Three-dimensional nanographene based on triptycene: Synthesis and its application in fluorescence imaging

Article abstract of DOI:10.1021/ol302833t

A novel kind of three-dimensional (3D) nanographene based on a triptycene structure bearing three hexa-peri-hexabenzocoronene (HBC) moieties was synthesized efficiently from triiodotriptycene. With the characteristic of intrinsic fluorescence, the 3D nanographene was used as a fluorescent agent for in vitro and in vivo fluorescence imaging with good antiphotobleaching ability and little toxicity.

Full text of DOI:10.1021/ol302833t

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Three-Dimensional Nanographene Based
on Triptycene: Synthesis and Its
Application in Fluorescence Imaging
Chun Zhang,*,† Ying Liu,† Xiao-Qin Xiong,† Lian-Hui Peng,† Lu Gan,*,†
Chuan-Feng Chen,*,‡ and Hui-Bi Xu†
College of Life Science and Technology, Huazhong University of Science and
Technology, and National Engineering Research Center for Nanomedicine, Hubei,
430074, China, and Beijing National Laboratory for Molecular Sciences, CAS Key
Laboratory of Molecular Recognition and Function, Institute of Chemistry, Chinese
Academy of Sciences, Beijing 100190, China
chunzhang@hust.edu.cn; lugan@hust.edu.cn; cchen@iccas.ac.cn
Received October 15, 2012
ABSTRACT
A novel kind of three-dimensional (3D) nanographene based on a triptycene structure bearing three hexa-peri-hexabenzocoronene (HBC) moieties
was synthesized efficiently from triiodotriptycene. With the characteristic of intrinsic fluorescence, the 3D nanographene was used as a
fluorescent agent for in vitro and in vivo fluorescence imaging with good antiphotobleaching ability and little toxicity.
Hexa-peri-hexabenzocoronene (HBC) and its derivatives,
namely nanographenes,1,2 have recently attracted great
interest because they are recognized as promising building
blocks for organic nanoelectronic and photovoltaic devices
with their large π-systems and strong assembly abilities. So
far, variousdevices basedon nanographene molecules with
different structures and functionalities have been well devel-
oped by organic synthetic or supramolecular protocols,
such as Aida’s nanotubes,3 Li’s graphene quantum dots,4
2,5
€
and Mullen’s graphene nanoribbons and large graphenes.
These HBC-based building blocks are all planar structures.
Although three-dimensional (3D) structures might afford
nanographenes, with new opportunities to improve their
properties to reduce the face-to-face interaction between
π-planes and increase their solubility like other organic
materials,6 no such molecules have hitherto been reported.
Moreover, as a consequence of the difficulty of water
† Huazhong University of Science and Technology.
‡ Beijing National Laboratory for Molecular Sciences.
(1) For recent reviews, see: (a) Feng, X.; Pisula, W.; Mullen, K. Pure
(3) (a) Zhang, W.; Jin, W.; Fukushima, T.; Saeki, A.; Seki, S.; Aida,
T. Science 2011, 334, 340. (b) He, Y.; Yamamoto, Y.; Jin, W.; Fukushima,
T.; Saeki, A.; Seki, S.; Ishii, N.; Aida, T. Adv. Mater. 2010, 22, 829.
(c) Zhang, W.; Jin, W.; Fukushima, T.; Ishii, N.; Aida, T. Angew. Chem.,
Int. Ed. 2009, 48, 4747.
€
€
Appl. Chem. 2009, 81, 2203. (b) Mullen, K.; Rabe, J. Acc. Chem. Res.
2008, 41, 511.
(2) For recent examples of nanographenes, see: (a) Cai, J.; Ruffieux,
(4) (a) Yan, X.; Cui, X.; Li, B.; Li, L.-S. J. Am. Chem. Soc. 2010, 132,
5944. (b) Yan, X.; Cui, X.; Li, L.-S. Nano Lett. 2010, 10, 1869.
P.; Jaafar, R.; Bieri, M.; Braun, T.; Blankenburg, M.; Muoth, M.;
€
Seitsonen, A. P.; Saleh, M.; Feng, X.; Mullen, K.; Fasel, R. Nature
2010, 466, 470. (b) Yang, S.; Feng, X.; Zhi, L.; Cao, Q.; Maier, J.;
€
(5) Wu, J.; Pisula, W.; Mullen, K. Chem. Rev. 2007, 107, 718 and
references therein.
