A self-assembled nanoprobe for long-term cancer cell nucleus-specific staining and two-photon breast cancer imaging

Article abstract of DOI:10.1039/c7cc09806e

Herein, a novel self-assembled nanoprobe for the long-term tracking of the nucleoli of cancer cells and for differentiating between clinical breast cancer tissues and para-carcinoma tissues has been developed.

Full text of DOI:10.1039/c7cc09806e

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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A self-assembled nanoprobe for the long term cancer cell nucleus-
specific staining and two-photon breast cancer imaging
Tang Gao a,1, Shuanglian Wangb,1, Wuwu Lvb, Mian Liub, Hongliang Zenga, Zhu Chena, Jie Donga,
Ziping Wua, Xueping Fengb,*, Wenbin Zenga,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Herein, a novel self-assembled nanoprobe for long-term tracking rotors, our group developed a novel series of AIEgens with
nucleolus of cancer cells and differentiating clinical breast cancer good photo-stability, high water solubility, and good
tissues and para-carcinoma tissues has been developed.
biocompatibility.[14] Based on the previous work, we intend to
develop novel fluorescent probes that can long term
selectively stain the nuclei of cancer cells. The physicochemical
properties of probes that facilitate nucleic acid binding include
cationic characteristics and planar aromatic systems. Probes
that allow access to cells are characterized by low protein and
lipid binding. Reduced non-nuclear site build up characteristics
include cationic high alkalinity and hydrophilicity.[15] The
quaternary ammonium salt, an electron accepting group with
positively charged, can promote the probe into the cell due to
lipophilicity and electrophoretic forces.[16] Furthermore,
AIEgens with superior photostability and excellent biological
application may aggregate in cell nucleus and emit strong
fluorescence,[17] which could be utilized for long-term tracing
cancer cells with low-toxicity.[18] Take above into consideration,
in this paper we designed and synthesized a novel water
soluble cationic bola-type small molecule 4-(1-(4-
chlorophenyl)-4,5-diphenyl-1H-imidazol-2-yl)-1-(12-(pyridin-1-
ium-1-yl)dodecyl)pyridin-1-ium bromide denoted as DPMPB
which can be self-assembled into positively charged
nanoparticles (DPMPB-FONs) with weak emission in aqueous
solution, and selectively stain the nuclei of cancer cells in a
long time (Figure 1). Remarkably, the applicability of DPMPB
to image breast cancer and para-carcinoma tissue highlighted
the potential as a guiding-agent in surgery.
As the most prominent organelle within cells, nucleolus
]
regulates ribosome synthesis and gene expression.[1 It
reported that the nuclei of cancer cells have distinguished
architecture compared with the nuclei of normal cells.[2
]
Therefore, fluorescence nucleus imaging in live cells is not only
to gain insights into the process of malignant transformation
of tumour, but also to provide a basis for developing new
diagnostic and therapeutic tools for cancer.[3] The well-known
organic dyes, including 4’,6-diamidino-2-phenylindole (DAPI),
propidium iodide (PI) and Hoechst, have been widely used to
stain nuclei of cancer or normal cells. However, these
fluorophores are generally mutagenic with potential health
hazards and often suffer from low water solubility, poor
photostability, which limit their further application such as
long-term imaging of nuclei.[4] Furthermore, though quantum
dots have been widely used to image nuclei, they have intrinsic
toxicity [5] and cannot penetrate into nucleus without surface
modification.[6] Small organic fluorophores, used for the cell
[8]
nucleolus imaging, such as styryl dyes,[7],
fluorescein,[9]
ruthenium (II)[10] and hemicyanine derivatives,[11] are often
used at very low concentration because of the limitation of
aggregation-caused quenching (ACQ) effect. Unfortunately,
these dyes are found to be easily photo-blench upon
[12]
photoexcitation,
which also inhibit their applications in
long-term monitoring the cell nucleus.[13] Therefore, it is highly
desirable to design novel fluorescent probes with high water
solubility, good photostability, low cytotoxicity and overcome
the effect of ACQ to achieve long-term cellular nucleus imaging.
