Red-Emissive Cell-Penetrating Polymer Dots Exhibiting Thermally Activated Delayed Fluorescence for Cellular Imaging

Article abstract of DOI:10.1021/jacs.1c06290

Fluorescence imaging in living cells is key to understanding many biological processes, yet autofluorescence from the sample can lower sensitivity and hinder high-resolution imaging. Time-gated measurements using phosphorescent metal complexes can improve imaging, at the cost of potential toxicity from the use of heavy metals. Here, we describe orange/red-emitting polymer dots (Pdots) exhibiting thermally activated delayed fluorescence (TADF) for time-gated imaging. Inspired by the cell invasion mechanism of the HIV TAT protein, the Pdots were formed from block copolymers composed of a hydrophilic guanidine-rich block as a cell-penetrating peptide mimic, and a rigid organic semiconductor block to provide efficient delayed fluorescence. These all-organic polymer nanoparticles were shown to efficiently enter HeLa, CHO, and HepG2 cells within 30 min, with cell viabilities remaining high for Pdot concentrations up to 25 mg mL-1. Pdot quantum yields were as high as 0.17 in aerated water, with the Pdot structure effectively shielding the TADF emitters from quenching by oxygen. Colocalization experiments revealed that the Pdots primarily accumulate outside of lysosomes, minimizing lysosomal degradation. When used for fixed cellular imaging, Pdot-incubated cells showed high signal-to-background ratios compared to control samples with no Pdot exposure. Using time-resolved spectroscopy, the delayed emission of the TADF materials was effectively separated from that of both a biological serum and a secondary fluorescent dye.

Full text of DOI:10.1021/jacs.1c06290

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Red-Emissive Cell-Penetrating Polymer Dots Exhibiting Thermally
Activated Delayed Fluorescence for Cellular Imaging
Cheyenne J. Christopherson,† Nathan R. Paisley,† Zhujun Xiao, W. Russ Algar,*
and Zachary M. Hudson*
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ABSTRACT: Fluorescence imaging in living cells is key to understanding many
biological processes, yet autofluorescence from the sample can lower sensitivity
and hinder high-resolution imaging. Time-gated measurements using phosphor-
escent metal complexes can improve imaging, at the cost of potential toxicity from
the use of heavy metals. Here, we describe orange/red-emitting polymer dots
(Pdots) exhibiting thermally activated delayed fluorescence (TADF) for time-
gated imaging. Inspired by the cell invasion mechanism of the HIV TAT protein,
the Pdots were formed from block copolymers composed of a hydrophilic
guanidine-rich block as a cell-penetrating peptide mimic, and a rigid organic
semiconductor block to provide efficient delayed fluorescence. These all-organic
polymer nanoparticles were shown to efficiently enter HeLa, CHO, and HepG2
cells within 30 min, with cell viabilities remaining high for Pdot concentrations up
to 25 mg mL−1. Pdot quantum yields were as high as 0.17 in aerated water, with
the Pdot structure effectively shielding the TADF emitters from quenching by
oxygen. Colocalization experiments revealed that the Pdots primarily accumulate outside of lysosomes, minimizing lysosomal
degradation. When used for fixed cellular imaging, Pdot-incubated cells showed high signal-to-background ratios compared to
control samples with no Pdot exposure. Using time-resolved spectroscopy, the delayed emission of the TADF materials was
effectively separated from that of both a biological serum and a secondary fluorescent dye.
thermal energy can facilitate reverse intersystem crossing
(RISC) from T1 to S1. This same property enables the
harvesting of both singlet and triplet excitons in devices such as
organic light-emitting diodes (OLEDs) and organic lasers.15,16
TADF probes with excitation and/or emission in the red to
near-infrared (NIR) are particularly advantageous, as they can
take advantage of the biological transparency window (650−
1350 nm) to further improve the signal-to-background
ratio.17,18
As TADF materials exploit long-lived triplet states, they
must be protected from quenching by oxygen to be useful in
practical bioimaging. Encapsulating the probe within a water-
soluble nanoparticle (e.g., one composed of proteins,
amphiphilic polymers, or mesoporous silica) is one way to
achieve this protection.13,19,20 These methods generally
produce nanoparticles with low cytotoxicity, though dye
leakage from the nanoparticle over time can reduce signal
1. INTRODUCTION
Fluorescence imaging is a critical tool for visualizing cellular
structures and processes in complex biological environments.