€
Mullen, K. Adv. Mater. 2010, 22, 838.
r
10.1021/ol302833t
XXXX American Chemical Society

solubilization, the biological applications of HBCs have
remained largely unexplored, especially in fluorescence
imaging.7 In this area, the problem of photobleaching for
fluorescent agents has become a great obstacle. Although
some antiphotobleaching fluorescent agents such as quan-
tum dots have been successfully developed, the potential
toxicity of heavy metals has greatly limited their biomedi-
cal applications. Searching for fluorescence imaging agents
with high stability and little toxicity is still a challenge.8
nanographene aqueous nanopaticles can be prepared for
fluorescence imaging with little cytotoxicity. Moreover,
the 3D nanographene displayed good antiphotobleaching
ability compared to other commercial fluorescent agents
such as LysoTracker Green. These results encourage further
studies of our 3D nanographene for biomedical applications
like graphene.13
Synthesis of the 3D nanographenes is outlined in Scheme 1.
Starting from triiodotriptycene 2,14 its palladium-catalyzed
Sonogashira coupling reaction with tert-butylphenylace-
tylene in the presence of Pd(PPh3)4, CuI, and NEt3 af-
forded triethynylation product 3 in good yield, which
was then subjected to a DielsꢀAlder reaction with tetra-
tert-butylphenylcyclopentadienone15 to result in the poly-
phenylene dendritic precursor 4 in 43% yield. Oxidative
cyclization of 4 with FeCl3/MeNO2 in CH2Cl2 resulted in
the formation of 3D nanographene 1 as a pale yellow solid
in 87% yield.
Scheme 1. Synthesis of 3D Nanographene 1
In the 1H NMR spectrum of 1, two sharp singlet signals
appeared at δ 8.26 and 8.91 ppm for the two methenyl
protons (Ha and Hb) of the triptycene scaffold, which were
assigned by 2D CꢀH COSY analysis (Figure S7). The
formation of 3D nanographenes was furthermore con-
firmed by the MALDI-TOF mass spectrum.16 As shown
in Figure S8, the mass spectrum of 1 revealed a peak at m/z
2429 for Mþ. Comparison of the MALDI-TOF spectrum
ofthe 3Dnanographene withthatofits precursor 4 (Figure
S9) indicated the elimination of 36 hydrogen atoms during
the Scholl oxidative condensation reactions occurred,
which is consistent with the differences between the mole-
cular formulas of 3D nanographene (C188H170) and its pre-
cursor 4 (C188H206). The infrared vibrational spectra showed
more simple absorption peaks in this range of the aromatic
CꢀH out-of-plane bending vibration (650ꢀ950 cmꢀ1) of 1
compared with 4, which afforded further evidence of the
formation of 3D nanographene (Figure S10).4b The ab-
sorption and emission spectra of the 3D nanographene
were obtained in dichloromethane solution. When 1 was
excited at 364 nm (ε = 3.05 ꢁ 105 Mꢀ1 cmꢀ1, 298 K), the
emission wavelength at 470, 490, and 500 nm with a
fluorescent quantum yield of 12.8% was observed
(Figures S11 and S12).
Triptycenes, with unique three-dimensional rigid frame-
works, have more applications in materials science9,10 and
molecular machines.11 Recently, we12 utilized the useful
building blocks to construct novel hosts and subsequently
develop a series of new supramolecular systems. Herein,
we describe the synthesis of triptycene derived three-
dimensional nanographene 1, which bears three HBC moi-
eties in the triptycene three-dimensional scaffold. With the
characteristic of intrinsic fluorescence, the three-dimensional
(6) (a) Mori, S.; Nagata, M.; Nakahata, Y.; Yasuta, K.; Goto, R.;
Kimura, M.; Taya, M. J. Am. Chem. Soc. 2010, 132, 4054. (b) Long,
T. M.; Swager, T. M. Adv. Mater. 2001, 13, 601.
€
(7) Yin, M.; Shen, J.; Pisula., W.; Liang, M.; Zhi, L.; Mullen, K.
J. Am. Chem. Soc. 2009, 131, 14618.
To direct the biological application of our 3D nanogra-
phene, water solubilization is necessary. Adding the THF
solution of 1 to a water containing poly(ethylene glycol)-
block-poly(propylyene glycol)-block-poly(ethylene glycol)
(pluronicF68) nonionic surfactant could prepare the aque-
ous nanoparticles of 1 after removing THF by rotary
evaporation (Figure 1a).17 For the aqueous nanoparticles
(8) Some recent examples of 2D nanographene/nanographene oxide
for fluorescence imaging with antiphotobleaching property: (a) Li,
J.-L.; Bao, H.-C.; Hou, X.-L.; Sun, L.; Wang, X.-G.; Gu, M. Angew.