Recently, based on tetraphenyl imidazole-cored molecular
Scheme 1. Schematic illustration of DPMPB as a fluorescent
nanoprobe for selective nucleus imaging in cancer cells.
The synthetic route to DPMPB was shown in Scheme S1
.
2-Hydroxy-1, 2-diphenylethanone (compound 1) was prepared
by thiamine hydrochloride-mediated benzoin condensation
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reaction. Oxidation of compound 1 afforded the corresponding studies. To explore the aggregation of DPMB in aqueous, we
benzil. 4-(1-(4-Chlorophenyl)-4, 5-diphenyl-1H-imidazol-2-yl) performed the experiments of dynamic light scattering (DLS)
pyridine (compound ) was achieved by a one-pot two-steps and scanning electron microscopy (SEM). The DLS studies
multicomponent reaction of 4-pyridinecarboxaldehyde, p- indicated the aggregates of DPMPB have an average diameter
chloroaniline, benzyl and ammonium acetate. Compound of 219 nm with PDI 0.160 (Figure 1c). As shown in Figure 1d
was reacted with 1-(12-bromododecyl)-4- methylpyridin-1-ium the compound DPMPB could self-assemble into spherical
bromide (compound to yield DPMPB The detailed nanoaggregates (DPMPB-FONs). The aggregates states with
syntheses and characterization of DPMPB and the faint emission may be ascribed to the formation of loosely
intermediates are given in the supporting information. packed with enough free volume to consume the radiative
3
3
,
4)
.
With DPMPB in hand, we firstly investigated its optical energy by the intramolecular rotation.[19] Furthermore, the
properties. Due to the hydrophilic nature of the Py salt, zeta potential experiments indicated that DPMPB-FONs had an
DPMPB has good solubility in polar solvents such as dimethyl average zeta potential of 3.70 mV in PBS (Figure S4), which
sulfoxide, acetonitrile, methanol and water. As shown in may be attributed to the presence of cationic pyridinium. This
Figure S1, DPMPB exhibits an absorption band at around 400 positive charge may improve the ability of DPMPB-FONs to
nm, irrespective of the type of solvent used. The absorption enter tumour cells through charge-mediated endocytosis. [20]
band at around 400 nm may attribute to the intramolecular
charge transfer (ICT) transition between the electron donating
imidazole and the electron-withdrawing Py salt. The
fluorescence emission spectra of DPMPB shown obvious
polarity dependence upon in different solvents upon excited
with 385 nm (Figure S2a). It showed strong emission at around
495 nm with larger Stokes shift (110 nm) in lower polarity
index media solution such as dioxane and tetrahydrofuran. By
contrast, the emission of DPMPB weakened and red shifted in
higher polarity index media (DMSO, CH3CN, CH3OH and H2O).
As anticipated, the solvent effect was derived from the ICT
effect. In order to further investigate the ICT interaction
between the pyridinium substituent and imidazole, the
computational calculations were performed. As shown in
Figure S2b, the electron- donating imidazole unit has a higher
electron in the highest occupied molecular orbital (HOMO).
While, in the lowest unoccupied molecular orbital (LUMO) the
electron density tends to be denser around the pyridinium
segment. It is clear that there is strong electron interaction to
Figure 1. (a) Fluorescent emission spectra of DPMPB (5 µM) in
THF-water mixtures with different THF fractions (ƒw). inset:
Photograph of DPMPB solid powder taken under portable UV
lamp 365 nm illumination. (b) Plots of fluorescent intensity vs.
THF fractions of DPMPB, (c) DLS size distribution of DPMPB-
FONs (5 µM) in an aqueous solution. (d) SEM image of DPMPB-
FONs.
allow the flow of electrons from imidazole unit to pyridinium
segment, resulting in a large ICT effect in DPMPB. After that,
we investigated the AIE properties of DPMPB. As shown in
Figure 1a, in dilute water solution, DPMPB showed a faint
green fluorescence. In contrast, upon increasing the THF
fraction to 90 Vol%, the emission of DPMPB increased.
