A cucurbituril/polysaccharide/carbazole ternary supramolecular assembly for targeted cell imaging

Article abstract of DOI:10.1039/c9cc01601e

A carbazole derivative (G) was synthesized as a photosensitizer to complex with cucurbit[8]uril, resulting in a nanocube with emission at 662 nm for lysosome cell imaging. Furthermore, the alkyl chain on G was included by α-cyclodextrine-modified hyaluronic acid for further assembly, affording a system targeted to cancer cells.

Full text of DOI:10.1039/c9cc01601e

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DOI: 10.1039/C9CC01601E
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A Cucurbituril/Polysaccharide/Carbazole Ternary Supramolecular
Assembly for Targeted Cell Imaging
Xuan Wu,a Yong Chen,a,b Qilin Yu,c Feng-Qing Li,a and Yu Liua,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A carbazole derivative (G) was synthesized as photosensitizer to Liu and coworkers reported a CB[8]-mediated NIR lysosome
complex with cucurbit[8]uril, resulting in the nanocube with the imaging system, in which the emission of the guest molecule
emission at 662 nm for lysosome cell imaging. Furthermore, the (4,4’-Anthracene-9,10-diylbis(ethene-2,1-diyl))bis(1-
alkyl chain on G was included by the α-cyclodextrine-modified ethylpyridin-1-ium) bromide) could shift to the NIR region after
hyaluronic acid for the further assembly, affording a system complexation with CB[8].17 However, there was no other
targeted to cancer cell.
binding site for the introduction of the secondary functional
motif. Following this line, we envisioned the addition of
another binding site onto the guest molecule, which would
enable a further assembly for combined function. To the best
of our knowledge, few researches have focused on both of the
CB[8]-based NIR imaging and targeted PDT systems. Based on
previous researches, molecules with strong intramolecular
push-pull effect could lower the energy gap between the S1
and T1 states, which could benefit the intersystem crossing
The far-red and near-infrared (NIR) (650-1000 nm) dyes1, 2
have been extensively studied and explored as the
photosensitizers in the fields of cancer diagnose and therapy,
for their less photo-damage to cells, lower scattering, deeper
light penetration, and better separation from medium auto-
fluorescence.3-7 Traditionally, the synthesis of this type of dyes
is always complex and tedious, especially for dyes containing
the targeted motifs, or some other functional groups for the
combined therapy. Therefore, how to construct a versatile
system in a convenient way remains a challenge. As an
effective approach to construct functional systems,
supramolecular assembly has attracted tremendous
attention.8-11 In these systems, the building blocks are
assembled into well-defined manners through non-covalent
interactions to achieve versatile functions. Therefore, with the
careful design of the building blocks, we could fabricate a
versatile assembly from simple molecules, thus enabling us to
construct a system with both NIR imaging and targeted
efficiency for the generation of singlet oxygen (1O2) or other
18-21
reactive oxygen species (ROS).
Herein, a supramolecular
system with NIR lysosome imaging and targeted PDT abilities
has been constructed in a two-step assembly. Firstly, a dipolar
fluorescence compound (G) with the carbazole as the electron
donor motif and the pyridinium as the electron accept motif
was synthesized, exhibiting an emission wavelength at 568 nm.
After the formation of supramolecular complex with CB[8], the
binary assembly could assemble into the nanocube with an
average diameter around 220 nm (Scheme 1). To our delight,
the emission peak exhibited the remarkable shift to 662 nm.
Besides,the ROS generation was also improved about 1.5 times
compared with G, and the cell assay certificated this binary
system could also be used as the lysosome imaging agent as
well as the photosensitizer for the cancer treatments.
Moreover, due to the long alkyl chain on the G, the α-
cyclodextrine-modified hyaluronic acid (HA-CD) was
introduced into the secondary assembly22, 23, resulting in the
formation of supramolecular nanoparticles without changing
the emission wavelength and ROS generation ability.
Therefore, the targeted PDT system could be constructed,
which exhibited low light cytotoxicity to normal cells (293T),
but high cytotoxicity to cancer cells (A549).
photodynamic therapy (PDT) properties in
convenient way.
a relatively
As is well known, cucurbit[8]uril12 (CB[8]) has been widely
studied for construction of emission-tunable systems due to its
ability to restrict the rotation of guest molecules, and form
extended conjugation system.13-16 Therefore, with careful
design of guest molecules, a NIR emission system could be
achieved by the complexation between CB[8] and the non-NIR
dye, which could be used as the first-stage assembly. Recently,
a. College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry,
Nankai University, Tianjin, 300071, China (E-mail: yuliu@nankai.edu.cn)
b. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin, 300071, China
Firstly, the host-guest property between G and CB[8] was
studied. Due to its poor solubility in the aqueous solution,
another similar compound (MG), with a shorter alkyl chain and
different counterion, was successfully synthesized. As shown in
c. Department of Microbiology, College of Life Sciences, Nankai University, Tianjin,
300071, China
Electronic Supplementary Information (ESI) available: [details of synthesis
procedure, UV-vis and fluorescence spectra, DLS and zeta potential results, TEM
and SEM images, fluorescence images of cells]. See DOI: 10.1039/x0xx00000x
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And the radius in D2O was calculated to be 1.55 nm by Stokes–
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DOI: 10.1039/C9CC01601E
Einstein relationship
Scheme 1. Schematic illustration of the construction of
targeting PDT system
figure 1, the upfield chemicalshift and broadening effect of the
protons He-h on the MG was observed in the presence of CB[8],
as well as the downfield shift of the protons Hb-d, resulting
from the inclusion of pyridium motif into the cavity of CB[8].
Moreover, the 2D NOESY spectroscopy also confirmed these
results by the observation of strong correlation signals
between the proton Hc and Hf (Figure S10, ESI†).
Figure 2. (a) Normalized UV-vis spectra of G with various molar
ratio of CB[8] ([G] = 2 × 10-4 M), (b) Normalized fluorescence
spectra of G with various molar ratio of CB[8] ([G] = 2 × 10-5
M). Insets in panel a are photos of the G solution before and
after the addiditon of CB[8].
(Table S1, ESI†), and the molecular size of MG was about 1.35
nm(Figure S14, ESI†). Therefore, there might exist a stable
oligomers at the molar ratio of 1:1 between CB[8] and G. And
the continual addition of CB[8] could destroy the oligomers,
thus resulting in the formation of the second isoabsorptive
point, which was consistent with UV-vis titration experiment.
Fluorescence spectroscopy has also been carried out to
determine the red-shift of G after formation of complexation
with CB[8]. As shown in figure 2b, the maximum emission
wavelength of G was at 568 nm with weak emission band,
whose fluorescence quantum yield was determined to be
0.69%, and fluorescence lifetime was 0.60 ns (Figure S15a,
ESI†). After complexation with CB[8] at the molar ratio of 1:1
([G]/[CB[8]]), the maximum emission wavelength exhibited a
remarkable red-shift to 662 nm in the NIR region (Figure 2b),
whose fluorescence quantum yield was 2.14%, and lifetime
was 4.21 ns (Figure S15b, ESI†). Through continually adding of
CB[8] at the molar ratio of 1:1.4 ([G]/[CB[8]]), a new emission
peak appeared at 568 nm, indicating the destruction of the
previous assembly. However, if the molar ratio between [G]
and [CB[8]] was 1:3, the emission peak would exhibit a blue
shift as well as the increase intensity at 568 nm. This result
1
Figure 1. Partial H NMR spectra (400M Hz, 298 K, D2O) of (a)
MG ([MG] = 2 mM), (b) MG + CB[8] ([MG] = [CB[8]] = 2 mM).
Furthermore, the Job’s plot indicated the 1:1 binding
stoichiometry between CB[8] and G (Figure S11, ESI†). And the
binding constant was determined to be (3.38 ± 0.23) × 106 M-1.
From the recorded spectra (Figure S12, ESI†), the obvious red-
shift in the absorption peak of G was observed upon the
gradual addition of CB[8], in which two isoabsorptive points
were observed at 466 nm and 482 nm. The changes in the
solution color also provided visible evidence for the above
mentioned change, from which the color of solution changed
from yellow to red upon the gradual addition of CB[8] (Figure
2a, inset). The occurrence of two isoabsorptive points during
the UV-vis titration attracted our attention. Therefore, the 2D
diffusion-ordered 1H NMR spectroscopy (DOSY) experiment
was performed, and the diffusion coeffcient of low
concentration (2 mM) was recorded. From the DOSY spectrum
(Figure S13, ESI†), only one set of signal could be observed.
2 | J. Name., 2012, 00, 1-3
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could also prove the formation of a stable oligomer at the relatively high concentration, the cell viability was more than
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molar ratio of 1:1 ([CB[8]]/[G]), but the continual addition of 95%, meaning the relatively low cytotoxiDcOityI: 1i0n.1d0a39rk/Cc9oCnCd0i1t6io01nE,
CB[8] would destroy the formed oligomer.
and acceptable biocompatibility. Upon the irradiation under
white light, cell viability was rapidly decreased. As illustrated in
figure S20b (ESI†), 82% of cells were killed in the presence of
GCB[8] under the light irradiated at the concentration of 2
μM ([CB[8]] = [G] = 2 μM), and nearly no alive cells could be
detected at the concentration of 4 μM. This phenomenon was
consistent with the previous results that the addition of CB[8]
could improve the ROS generation ability. This result also
certificated the NIR and enhanced PDT system were
conveniently constructed through the first-step assembly.
After the successful construction of binary assembly, the
HA-CD was introduced for the further assembly, thanks to the
host-guest interaction with the alkyl chain and the
electrostatic interaction. Therefore, the obtained ternary
system would be endowed the targeting property to the
cancer cells.24, 25 Firstly, the Job’s plot indicated the 1:1 binding
stoichiometry between α-CD and G (Figure S21, ESI†), and the
binding affinity was determined to be (5.9 ± 0.4) × 103 M-1
(Figure S22, ESI†). Furthermore, circular dichroism spectrum
confirmed the inclusion of alkyl chain into the cavity of α-CD
(Figure S24, ESI†). The similar phenomena could also be
observed in the process of UV-vis titration between α-CD and
GCB[8] (Figure S23, ESI†), where only the adsorption
intensity decreased upon the gradual addition of α-CD,
meaning the previous binding model could not be destroyed.
This result provided a foundation for the construction of the
targeted ternary system.
Due to the amphipathy of G, the self-assembly morphologies
in presence and absence of CB[8] were investigated. From the
TEM and SEM images (Figure 3a and 3c), the G could self-
assemble into nanofibers in aqueous solution. While for the
GCB[8] complexation, they could self-assemble into
nanocubes (Figure 3b, and 3d) with the broad diameter
distribution ranging from 200-600 nm, and the average
diameter was determined to be around 220 nm by DLS analysis
(Figure S16b, ESI†). Moreover, the obtained nanocube had a
positively-charged surface, whose value was measured to be
+49.6 mV (Figure S17b, ESI†). Moreover, their ROS and 1O2
generation ability were studied by using commercially
available probes (details in figure S18, ESI†), and both ROS and
1O2 generation abilities were enhanced after the addition of
CB[8], indicating their potential application in PDT. Based on
the above results, it was concluded that the NIR emission
assembly with enhanced PDT efficiency was simply
constructed by the host-guest interaction between CB[8] and
G.
The HA-CD was successfully synthesized with the degree of
substitution of 11%. The addition of HA-CD to GCB[8]
solution had no influence on its fluorescence emission and UV-
vis adsorption properties concluded from figure S25 (ESI†).
Following, the TEM and SEM images indicated the ternary
system could form the nanoparticles with the average
diameter around 500 nm (Figure S26 and S27a, ESI†) and a
negative charge distribution (-18 mV) on the surface (Figure
S27b, ESI†), indicating the distribution of HA-CD on the
surface, which would enable the obtained ternary system with
the targeting property. Moreover, the ROS generation ability
was also reserved by using the H2DCF-DA as the ROS indicator
(Figure S28, ESI†).
To our delight, the introduction of HA-CD could reduced the
uptake of HA-CD@GCB[8] into the normal cells (293T,
Purchased from Cell Center of Peking Union Medical College
Hospital, Chian), concluded from the fluorescence images of
cells (Figure S29, ESI†). Then the MTT assay was carried out to
further evaluate the targeted efficiency. As exhibited in figure
4, the GCB[8] could induce the half of 293T cells dead even in
a relatively low concentration, meanwhile, the addition of HA-
CD could reduce its photodamage to the normal cells, more
than 90% remained alive at the same condition. What’s more,
this ternary system could also remain their phototoxicity to
the cancer cells (A549). And these results certificated the
successful construction of functional NIR imaging and targeted
PDT system by multi-step assembly.
Figure 3. (a) TEM image of G, (b) TEM image of GCB[8], (c)
SEM image of G, and (d) SEM image of GCB[8].
To examine the biological application of the obtained
supramolecular assembly, the cell imaging experiment was
carried out. After being incubated with GCB[8] solution
followed by the observation under a laser scanning confocal
microscope, the bright green fluorescence within A549 cells
(Purchased from Cell Center of Peking Union Medical College
Hospital, China) can be observed in the dye channel. Further
colocalization experiment by co-incubating with LysoTracker
(Purchased from Invitrogen Co., Ltd., USA) indicated the
formed nanoparticles could stain lysosome specifically (Figure
S19, ESI†). Then the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT, purchased from Amresco
Co.,Ltd., USA) assay was carried out to evaluate its anti-cancer
ability. The A549 cancer cells were incubated with G, CB[8] and
GCB[8] respectively, then their viability was calculated in
different conditions. As shown in figure S20a (ESI†), less than
5% of A549 cells were killed in the dark environment. Even in a
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J. Name., 2013, 00, 1-3 | 3
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We thank the National Natural Science Foundation of China
DOI: 10.1039/C9CC01601E
(grant nos. 21672113, 21432004, 21772099, 21861132001 and
91527301) for financial support.
Herein, a ternary supramolecular assembly was successfully
constructed by G, CB[8] and HA-CD in a multi-step assembly
manner, which could be used as the targeted NIR lysosome
probe and PDT agent. Firstly, a carbazole derivative (G) with
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Notes and references
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Figure 4. The concentration dependent MTT assay of HA-
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which was a non-NIR emission dye with the emission
wavelength at 568 nm. After the formation of host-guest
complexation with CB[8], its fluorescence spectrum exhibited
remarkbale red shift to 662 nm. Moreover, this binary
assembly could be used for targted NIR lysosome imaging and
act as the efficient PDT agent. Finally, the HA-CD was
incorporated for the secondary assembly, due to the strong
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
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Graphical Abstract
A ternary supramolecular assembly with near-infrared lysosome and targeted photodynamic therapy
properties was successfully orthogonally constructed in a two-step assembly manner.