Chem., Int. Ed. 2012, 52, 1830. (b) Pan, D.; Guo, L.; Zhang, J.; Xi, C.;
Xue, Q.; Huang, H.; Li, J.; Zhang, Z.; Yu, W.; Chen, Z.; Li, Z.; Wu, M.
J. Mater. Chem. 2012, 22, 3314.
(9) For recent reviews, see: (a) Swager, T. M. Acc. Chem. Res. 2008,
41, 1181. (b) Chong, J. H.; MacLachlan, M. J. Chem. Soc. Rev. 2009, 38,
3301 and references therein.
(10) For recent examples, see: (a) Zhang, C.; Liu, Y.; Li, B.; Tan, B.;
Chen, C.-F.; Xu, H.-B.; Yang, X.-L. ACS Macro. Lett. 2012, 1, 190.
(b) VanVeller, B.; Robinson, D.; Swager, T. M. Angew. Chem., Int. Ed.
2012, 51, 1182. (c) Mastalerz, M.; Schneider, M. W.; Oppel, I. M.; Presly,
O. Angew. Chem., Int. Ed. 2011, 50, 1046.
(11) Kelly, T. R.; Cai, X.; Damkaci, F.; Panicker, S. B.; Tu, B.;
Bushell, S. M.; Cornella, I.; Piggott, M. J.; Salives, R.; Cavero, M.;
Zhao, Y.; Jasmin, S. J. Am. Chem. Soc. 2007, 129, 376.
(13) (a) Robison, J. T.; Tabakman, S. M.; Liang, Y.; Wang, H.;
Casalongue, H. S.; Vinh, D.; Dai, H. J. Am. Chem. Soc. 2011, 133, 6825.
(b) Liu, Z.; Robinson, J. T.; Sun, X.; Dai, H. J. Am. Chem. Soc. 2008,
130, 10876.
(14) Zhang, C.; Chen, C.-F. J. Org. Chem. 2006, 71, 6626.
(15) Draper, S. M.; Gregg, D. J.; Madathil, R. J. Am. Chem. Soc.
2002, 124, 3486.
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(16) Tomovic, Z.; Watson, M. D.; Mullen, K. Angew. Chem., Int. Ed.
2004, 43, 755.
(17) (a) Xiao, J.; Yin, Z.; Li, H.; Zhang, Q.; Boey, F.; Zhang, H.;
Zhang, Q. J. Am. Chem. Soc. 2010, 132, 6926. (b) Liu, Q.; Yang, T.;
Feng, W.; Li, F. J. Am. Chem. Soc. 2012, 134, 5390.
(12) (a) Zhu, X.-Z.; Chen, C.-F. J. Am. Chem. Soc. 2005, 127, 13158.
(b) Zong, Q.-S.; Zhang, C.; Chen, C.-F. Org. Lett. 2006, 8, 1859. (c)
Zhang, C.; Chen, C.-F. J. Org. Chem. 2007, 72, 3880. (d) Zhang, C.;
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B
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with a concentration of 5 μg/mL, dynamic light scattering
analysis displayed that the hydrodynamic size of these
nanoparticles was maintained at 100 nm as shown in
Figure 1b. The UV spectrum of the nanoparticles dis-
played the maximal absorption peak at 364 nm, which is
similar to their dichlormethane solution. Excitation of the
nanoparticles (5 μg/mL) in water at 364 nm resulted in a
fluorescence emission at 475 and 504 nm with a shoulder at
557 nm (Figure 1c). Figure 1d shows a typical transmission
electron micrograph (TEM) image of 3D nanographene
nanoparticles with a size distribution of 40ꢀ50 nm.
