Aggregation-induced near-infrared emitting platinum(ii) terpyridyl complex: Cellular characterisation and lysosome-specific localisation

Article abstract of DOI:10.1039/c8cc06839a

A novel near-infrared luminogen (TPE/TPY-Pt-PA/PEG) based on the terpyridyl Pt(ii) complex with a tetraphenylethene periphery is synthesised and characterised. It displays aggregation-induced emission with the formation of self-assembled nanostructures. T

Full text of DOI:10.1039/c8cc06839a

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Aggregation-induced near-infrared emitting platinum(II)
terpyridyl complex: cellular characterisation and lysosome-
specific localisation†
Received 00th January 20xx,
Accepted 00th January 20xx
Jiatao Wu,a,b Yaqiong Lic, Chunyan Tan,d,e Xin Wang,a Youming Zhang,a,b Jun Song,*a Junle Qu,*a
and Wai-Yeung Wong*a,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
A novel near-infrared luminogen (TPE/TPY-Pt-PA/PEG) based on
the terpyridyl Pt(II) complex with a tetraphenylethene periphery is
synthesised and characterised. It displays aggregation-induced
emission with the formation of self-assembled nanostructures.
The nanoparticles fabricated with TPE/TPY-Pt-PA/PEG show
excellent biocompatibility with high specificity to lysosomes in
HeLa cells.
possible applications for
Near-infrared (NIR) fluorophores have received a great deal of
attention during the past decades because of their extensive
applications in organic light-emitting diodes,1 therapy2 and
bioimaging.3, 4 Although the most widely used commercial NIR
fluorescent probes are organic fluorophores,5 transition-metal
complexes have highly promising prospects in imaging6
because of several important advantages, including
outstanding photostability, long excited-state lifetimes, large
Stokes shift and high emission quantum yields.7
Scheme 1 Schematic illustration of AIE active TPE/TPY-Pt-PA/PEG.
Among the various transition-metal complexes applied in
cellular imaging,8-10 Pt(II) complexes show ligand to ligand
charge transfer (LLCT), metal-to-ligand charge transfer (MLCT)
and metal-to-metal–ligand charge transfer (MMLCT), which
can emit strong emission from the visible to the NIR region.
Because of their square-planar geometry, Pt(II) complexes
possess attractive biosensing properties, which are due to
their high stacking tendency to form high-order
supramolecular assemblies.11-14 To date, several NIR Pt(II)
complexes that have been developed hold great promise for in
vivo imaging.6 However, low water solubility, aggregation-
induced quenching and high phototoxicity still reduce their
imaging.15, 16 Recently, fluorescent probes with aggregation-
induced emission (AIE) characteristics have emerged as an
extraordinary class of luminogens for biosensing.17 AIE
materials are weakly emissive or non-emissive in dilute
solutions and become highly emissive in an aggregated state.
Nevertheless, AIE NIR luminogens based on terpyridyl Pt(II)
complexes for bioimaging applications have been rarely reported.
In view of the excellent nature of these two functionalities
and their high preference for self-assembly, we have combined
the AIE-active tetraphenylethylene (TPE)18 with the terpyridyl
Pt(II) complex to fabricate an AIE fluorophore (TPE/TPY-Pt-
PA/PEG), leading to bathochromic shifts of both excitation and
emission towards more biologically interesting NIR windows.
The AIE luminogen is composed of a terpyridyl Pt(II) core
which displays strong intermolecular interactions and emissive
MMLCT excited states in the aggregate state, a TPE unit which
can not only extend π-conjugation of the cyclometalated
ligand, but also weaken the strong π–π stacking interactions,
and a hydrophilic pentaethylene glycol unit that ensure its
biocompatibility (Scheme 1). The synthetic route for TPE/TPY-
Pt-PA/PEG is shown in Scheme S1 (ESI†). In acetonitrile,
TPE/TPY-Pt-PA/PEG is non-emissive, but addition of water
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water content (Fig. S6a), TPE/TPY-Pt-PA/PEG exhibits a slight
bathochromic shift
Fig. 2. (a) Photoluminescence (PL) spectra of TPE/TPY-Pt-PA/PEG in
acetonitrile/water mixture with different water percentages. (b) Plot of PL
intensities of TPE/TPY-Pt-PA/PEG in acetonitrile/water mixture at 730 nm versus
water content. Inset: images of TPE/TPY-Pt-PA/PEG in acetonitrile (0% water)
and TPE/TPY-Pt-PA/PEG in acetonitrile (70% water), with emission taken under a
UV lamp with 355 nm excitation. Conditions: [TPE/TPY-Pt-PA/PEG] = 10 μM,
excitation wavelength (λex) = 465 nm.
Fig. 1. UV-vis absorption spectra of TPE/TPY-Pt-PA/PEG in acetonitrile (40 μM)
with increasing water content (a) from 10% to 50% and (b) from 50% to 90% (c)
Photo showing the colour changes of TPE/TPY-Pt-PA/PEG in acetonitrile at
different water proportions (left to right: 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% and 90% water content).
at the MLCT/LLCT absorption band with an emergence of a
low-lying energy absorption tail at about 480−525 nm (Fig. 1a).
TPE/TPY-Pt-PA/PEG gives the largest hydrodynamic diameter
at 60% water content and the size of the nanoaggregates
shows a clear decline with further increasing water content
(Fig. S6a). This phenomenon is resulted from the tendency to
form more compact nanostructures for minimizing contact of
the hydrophobic groups with water. However, the levelling-off
absorption tail is still increasing with the weakening of the
MLCT/LLCT band (Fig. 1b), suggesting that the weak absorption
tail can be assigned as MMLCT absorption due to the
formation of molecular aggregates via Pt–Pt interactions, π−π
interactions or both.
enhances significantly its NIR emission centred at 730 nm; this
can be attributed to the MMLCT emission by the aggregated
molecules, along with the restriction of intramolecular rotation
of the TPE phenyl rings which facilitate radiative decay
channels. The nanoparticles (NPs) fabricated by encapsulating
TPE/TPY-Pt-PA/PEG with Pluronic F127 (F127), a commonly
used amphiphilic triblock copolymer for drug delivery, lead to
the switch-off of toxicity caused by the isolation of platinum(II)
complexes and difficult accessibility of the metal center.
TPE/TPY-Pt-PA/PEG@F127
NPs
exhibit
excellent
biocompatibility with good performance in lysosome-specific
localisation, which demonstrates an excellent molecular
modification strategy for Pt(II) complexes for cell imaging.
The optical properties of TPE/TPY-Pt-PA/PEG have been
investigated by UV–vis absorption in different solvents (Fig.
S4). The photophysical data are summarized in Table S1. There
are two main absorption bands: one located at 263-330 nm
corresponding to intraligand [π→π*] transitions of the alkynyl
and terpyridine ligands, and the other located at 375-490 nm
attributed to a mixture of [dπ(Pt) → π(terpyridine)] and
The aggregation induced photoluminescence of TPE/TPY-Pt-
PA/PEG in acetonitrile with different water percentages is
characterised in Fig. 2. Although the weak absorption tail at
480–525 nm emerges at 20% water content, TPE/TPY-Pt-
PA/PEG remains non-emissive. Upon a further increase of the
water percentage, a NIR emission band centred at 730 nm is
observed and reaches a maximum intensity at a water
percentage of 70%. On the basis of the previous spectroscopic
studies,22-24 this emission band can be attributed to the
MMLCT emission with enhanced Pt–Pt and/or π−π stacking
interactions facilitated by the solvent-induced aggregation.
Moreover, the aggregation-induced near-infrared emission is
also contributed by the restriction of intermolecular rotation
of the TPE moiety, which would slow down the non-radiative
pathway and facilitate the radiative process. However, the PL
intensity of TPE/TPY-Pt-PA/PEG shows a clear decline trend
with further increasing water content. A possible reason for
this phenomenon is the formation of high-quality nanocrystal
particles at 70% water percentage with a low polydispersity
index (PDI) of 0.05 (Figure S6b), suggesting that the
nanoparticles possess the characteristic of high homogeneity.
When the water content exceeds 70%, the TPE/TPY-Pt-PA/PEG
gradually aggregates from homogeneous crystal particles to
[π(alkynyl)
→
π*(terpyridine)] transitions.19-21 TPE/TPY-Pt-
PA/PEG is non-emissive in good solvents, probably as a result
of the fast rotation of the phenyl rings on the TPE moiety.
Nevertheless, TPE/TPY-Pt-PA/PEG dissolved in water or ethyl
acetate exhibited novel broad absorption bands at around
520-650 nm, which may be induced by molecular aggregates.
In addition, the concentration-dependent UV−vis absorpꢀon
spectra of TPE/TPY-Pt-PA/PEG in acetonitrile (Figures S5)
reveal a deviation from Beer’s Law at 480 nm which can be
assigned to MMLCT transitions, demonstrating that TPE/TPY-
Pt-PA/PEG would self-assemble into superstructures in a poor
solvent.
Since TPE/TPY-Pt-PA/PEG tends to form molecular
aggregates in water (Fig. S4), the UV-vis absorption spectra
and dynamic light scattering (DLS) were used in the self-
assembly studies of TPE/TPY-Pt-PA/PEG in acetonitrile mixture
(40 μM) with increasing water content. Though the
nanoaggregates of TPE/TPY-Pt-PA/PEG become apparent at 20%
amorphous particles with PDI changing from 0.05 to 0.31,
resulting in the reduction of PL Intensity.25,
26
Moreover,
TPE/TPY-Pt-PA/PEG would form more compact nanostructures
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at high water content, and only the molecules on the surface concentration of 20 μM. Even at 100 μM, the cell viability was
of the nanoaggregates emitted light upon excitation, which is around 86%, indicating that TPE/TPY-Pt-PA/PEG@F127 NPs
also an important reason for the decrease of PL intensity.27
have excellent biocompatibility. The NPs for in vitro cellular
imaging were studied with an Olympus model IX71 inverted
microscope. LysoTracker Green was chosen as a commercial
Fig. 3. (a) Particle size distribution of TPE/TPY-Pt-PA/PEG@F127 NPs measured
by DLS. Inset: TEM images of AIE NPs with different scale bars. (b) Normalised
UV–vis absorption and PL spectra of TPE/TPY-Pt-PA/PEG@F127 NPs (red) and
TPE/TPY-Pt-PA/PEG nanoaggregates (blue) in water. [TPE/TPY-Pt-PA/PEG
nanoaggregates] = 20 μM; [TPE/TPY-Pt-PA/PEG@F127 NPs] = 10 μM; λex = 465
nm.
Fig. 4. Confocal laser scanning microscope images of HeLa cervical cancer cells
incubated with (a) 100 nM LysoTracker Green (λex = 488 nm, 500–580 nm emission
filter) for 30 min and (b) 20 μM TPE-PEG-F127 NPs (λex = 465 nm, 600–750 nm emission
filter) for 4 h. (c) The merged images of (a) and (b).
The aggregation-induced NIR emission of TPE/TPY-Pt-
PA/PEG inspires us to explore its cell imaging applications.
Since TPE/TPY-Pt-PA/PEG gradually formed unstable
reference to compare the cellular localisation and bioimaging
efficiency (Fig. 4a). Intracellular NIR emissions observed upon
excitation at 488 nm confirm that these TPE/TPY-Pt-
PA/PEG@F127 NPs can be internalised by living cells (Fig. 4b).
The intensity profiles across HeLa cells stained with TPE/TPY-
Pt- PA/PEG@F127 NPs and LysoTracker Green displayed 88.3%
overlap coefficient (Fig. 4c, calculated using Olympus Fluoview
ver. 4.2a viewer), indicating that the NPs can preferentially
localise in the lysosomes of HeLa cells. The lysosome-specific
localisation properties may be derived from the acid–base
properties of these terpyridyl complexes.31 Upon
internalisation by endocytosis, TPE/TPY-Pt-PA/PEG@F127 NPs
are transported from endosomes to lysosomes, where a
significant portion of these complexes become protonated and
develop a net positive charge, which causes them to be
trapped inside the lysosomes.
aggregates
in
aqueous
media,
TPE/TPY-Pt-PA/PEG
nanoparticles (abbreviated as TPE/TPY-Pt-PA/PEG@F127 NPs)
were fabricated with biocompatible F127 to enhance the
stability.28, 29 As shown in
Fig. 3a, the average hydrodynamic diameters for the NPs are
116 nm as measured by DLS; the PDI is 0.31. The percentage of
Gaussian-shape distribution intensity at ca. 20 nm and ca. 110
nm is 7.1% and 92.9% respectively, which suggests that
TPE/TPY-Pt-PA-PEG@F127
nanoparticles
are
mainly
distributed with the effective diameter at about 110 nm. This
was further confirmed by the rod-like shape of the NPs in
transmission electron microscopy (Fig. 3a). TPE/TPY-Pt-
PA/PEG@F127 NPs were chemically stable for several weeks at
room temperature, demonstrating that they were well
dispersed in aqueous solution and did not form precipitates.
The photophysical properties of TPE/TPY-Pt-PA/PEG in
different states are reported in Table S2. The quantum yield in
aqueous solution is greater for TPE/TPY-Pt-PA/PEG
nanoaggregates as compared with the solid-state thin film and
TPE/TPY-Pt-PA/PEG@F127 NPs. This can be ascribed to the
high-quality nanocrystal stacking structure at 70% water
percentage. The TPE/TPY-Pt-PA/PEG nanoaggregates showed
two absorption maxima at 290 and 445 nm, while the UV-vis
absorption spectra of AIE NPs showed slightly red-shifted
absorption peaks at 293 and 446 nm (Fig. 3b). The peak
emission wavelengths were 730 and 736 nm for TPE/TPY-Pt-
PA/PEG nanoaggregates and TPE/TPY-Pt-PA/PEG@F127 NPs,
respectively. The excitation and emission profiles of TPE/TPY-
Pt-PA/PEG@F127 NPs match well the commercial laser
sources and fluorescence-imaging systems, making it
promising for bioimaging applications.30
In summary, we have combined the AIE-active TPE with the
terpyridyl Pt(II) complex to convert the platinum(II) terpyridyl
complex into an AIE-active fluorophore (TPE/TPY-Pt-PA/PEG)
with bathochromic shifts of both excitation and emission
towards more biologically interesting spectral windows.
TPE/TPY-Pt-PA/PEG have shown AIE NIR emission and
TPE/TPY-Pt-PA/PEG@F127 NPs were found to selectively stain
lysosomes in cancer cells with excellent biocompatibility,
making them promising candidates for long-term cell tracking.
Further studies using the AIE NPs will shed light on the
involvement of lysosomes in animal models of lysosomal
storage diseases.
Parts of this work were supported by the National Basic
Research Program of China (2015CB352005); the National
Natural Science Foundation of China (61775145, 31771584,
61605124, 61620106016, 61525503, 61378091, 61405123,
61705143); the Guangdong Natural Science Foundation
Innovation Team (2014A030312008); Hong Kong, Macao, and
Taiwan cooperation innovation platform & major projects of
international cooperation in Colleges and Universities in
Guangdong Province (2015KGJHZ002); China Postdoctoral
Science Foundation Funded Project (2017M622748,
A critical consideration for achieving long-term cell tracking
is an understanding of the cytotoxicity of NPs. The MTT assay,
a colourimetric assay for assessing cell metabolic activity, was
used to evaluate the cytotoxicity of TPE/TPY-Pt-PA/PEG@F127
NPs to HeLa cells in the concentration range of 0–100 μM (Fig.
S7). Cell viability rates were above 96% at the working
2018M630979);
Shenzhen
Basic
Research
Project
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A table of contents entry
An aggregation-induced near-infrared emitting terpyridyl Pt(II) complex with excellent
biocompatibility shows high specificity to lysosomes in HeLa cells.