Although the autofluorescence of biological samples can
interfere with obtaining high-resolution images, the use of
probes with long-lived luminescence can be used to easily
distinguish the desired signal using time-gated imaging.1−3 By
applying a brief delay to the detector activation following
pulsed excitation, the short-lived autofluorescence can be
effectively removed, improving image contrast and resolution
through higher signal-to-background ratios. Materials contain-
ing Ru(II), Ir(III), Pt(II), or Ln(III) have been used
successfully as phosphorescent probes for time-gated imaging,
but there are concerns surrounding the potential toxicity of
heavy metal ions that dissociate from a complex within a
biological system.4−6
Alternatively, materials exhibiting thermally activated
delayed fluorescence (TADF) can provide long-lived emission
using purely organic materials.7−9 As a result of rapid
interconversion between the lowest singlet (S1) and triplet
(T1) excited states, TADF materials exhibit both short (ns)
and long (μs−ms) emission lifetimes, facilitating their use in
time-resolved fluorescence imaging.10−14 TADF is achieved by
minimizing the singlet−triplet gap (ΔEST) such that ambient
Received: June 21, 2021
Published: August 12, 2021
https://doi.org/10.1021/jacs.1c06290
J. Am. Chem. Soc. 2021, 143, 13342−13349
© 2021 American Chemical Society
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Figure 1. (A) Polymerization of mCPN-based polymers. SEC traces of (B) mCPN90-co-DMAcN10 and (C) mCPN91-co-DPAcN9.
Photoluminescence emission spectra of (D) mCPN90-co-DMAcN10 and (E) mCPN91-co-DPAcN9 in toluene (0.01 mg mL−1) or as thin films
(λex = 340 nm). Photoluminescence decays of (F) mCPN90-co-DMAcN10 and (G) mCPN91-co-DPAcN9 films under air and inert conditions (λex
=
313 nm). Temperature-dependent photoluminescence decays of (H) mCPN90-co-DMAcN10 and (I) mCPN91-co-DPAcN9 films (λex = 313 nm).
localization and damage cells through the production of singlet
oxygen.21−24 Alternatively, semiconducting polymers that
exhibit TADF may be combined with a hydrophilic polymer
either covalently or by coprecipitation.25 This gives lumines-
cent nanoparticles termed polymer dots (Pdots), reducing or
eliminating dye leakage.
without damaging the cell would thus be highly desirable but
have not been reported to date.
One well-known strategy for achieving intracellular delivery
takes inspiration from the HIV TAT protein, which contains a
protein transduction domain rich in cationic lysine and
arginine residues. This motif allows the TAT protein to
efficiently penetrate the cell membrane and has inspired the
synthesis of cation-rich synthetic polymers that mimic this
biological activity.35−39 These cell-penetrating polymers have
been used as vectors for a diverse array of cargo, including
nucleic acids, fluorophores, and drug molecules.40,41 In 2018,
Zhao and co-workers used an amphiphilic copolymer
containing a hydrophobic phenylalanine block and a cell-
penetrating peptide to give water-soluble nanoparticles.42
Fluorescent nanoparticles were prepared by encapsulating
small-molecule TADF dyes, and exhibited photoluminescence
quantum yields (ΦPL) ranging from 0.8% to 12%. The
amphiphilic nature of the encapsulating polymer allowed
transport of the TADF dyes across the cell membrane. While
this approach enabled effective intracellular time-gated
imaging, the small molecule dyes showed a large reduction
in ΦPL when encapsulated into the phenylalanine core of the
nanoparticles.