With the characteristic of intrinsic fluorescence, the
aqueous nanoparticles of 3D nanographene could be used
as a fluorescent agent for cellular imaging with green
fluorescence, as well as red fluorescence for human hepa-
tocellular carcinoma HepG2 cells after 6 h of incubation
with a concentration of 5 μg/mL (Figure S13). To further
determine the cellular uptake process, the cellular imaging
experiments were performed for human ovarian cell line
A2780 and mouseleukemicmonocyte macrophage cellline
RAW264.7 using an Andor Revolution spinnig disk con-
focal microscope when the cells were treated with aqueous
nanoparticles (5 μg/mL) for multiple time points (i.e., 1, 3,
and 6 h). As shown in Figure 2a, 3D nanographene
nanoparticles can be readily detected in both A2780 and
RAW267.4 cells after 1 h of incubation. As time went on,
the fluorescence intensity increased and more nanoparticles
were internalized into the cells. To examine the potential
for in vivo imaging, subcutaneous injection of 3D nano-
graphene 1 (20 μL aliquots, 0.1 mg/mL) into the left flank
ofa nude mouse was administered. The signals were clearly
observed when the mouse was imaged in a fluorescence
mode (green fluorescent protein (GFP) excitation filter,
445ꢀ490 nm and dsRed emission filter, 570ꢀ650 nm)
without any skin autofluorescence (Figure 2b). We next
examined the biodistribution of 3D nanographene 1 after
intravenous injection (100 μL aliquots, 0.1 mg/mL). The
results showed that 3D nanographene 1 mainly accumu-
lated in the liver (Figure 2c).
Figure 1. (a) Photographs under daylight (left) and under
UV light (right), (b) hydrodynamic size with concentration of
5 μg/mL, (c) UV (blue line) and fluorescent (red line) spectra,
and (d) TEM image of 3D nanographene nanoparticles.
For fluorescence imaging agents, the shortcoming of
photobleaching greatly limits their biomedical application.
To examine the intracellular photostability of 3D nano-
graphene 1, HepG2 cells were incubated with either the
commonly used LysoTracker Green or 3D nanographene
1 for 1 h and were imaged over time during continuous
excitation under a confocal microscope. At the zero time
point, the fluorescence intensities of LysoTracker Green
and 3D nanographene 1 were similar. After approximately
6 min of irradiation, the LysoTracker Green signal was
almost completely photobleached, while the signal of 3D
nanographene 1 was still readily detected (Figures 3 and
S14). Furthermore, the cytotoxicity of 3D nanographene 1
was evaluated by the 3-(4, 5-dimethyl-2-thiazolyl)-2,
5-diphenyltetrazolium bromide (MTT) assay in A2780 and
RAW264.7 cells. As shown in Figure S15, 3D nanogra-
phene 1 induced time- and concentration-dependent cyto-
toxicity; it only induced a less than 10% reduction in cell
viability for A2780 and RAW264.7 cells, even at a con-
centration of 20 μg/mL for 6 h.
Figure 2. (a) In vitro cellular imaging with 3D nanographene
nanoparticles in A2780 and RAW264.7 cells after 1 h, 3 and 6 h
incubation. Scale bar: 50 μm. (b) In vivo fluorescence image of
3D nanographene nanoparticles (20 μL of 0.1 mg/mL) injected
subcutaneously on the left flank of a mouse. (c) Fluorescence
images showing the biodistribution of 3D nanographene nano-
particles in a mouse 1 h after injection. K, Lu, Sp, Li and H
indicate kidney, lung, spleen, liver and heart, respectively.
Org. Lett., Vol. XX, No. XX, XXXX
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nonionic surfactant to provide water dispersal and low
toxicity. Much can be done in the future to load some
anticancer drugs in the expanded “internal molecular free
volume” of triptycene which can be used as a multifunc-
tional theranostic platform for cancer treatment.
Acknowledgment. This work was supported by the
National Natural Science Foundation of China (20902031),
the National Basic Research Program (2012CB932500,
2011CB933103, and 2011CB932501), the Fundamental Re-
search Funds for the Central Universities (HUST 2011TS148),
and Wuhan ChenGuang Program (201150431118). We
also thank the Analytical and Testing Center of Huazhong
University of Science and Technology for related analysis.
Figure 3. In vitro photostability after incubation with either
LysoTracker Green or 3D nanographene nanoparticles in
HepG2 cells. Scale bar: 50 μm.
Supporting Information Available. Details of experimen-
tal materials and measurements, synthetic procedures for 1,
FT-IR and UV spectra of 1 and 4, Fluorescent emission
spectra of 1, in vitro and in vivo fluorescence imaging,
photostability and MTT experiments. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
In conclusion, we have synthesized a novel 3D nano-
graphene 1 based on a triptycene scaffold and charac-
terized its structure by NMR, MALDI-TOF MS, UV,
fluorescence, and IR spectra. With its intrinsic fluores-
cence, 3D nanographene 1 can be employed for in vitro and
in vivo fluorescence imaging, upon functionalization by a
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX