are cell permeable
and accumulate exclusively in the cytosol without any
measurable inhibition of cell viability. In addition, CPNs
exhibit high resistance to photobleaching, in contrast to
commercially available dyes.
A PPE was synthesized as a representative CP by the
palladium/copper-catalyzed cross-coupling reaction in
a
mixed solvent of DMSO and morpholine (1:1 v/v). We
designed monomers to minimize p-stacking of aromatic
backbones in aqueous media by introducing amine groups
at the end of ethylene oxide linkers (Scheme 1). Primary
amine groups in the PPE are of particular interest because
they increase aqueous solubility of PPE upon protonation and
come the stability and toxicity
issues in live-cell imaging are in
high demand.[7–10]
Conjugated polymers (CPs)
are attractive materials that
meet the optical requirements
suitable for fluorescence micro-
scopic imaging.[11] CPs exhibit
high
fluorescence
quantum
yield, large extinction coeffi-
cients, and efficient optical
signal transduction. In addition,
the synthetic versatility of CPs
Scheme 1. Synthesis of PPE from diiodoarene 1 and diyne 2. See Experimental Section for details.
allows a wide selection of functional groups and coupling
chemistries for attachment of biological molecules. In spite of
these promising properties, intrinsic hydrophobicity originat-
ing from the p-conjugated aromatic backbone limits the
potential applications of CPs in biological systems. By
introducing charged functional groups in the CPsꢀ side
chains, the detection of nucleic acids,[12] proteins,[13,14] bac-
teria,[15] or cancer cells[16] in vitro has been demonstrated.
Herein, we introduce fluorescent conjugated polymer
nanoparticles (CPNs) that are capable of fluorescence imag-
ing of live cells. CPNs are stable, nanometer-sized fluorescent
particles fabricated by a simple solvent exchange in a CP
provide a site for coupling of biologically active molecules.
Moreover, protonation of the amine group increases fluores-
cence intensity because of reduced chain–chain interac-
tions.[17,18]
We reported that the phase-inversion precipitation of PPE
in a poor solvent allowed formation of stable particles.[19] PPE
formed variously sized particles in aqueous phases, depending
on both PPE concentration and salinity. Herein, sequential
ultrafiltration with acetic acid, ethylenediaminetetraacetic
acid (EDTA), and water were used for the fabrication of
stable nanometer-sized particles. Acetic acid aided removal of
metal-ion contamination (palladium and copper) and reduced
PPE aggregation in the CPNs by generating repulsive forces.
Features in the fluorescence spectrum, such as broadened
emission, indicated that the particle formation was driven by
aggregation of PPEs. However, CPN exhibits considerable
quantum yields (QY; 0.17) in water, unlike other secondary-
or tertiary-amine-containing PPEs, which exhibit very low
QY in water owing to severe aggregation.[20,21] Dynamic light-
scattering experiments indicated the formation of nanopar-
ticles with an average size of 97 nm and polydispersity index
of 0.13.[22] Transmission electron microscopic (TEM) images
also indicate formation of nanoparticle clusters with broad
[*] Dr. J. H. Moon, W. McDaniel, P. MacLean, Dr. L. F. Hancock
ICx Technologies
215 First St., Suite 104, Cambridge, MA 02142 (USA)
Fax: (+1)617-441-8874
E-mail: Joongho.moon@icxt.com
Homepage: http://www.icxt.com
[**] This work was partially supported by a NIH grant
(1R43 GM075516-01)
Supporting Information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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8223

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Figure 1. Fluorescence images of live (a) and fixed (b) cells. a) BALB/
C 3T3 cells were incubated sequentially with CPNs (green) and
Hoechst dye (blue). The image is a composite of two micrographs
using GFP (for CPNs) and DAPI/Hoechst/AMCA (for Hoechst) filter
sets. b) Live BALB/C 3T3 cells were incubated with CPNs and fixed for
confocal microscopic study. Variously sized spherical dots were found
in cytosol. In both cases, CPNs accumulated in the cytosol, not in the
nucleus.
Figure 2. Cell viability results at various concentrations of CPN. BHK
cells were incubated with various concentrations of CPN for up to
~
&
*
^
seven days. no CPN; 33 mm of CPN; 66 mm; 132 mm;
& 264 mm of CPN.
inhibition observed even at 264 mm of CPNs over the course
of a one-week cell culture.
size distribution resulting from various aggregations of CPNs
in a TEM grid (see Supporting Information).
We compared the photostability of CPNs with represen-
tative fluorescent dye fluorescein. Live 3T3 cells were
incubated with fluorescein-labeled TAT (FITC-TAT) fol-
lowed by paraformaldehyde fixation. TAT is a peptide
fragment (47–57) of HIV transactivator protein that pene-
trates cellular membranes.[26] Under a fixed confocal laser
setup, fluorescence images of CPN and FITC were collected
for 200 seconds at 6-second intervals by excitation at 488 nm.
Normalized fluorescence intensity of individual images were
plotted as a function of exposure time (Figure 3). CPN
maintained fluorescence intensity over the exposure periods,
whereas FITC exhibits fast photobleaching. Moreover, fluo-
rescence intensity of CPN collected at 20-times-higher laser
intensity than the normal operational laser power (4%) also
exhibited no noticeable increase in the photobleaching (open
circles in Figure 3); indicating CPN is a photostable fluoro-
phore. It is not clear why CPNs possess better photostability
than the FITC dye. We assume that when the nanoparticles
The CPNs were further diluted with a cell-culture media
(Dulbeccoꢀs modified eagle media, Invitrogen) for live-cell
imaging. Various cells, including baby-hamster kidney (BHK)
and BALB/C 3T3 (mouse embryonic fibroblast), were incu-
bated with the CPNs in culture media for various time periods
(from one hour to several days) to examine the cell perme-
ability, photostability, and cellular toxicity of CPNs. Figure 1
shows microscopic images of live BALB/C (a) and fixed 3T3
cells (b) stained by the CPNs overnight.[23] CPNs were found
exclusively in the cytoplasm, and especially around the
perinuclear region. To identify the location of the CPNs, we
co-stained the cells with CPNs then a lysosome-specific
fluorophore (LysoTracker Red DND-99, Invitrogen). The
fluorophores with weakly basic amines are likely to accumu-
late in cellular compartments with low internal pH values,
such as lysosomes.[24] Although CPN has many amine groups,
it accumulated randomly throughout the cytosol, which was
determined by the lack of overlap with the LysoTracker (see
Supporting Information). It is noteworthy that fast photo-
bleaching of the molecular fluorophore was observed during
fluorescence imaging of the live cells, whereas CPNs had a
fairly stable signal during the imaging (see Supporting
Information for a stability comparison). A confocal micro-
scopic study of fixed 3T3 cells further suggests that CPNs
were accumulated in vesicular structures, such as early or late
endosomes (Figure 1b).[25]
The quantitative CPN effect on cell viability was mea-
sured using a commercial viability assay kit. BHK cells were
inoculated into 96-well cell-culture plates (Corning) at
500 cells per 100 mL, followed by incubation over time in
the presence of varying amounts of CPNs. Live cells were
quantified at various time points using the CellTiter-Glo assay
kit (Promega Corp.), which measures ATP as an indicator of
metabolically active cells. The results for the CPN-containing
culture wells were compared to control wells incubated in the
absence of CPNs. Figure 2 shows the average luminescence
values for each time point with only minimal viability
*
*
Figure 3. Photostability comparison of CPN ( and ) compared to
fluorescein (FITC, &). Fluorescence intensity is normalized to the
initial intensity. CPN excited at 80% of 488 nm laser power; CPN
excited at 4% of the power; & FITC excited at 4% of the power.
*
*
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angewandte
Chemie
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In conclusion, we have presented a potential fluorescent
nanoprobe with fluorescent conjugated polymers that is easily
synthesized and has promise for studies in live cells with no
viability inhibition. CPN has a reasonably high quantum yield
and better photostability than representative fluorophores.
Knowledge about both cellular uptake mechanism and
location of CPNs will provide a foundation for various
fluorescence-imaging-based applications including delivery of
bioactive molecules in cells.
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Experimental Section
Synthesis and purification of PPE: 1 (42 mg, 93.3 mmol) and 2 (50 mg,
93.3 mmol) were weighed into a 40-mL glass vial. CuI (12 mg) was
weighed into a second vial, and both vials were transferred into the
glove box. Morpholine was added to the CuI-containing vial to make
a 10 mgmLÀ1 solution. [Pd(PPh3)4] (11 mg) was dissolved in morpho-
line to make a 5 mgmLÀ1 solution. DMSO (5 mL), morpholine
(3.8 mL), some of the CuI solution (124 mL), and some of the
[Pd(PPh3)4] solution (1.08 mL) were added to the vial containing the
monomers. The vial was then placed in an 808C oil bath and vial
contents allowed to stir for 16 h. The solution turned clear orange
with some gels in solution and some on the vial wall at the meniscus.
The probe was diluted with glacial acetic acid (300 mL), which
dissolved all of the gel. Using a solvent-resistant stir cell fitted with a
10000-molecular-weight cut-off (MWCO) membrane, the solution
was concentrated, combined with acetic acid (500 mL), and purified
by filtration. It was then concentrated to approximately 50 mL,
diluted into 300 mL of 0.1 mm EDTA, then dialyzed against of EDTA
(500 mL, 0.1 mm). It was again concentrated to approximately 50 mL
then diluted by adding to water (300 mL), and dialyzed against 1 L of
water. The solution was finally concentrated to approximately 75 mL,
filtered through glass wool (1.0 mm) and a syringe filter (0.45 mm). The
filtrate was diluted to a final volume of 90 mL (0.6 mgmLÀ1 as
determined by lyophilization). Yield: 80%. 1H NMR (400 MHz,
D2O): d = 6.996 (br s, 4H, aromatic), 4.266 (br s, 8H, PhOCH2), 3.955–
3.123 ppm (br m, 38H).
[22] The particle size was averaged from three independent measure-
ments during a two-week storage period in water. Polydispersity
index (PDI) is a parameter calculated from a cummulant
analysis of dynamic light scattering intensity measured autocor-
relation function. Percent polydispersity is a square of coef-
ficient of variation and defined as (PDI)1/2 100. From the
relationship, particle size showed 36% polydispersity, indicating
broad size distributions. We are investigating photophysical
behavior as a function of particle sizes and functional groups.
[23] We observed fluorescence signals from 1-hour incubation (see
Supporting Information). However, photostability of CPNs from
the 1 hour incubation was poorer compared to the overnight
incubation. We are under investigation of CPN photostability as
function of both concentration and particle size.
[24] F. Galindo, M. I. Burguete, L. Vigara, S. V. Luis, N. Kabir, J.
Gavrilovic, D. A. Russell, Angew. Chem. 2005, 117, 6662;
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Cell viability measurements: BHK cell suspensions (5 ”
103 cellsmLÀ1, or 0 cellsmLÀ1 as a negative control) were prepared,
seeded at 95mLwellÀ1, in 96-well tissue culture (TC)-treated plates.
Cells were incubated overnight (378C/5% CO2) to allow attachment
to occur, and then spiked with CPN (5 mL, diluted in PBS) at varying
concentrations to yield the desired final CPN concentration in culture
(0, 2.5, 5, 10, 20 mg CPN per 100 mL well). Cells were then incubated
(378C/5% CO2) and assayed at various times using the CellTiter-Glo
assay kit (Promega). Four replicates at each CPN concentration were
assayed at each time (1 hour, 1 day, 2 days, 3 days, and 4 days)
according to the manufacturerꢀs instructions (analysis by lumines-
cence, Perkin–Elmer Victor 2 reader).
Received: May 4, 2007
Revised: July 16, 2007
Published online: September 21, 2007
[25] We are currently co-staining with various vesicular specific
antibodies to identify CPNꢀs location.
[26] E. Vives, B. Lebleu in Cell-Penetrating Peptides: Processes and
Applications (Ed.: U. Langel), CRC, Boca Raton, FL, 2002,
pp. 3 – 21.
Keywords: fluorescent probes · imaging agents ·
live cell imaging · nanotechnology
.
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Angew. Chem. Int. Ed. 2007, 46, 8223 –8225
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
8225