Pdots are water-soluble nanoparticles containing ≥50% of
semiconducting polymer by weight or volume and have
emerged as powerful probes for bioimaging, biosensing, and
photodynamic therapy.20,26−28 Characterized by improved
photostability and high brightness, Pdots can also be
functionalized for selective cell targeting, providing a versatile
platform for theranostics.29−31 As a result, Pdots have recently
found application in single-particle tracking in cells, lateral flow
or blot-style assays, and immunolabeling of cancer cells.20,32,33
As they combine many chromophores into a single nano-
particle, some Pdots can even exhibit sufficient brightness to
enable visualization with a hand-held smartphone camera.34
While Pdots have been increasingly studied for the labeling
of cell membranes, this strategy provides limited or no
information about intracellular structures or biological
processes. TADF Pdots capable of crossing the cell membrane
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Table 1. Polymer Photophysical Data
Air
τsoln
Inert
τsoln
b
b
Polymer
λem,max (nm)
ΦPL
τsolid
ΦPL
τsolid
a
b
a
b
b
a
a
b
b
a
a
mCPN90-co-DMAcN10
642 /605
0.06 /0.30
13.2 ns
636 ns
7.10 ns
1.35 μs
17.3 ns
14.5 μs
13.8 ns
26.6 μs
13.5 ns
7.37 μs
10.0 ns
18.8 μs
−
0.16 /0.56
13.5 ns
2.89 μs
13.9 ns
14.4 μs
9.9 ns
28.1 μs
−
a
a
b
a
a
a
a
mCPN91-co-DPAcN9
610 /582
0.03 /0.24
0.17 /0.77
9.70 ns
19.2 μs
17.0 ns
15.2 μs
14.2 ns
28.1 μs
a
a
c
BGN10-b-M20Pdot
604
0.11
0.15
0.15
0.19
c
BGN10-b-P20Pdot
582
−
−
a
b
c
0.01 mg mL−1 in toluene. On glass. 0.01 mg mL−1 in water.
Scheme 1. Synthesis of BGNa-b-M20/P20 Diblock Copolymers
Here, we prepare diblock copolymers composed of a cell-
penetrating peptide mimic and red-emitting TADF block for
intracellular, time-gated imaging. Red TADF monomers are
prepared using a 1,8-napthlalimide (NAI) acceptor and
dimethyl- or diphenylacridine donors, taking inspiration from
emitter materials recently used by Yang to prepare red OLEDs
with some of the highest efficiencies known. A guanidinium-
rich oxonorbornene monomer is used to give a polycationic
motif that easily penetrates cell membranes and can be
appended to the emissive block via ring-opening metathesis
polymerization (ROMP). Using this strategy, we form red-
emitting, cell-penetrating Pdots with high ΦPL in water, no
emitter leakage, and low cytotoxicity. These results are
supported with nanoparticle tracking analysis, time-resolved
spectroscopy, atomic force microscopy, cell viability assays,
colocalization analysis, and cellular imaging by microscopy.
Cells incubated with Pdot solutions showed up to 629%
increased fluorescence within 30 min compared to control cells
not exposed to Pdots. Time gating was then used to easily
distinguish the red emission from the Pdot probe from both
autofluorescence from a biological serum and a secondary
fluorescent dye.
Pdots. To study the photophysical properties of these
materials, the high molecular weight copolymers mCPN90-co-
DMAcN10 and mCPN91-co-DPAcN9 were synthesized via
ROMP containing 12 wt % of TADF dopant, where the
subscripts indicate the degree of polymerization (DP). The
polymers had Mn values of 54 900 and 63 700 respectively by
size exclusion chromatography (SEC) and narrow dispersities
Đ of 1.08. Purity and dopant incorporation were confirmed by
1H NMR, with DMAcN and DPAcN incorporated into the
respective polymers at 10 and 9 mol %, in agreement with feed
ratios (Figures S1, S2).
2.2. Photophysical Characterization. The absorption
spectra of both polymers (Figure S3) are dominated by
mCPN-based absorption with maxima at 340 nm, although a
much weaker absorption band attributed to charge-transfer
transitions in the dopants is observed at 450−500 nm. The
emission spectra for mCPN90-co-DMAcN10 and mCPN91-co-
DPAcN9 in toluene show strong dopant emission, with
maxima at 642 and 610 nm, respectively (Figure 1D, E;
Table 1). In the solid state, the emission of spin-cast films of
both polymers is blue-shifted by 30−40 nm. Thin films of
mCPN90-co-DMAcN10 and mCPN91-co-DPAcN9 exhibit high
Φ
PL values of 0.56 and 0.77, in good agreement with the small-
molecule design reported by Yang.43
2. RESULTS AND DISCUSSION
The emission lifetimes of both polymers were further
investigated using time-correlated single photon counting
(TCSPC) and multichannel scaling (MCS) measurements.
Biexponential fits accurately modeled the emission from both
mCPN90-co-DMAcN10 and mCPN91-co-DPAcN9, which ex-
hibited both prompt (10−9 − 10−8 s) and delayed (10−6−10−4
s) emission in solution and the solid state. As suggested by the
ΦPL data, mCPN91-co-DPAcN9 has a larger delayed emission
lifetime than mCPN90-co-DMAcN10 (Figure 1F, G; Figures S4,
S5). Under atmospheric conditions, quenching of the delayed
emission is evident in both cases, and the delayed lifetimes
2.1. Synthesis of TADF Homopolymers. The norbor-
nene-functionalized monomers DMAcN and DPAcN were
synthesized by combining 9,9-dimethyl-9,10-dihydroacridine
or 9,9-diphenyl-9,10-dihydroacridine donors with an NAI
acceptor (Figure 1, Scheme S1). These donor−acceptor pairs
are well-known to give red TADF with high ΦPL, and have
previously been exploited in high-efficiency red OLEDs and
ratiometric temperature probes.43−45 A carbazole-based host
monomer mCPN was also synthesized (Scheme S2) to act as a
rigid matrix for the TADF dopants, in order to reduce triplet−
triplet annihilation and excited state quenching in the eventual
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Table 2. Block Copolymer and Pdot Size Data
BGN Block
TADF Block
Diblock
Pdot
a
a
b
c
a
d
d
Polymer
DP
Mn (exp)
Mn (theo)
Đ
DP
Mn (exp)
Mn (theo)
Đ
μ (nm)
σ (nm)
BGN10-b-M20
BGN20-b-M20
BGN10-b-P20
BGN20-b-P20
10
20
10
20
7800
16 800
7700
9200
17 700
9200
1.15
1.23
1.13
1.16
20
20
20
20
10 700
12 300
11 400
11 400
11300
11300
11300
11300
1.17 (1.23)
1.26 (1.32)
1.25 (1.28)
1.26 (1.24)
107
93
98
40
21
24
25
15 100
17 700
98
a
b
c
1
1
Calculated using SEC. Đ before preparatory SEC in parentheses. mCPN:TADF molar ratio is 9:1 in all cases by H NMR. Calculated by H
d
NMR. Calculated using NTA.
decrease significantly (Table 1). In all cases, oxygen had little
effect on the prompt lifetimes observed. Temperature-depend-
ent lifetime measurements revealed a significant reduction in
delayed fluorescence of both polymers upon cooling to 77 K,
consistent with a TADF emission mechanism for DMAcN and
DPAcN.
2.3. Synthesis of Cell-Penetrating Polymers. A bis-
guanidinyl norbornene monomer BGN was then synthesized
in order to give hydrophilic polymer blocks that can serve as
cell-penetrating peptide mimics (Scheme S3).36,37 With pKa
values of ∼13.6,46 the guanidine groups are strongly
protonated in even basic aqueous solution, giving a synthetic
polycation that resembles arginine-rich polypeptides. To
facilitate ROMP, the guanidine groups were first protected
using tert-butoxycarbonyl (Boc) groups, which can be easily
removed under mild acidic conditions following the polymer-
ization. An oxonorbornene group was also used in the BGN
monomer in place of norbornene to improve the hydrophilicity
of this block still further.
Block copolymers containing a hydrophobic TADF-emitting
block and a hydrophilic polyguanidine block were then
synthesized via ROMP (Scheme 1). A target DP of 20 was
chosen for the TADF block, whereas DPs of both 10 and 20
were targeted for the BGN block to study the effect of chain
length on their cell-penetrating properties. The more reactive
oxonorbornene block was formed first by polymerizing BGN at
room temperature, after which the reaction was cooled to −20
°C and both mCPN and dopant monomer (12 wt %) were
added. If this cooling step is not performed, a high molecular
weight shoulder is observed in the SEC trace of all mCPN-
containing polymers. Purification by preparatory SEC yielded
Boc-protected polymers BGN10-b-(mCPN18-co-DMAcN2),
BGN20-b-(mCPN18-co-DMAcN2), BGN10-b-(mCPN18-co-
DPAcN2), and BGN20-b-(mCPN18-co-DPAcN2). For brevity,
we abbreviate mCPN18-co-DMAcN2 as ‘M20’ and mCPN18-co-
DPAcN2 as ‘P20’, and denote these polymers as BGN10-b-M20,
BGN20-b-M20, BGN10-b-P20, and BGN20-b-P20, respectively.
The Boc groups were then removed to expose the water-
soluble guanidines by stirring in a 1:1 mixture of MeOH and
0.2 M aqueous HCl, after which the polymer solutions were
dialyzed with Milli-Q water to remove residual small molecules
and isolated by lyophilization.
2.4. Pdot Preparation and Characterization. Pdots
(Figure 2) were formed by dissolving the polymers in THF,
followed by rapid addition to Milli-Q water under sonication
and then removal of the THF in vacuo. All Pdots had mean
diameters μ between 93 and 107 nm, with standard deviations
σ of 21−40 nm and no distinct trend in size observed for
different block ratios (Figure S11; Table 2), as determined
from nanoparticle tracking analysis (NTA). We refer to these
nanoparticles as Pdots because of the large mass fraction of
semiconducting polymer, though an upper limit of 30 nm
diameter has been described in some definitions.25 A spherical
morphology was further confirmed by atomic force microscopy
(Figure S12).
While a large red shift in emission would normally be
expected for a donor−acceptor material in polar solvents, the
emission maxima of BGN10-b-M20 and BGN10-b-P20 Pdots
(582 and 604 nm) are blue-shifted relative to the copolymers
in toluene, more closely resembling the polymers in the solid
state (Table 1). This result suggests that the Pdot morphology
and rigid mCPN matrix effectively shields the TADF emitters
from the surrounding medium.
Under inert conditions, the ΦPL of the Pdots in water are
comparable to those of the mCPN-based copolymers in
toluene solution, with values of 0.15 for BGN10-b-M20 and 0.19
for BGN10-b-P20. This compares favorably with the value of
ΦPL = 0.025 obtained in an earlier report, in which a small-
molecule NAI-based TADF emitter was encapsulated by
noncovalent interactions within the hydrophobic core of a
The molecular weight and dispersity of the diblock
copolymers was determined before Boc deprotection, as the
free guanidine groups interact significantly with the SEC
columns. The Mn of the BGN block was determined by SEC
using triple detection (Figure S6), and the Mn of the TADF
block was then calculated using the relative integration of
Figure 2. (A) Water-soluble Pdots formed from BGNa-b-M20 (R =
CH3) or BGNa-b-P20 (R = Ph). Orange = BGN block, red = TADF
block. Photoluminescence lifetimes of (B) BGN10-b-M20 and (C)
BGN10-b-P20Pdots under air and inert conditions in water (0.01 mg
mL−1; λex = 313 nm).
1
signature peaks from each block by H NMR (Figures S7−
S10). Molecular weights for all polymers were in good
agreement with theoretical values calculated from the
monomer feed ratios, and Đ was ≤1.26 in all cases (Table 2).
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nanoparticle.42 We attribute this increase in ΦPL to the use of
the rigid mCPN matrix, which can help to exclude O2.
Additionally, covalent incorporation of the TADF emitters into
the polymer eliminates the potential for dye leakage from the
Pdot. Importantly, while ΦPL decreased by 70−80% for
mCPN90-co-DMAcN10 and mCPN90-co-DPAcN10 upon ex-
posure to air in toluene, ΦPL for the Pdots decreases only by
21−27%, suggesting that the Pdot morphology helps to
exclude oxygen and prevent emission quenching. This is
further supported by the Pdot delayed fluorescence lifetimes,
which exhibit a reduction of <10% under air compared to inert
conditions (Figure 2B, C; Table 1).
2.5. Cytotoxicity. HeLa, CHO, and HepG2 cells were each
incubated with Pdot solutions of concentrations up to 25 μg
mL−1 at 37 °C/5% CO2; cell viabilities were measured for each
cell line after 1 or 24 h of exposure to Pdot solutions. HeLa
and HepG2 cell lines showed high viability (>70%) after 24 h
incubation with Pdot concentrations up to 5 μg mL−1, and a
significant decrease in viability at increasing concentrations
(Figures S13, S14). CHO cells showed poor viability above
1.25 μg mL−1 after 24 h. After 1 h, however, all three cell lines
showed high viability with Pdot concentrations up to 10 μg
mL−1, with cell viabilities of up to 90%, 97% and 83% for
CHO, HeLa, and HepG2 cells, respectively. Notably, HeLa
cells also exhibited up to 96% viability at 25 μg mL−1. These
cytotoxicity results compare favorably with related systems
employing guanidine-based peptide mimics, particularly at
concentrations and time scales suitable for imaging experi-
ments.47,48
2.6. Cellular Uptake Studies. Cells were incubated with
Pdot solutions at concentrations ranging from 5 to 25 μg mL−1
for 30 min at 37 °C/5% CO2. After washing with PBS and
medium, their mean cellular fluorescence in the B610-ECD-A
(610/20 nm bandpass) channel was measured and compared
to control cells with no Pdot exposure using flow cytometry.
The polymers with the shorter guanidine block (BGN10-b-
M20 and BGN10-b-P20) showed higher uptake in the human
cancer cell lines, HeLa and HepG2. Compared to a control
sample, HeLa cells exhibited up to a 259% and 171% increase
in mean cellular fluorescence for short and long guanidine
block lengths, respectively (Figure S15). HepG2 cells
incubated with the shorter polymer showed an increase in
mean cellular fluorescence of up to 629%, compared to only a
218% increase compared to control for the longer polymers
(Figure 3A−D). This difference in uptake efficiencies for
shorter vs longer guanidine blocks in HeLa and HepG2 cells is
consistent across all concentrations tested.
Conversely, the longer guanidine block copolymers (BGN20-
b-M20 and BGN20-b-P20) showed an increased uptake in CHO
cells, with cells incubated with the longer polymers showing an
increase in mean cellular fluorescence of up to 403%, and a
303% increase with the shorter polymer (Figure S16). These
observations are consistent with the work of Tew and co-
workers, who have demonstrated that the level of uptake into a
cell is dependent on the amount of guanidine in the
nanoparticle, and that different cell types exhibit different
sensitivities and uptake values depending on the guanidine
concentration and block length.47 This previous work also
confirmed internalization of the nanoparticles by confocal
microscopy; thus, we extrapolated that our guanidine-
containing Pdots would also be internalized. Differences in
mean cellular fluorescence were also dose-dependent, as Pdot
solutions of 25 μg mL−1 resulted in a mean cellular
Figure 3. (A - D) Mean cellular fluorescence of HepG2 cells after
incubation for 30 min with (A) BGN10-b-M20, (B) BGN20-b-M20, (C)
BGN10-b-P20, and (D) BGN20-b-P20 (0−25 μg mL−1). Measured by
flow cytometry using the B610-ECD-A channel. Colocalization
images of HepG2 showing (E) Differential interference contrast
(DIC) image, (F) merged fluorescence channels, (G) BGN10-b-
M20Pdot channel, and (H) Lysotracker blue channel. Scale bar = 15
μm.
fluorescence up to 370% higher than 5 μg mL−1 (Table S1).
No consistent difference in uptake values was observed
between Pdots with DMAcN or DPAcN dopants.
These results imply that HepG2 cells have the highest level
of uptake into the cells, while HeLa showed the least amount
of uptake. This is consistent with the high cell viability
observed in HeLa cells, as fewer Pdots are entering the cells
resulting in reduced cell death. However, HepG2 cells also
demonstrate relatively high cell viability (83%) after 1 h at
concentrations up to 10 μg mL−1. This cell line showed the
most promise for further investigation, giving the largest
increase in mean cellular fluorescence after only 30 min of
incubation time.
2.7. Colocalization Analysis. Colocalization analysis was
then performed by incubating HepG2 cells with BGN10-b-M20
Pdots alongside Lysotracker Blue, a small-molecule dye that
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Figure 4. Photoluminescence and DIC images of HepG2 cells incubated with (A, C) BGN10-b-M20 (25 μg mL−1) and (B, D) control (no Pdot
exposure). Scale bar is 20 μm. For image C: SBR = 2.0 0.3; SNR = 14 2 (from 20 cells). Experimental and control cellular images were
adjusted to the same brightness and contrast settings. (E) Prompt (0 μs delay; 500 μs integration time) and time-gated (50 μs delay; 500 μs
integration time) photoluminescenceemission spectra for fluorescein dye, Pdot solution, and a mixture of fluorescein and BGN10-b-M20 Pdots. (G)
Prompt (0 μs delay; 500 μs integration time) and time-gated (50 μs delay; 500 μs integration time) photoluminescence emission spectra for
BGN10-b-M20Pdots diluted in bovine serum.
accumulates primarily in lysosomes and other acidic vesicles.
The relative localization of the Pdots compared to the
Lysotracker dye was anticipated to provide insight into the
uptake mechanism. In addition to internalization by
endocytosis, cell-penetrating nanoparticles have also been
reported to penetrate cells by membrane disruption.42,49,50
The colocalization of Pdots and Lysotracker Blue was observed
to be low, with a Pearson’s correlation coefficient of r = 0.427
(Figure 3E−H). This result implies that the Pdots do not
localize in the lysosomes, consistent with previous studies
using similar cell-penetrating moieties.36,42 This also suggests
that the Pdots may be internalized through membrane
disruption rather than endocytosis, or that several uptake
mechanisms are present.49−51 The fact that the Pdots do not
localize in acidic vesicles like lysosomes would be expected to
reduce lysosomal degradation and exocytosis. This improves
their potential for cargo delivery applications, as lysosomal
degradation or sequestration of the cargo can potentially be
avoided.
CHO, HeLa and HepG2 cells respectively (Figure 4A-D; S18−
22), and a signal-to-noise ratio (SNR) of 28 6, 16 1, and
28 5 for the same cell lines, at 20× magnification. In all three
cell lines, control cells show negligible fluorescence in the
observed channel (Figure S17 − S21).
To illustrate the potential of TADF Pdots in time-gated
experiments, emission spectra were obtained in the presence of
BGN10-b-M20 Pdots and either fluorescein dye or bovine
serum, with and without time-gated detection. Prompt PL
measurements show clear contributions from both the
fluorescein and the Pdots (Figure 4E) or from the Pdots and
serum autofluorescence (Figure 4G). However, time-gated
images at 50 μs display only emission from the Pdots in both
cases (Figure 4F, H). Importantly, these measurements were
done in aerated solution, again showing the ability of the Pdot
morphology to shield the TADF emitter from oxygen and
demonstrating their applicability in live cell imaging.
3. CONCLUSIONS
2.8. Cellular Imaging and Time-Resolved Emission
Measurements. Cellular imaging was performed with
BGN10-b-M20 Pdots, as the more red-shifted emission
maximum of this dopant allowed for a greater signal-to-
background ratio. Cells were seeded on a gelatin-coated
coverslip and incubated with Pdot solutions of 10 μg mL−1 for
30 min at 37 °C/5% CO2 in medium before being washed
thoroughly with PBS and fixed with 4% w/v paraformaldehyde.
Cells were imaged using a 350/50 nm bandpass excitation filter
and a 550 nm long-pass filter. Cells incubated with Pdot
solutions showed a signal-to-background ratio (SBR) of 3.2
0.7 (74 cells), 1.4 0.1 (63 cells) and 2.2 0.4 (28 cells), for
Here we have described red-emissive, metal-free, cell-
penetrating polymers for time-gated cellular imaging using
polycationic peptide mimics. They show low cytotoxicity in
multiple cell lines, use covalently bonded dyes to eliminate dye
leakage, and exhibit quantum yields up to 19%. Using ROMP,
the donor−acceptor type DMAcN and DPAcN TADF
dopants were incorporated into random copolymers with a
rigid mCPN host, as well as diblock copolymers with a BGN
cell-penetrating peptide mimic. The diblock copolymers were
then used to prepare Pdots in water, which retained high ΦPL
and long emission lifetimes even in aerated solution. The Pdot
morphology effectively shielded the emitter from quenching by
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Article
oxygen, enabling their use in time-gated emission measure-
ments.
ACKNOWLEDGMENTS
■
The authors thank the Natural Sciences and Engineering
Council of Canada (NSERC), the British Columbia Knowl-
edge Development Fund (BCKDF), and the Canada
Foundation for Innovation (CFI) for financial support.
C.J.C. and N.R.P. thank NSERC for Postgraduate Scholar-
ships. W.R.A. and Z.M.H. are grateful for support from the
Canada Research Chairs program.
When incubated with CHO, HeLa, and HepG2 cells, the
Pdots were effectively taken up in 30 min and exhibited
minimal cytotoxicity after 1 h, at concentrations up to 25 mg
mL−1. The Pdots primarily accumulated outside of lysosomes
and other acidic vesicles, increasing their potential for cargo
delivery applications as lysosomal degradation is minimized.
Additionally, imaging using Pdots with all three cell lines
showed high contrast compared to control cells. Time-resolved
emission measurements showed the long fluorescence lifetime
of the TADF emitters could be harnessed to improve signal-to-
background ratio when imaging live cells, eliminating back-
ground fluorescence from both a biological serum and an
added fluorescein dye. Moving forward, TADF Pdots show
promise for theranostic applications that facilitate both imaging
and the delivery of cargo, as the semiconductor core of the
Pdots should efficiently encapsulate hydrophobic drug
molecules.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c06290.
Synthetic procedures, monomer and polymer character-
ization data (NMR, HRMS, GPC), additional optical
and electrochemical data, flow cytometry data, and
cellular imaging experiments (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
W. Russ Algar − Department of Chemistry, The University of
British Columbia, Vancouver, British Columbia, Canada
V6T 1Z1; orcid.org/0000-0003-3442-7072; Phone: +1-
604-822-3266; Email: algar@chem.ubc.ca; Fax: +1-604-
822-2847
Zachary M. Hudson − Department of Chemistry, The
University of British Columbia, Vancouver, British Columbia,
Canada V6T 1Z1; orcid.org/0000-0002-8033-4136;
Phone: +1-604-822-3266; Email: zhudson@chem.ubc.ca;
Fax: +1-604-822-2847
Authors
Cheyenne J. Christopherson − Department of Chemistry,
The University of British Columbia, Vancouver, British
Columbia, Canada V6T 1Z1
Nathan R. Paisley − Department of Chemistry, The University
of British Columbia, Vancouver, British Columbia, Canada
V6T 1Z1
Zhujun Xiao − Department of Chemistry, The University of
British Columbia, Vancouver, British Columbia, Canada
V6T 1Z1
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c06290
Author Contributions
†C.J.C. and N.R.P. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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