Furthermore, when the fraction of THF up to 100% a strong
emission peak at 495 nm was detected and bright green
fluorescence was observed under 365 nm UV illumination from
a hand-held UV lamp (Figure 1b inset). The high THF fraction
induces a dramatic increase in fluorescence, which confirms
the AIE property of DPMPB. In order to prove the high
emission in high THF portion solution was attributed to AIE
It is reported that the difference in mitochondrial
membrane potential between normal epithelial cells and
carcinoma cells is at least 60 mV. In addition to mitochondrial
membrane potential, plasma membrane potential is also
[21]
higher in carcinoma cells than in normal epithelial cells.
Thus, due to the stronger electrostatic interaction and charge-
mediated endocytosis, we hypothesized that DPMPB-FONs will
be more internalized and accumulated in cancer cells than
normal cells. To verify this hypothesis, nasopharyngeal
carcinoma cells C666-1, CNE2, adenocarcinoma gastric cells
AGS and normal gastric mucosa cells GES-1 were incubated
with DPMPB-FONs under the same conditions. As shown in
Figure S5, strong greenish fluorescence was observed in
cancerous cells (C666-1, CNE2 and AGS) after incubation of 30
min. In sharp contrast, the faint fluorescence was observed in
effect. By increasing the concentration of DPMPB from 5 μM
to 90 μM, the fluorescence intensity increased, suggesting that
DPMPB started to form aggregates (Figure S3). Such emission
is possibly attributed to the aggregation of DPMPB and
activates the RIR process. The quantum yield (QY) of DPMPB in
pure THF was 14.2%. Compared to the quenched in the solid
state of traditional fluorescent dyes, DPMPB solid powder
emits the stronger green light under the excitation of
ultraviolet light
(Figure 1a inset). The excellent optical
property makes DPMPB suitable for fluorescence imaging
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GES-1 cells (Figure S5d). In addition, after incubation of 22 and
29 hours cancer cells (C666-1, CNE2 and AGS) still exhibited
strong fluorescence(Figure S6), and the results showed that
the DPMPB-FONs could be used for long-term fluorescence
imaging of cancer cells. The location and distribution of
DPMPB-FONs in living cells was further explored by confocal
scanning laser microscopy. Four kinds of cells, including three
cancer cells (C666-1, CNE2 and AGS) and one normal cell (GES-
1), were employed as a cell model and were co-stained with
DPMPB-FONs and DAPI for 4 h. As shown in Figure 2, in all
three kinds of cancer cells, the green fluorescence from
DPMPB-FONs are overlaps well with the blue fluorescence of
DAPI. In contrast, no greenish emission of DPMPB-FONs was
observed in living cells of GES-1 (Figure S7).These results
indicated that DPMPB-FONs not only can distinguish between
cancer cells and normal cells, but also specifically target the Figure 2. Confocal laser fluorescence microscopic images of
nucleolus of cancer cells. Nucleuses, the most prominent C666-1, CNE2 and AGS cells treated with 5 μM DPMPB-FONs
cellular organelle, are closely associated with diseased and DAPI (1.0 μM), respectively. a), e) and i) Fluorescence
phenotypes, [22] and many of anticancer drugs are target to the imaging of DPMPB-FONs in C666-1, CNE2 and AGS cells,
nucleus. Thus, fluorescent probes targeted to nucleus for a respectively collected at 480-530 nm and excited at 400 nm. b),
long period time are very helpful to acquire crucial diagnostic f) and j) Fluorescence image of DAPI in C666-1, CNE2 and AGS
and prognostic information.[23] In our study, the long-term cells, respectively collected by a 450-470 nm band path filter
tracking nucleolus ability of DPMPB-FONs was also with excitation at 358 nm. c), g) and k) Merged image of C666-
determined. As seen from Figure S8, we can see that the green 1, CNE2 and AGS cells, respectively. d) , h) and l) Correlation
fluorescence was collected from the nucleus region and plot of DAPI and DPMPB-FONs intensities. Scale bar: 7.5 μm.
overlap well with the blue fluorescence of DAPI until
Surgical operation is the primary treatment modality for
incubation of 112 hrs. Furthermore, in such a long incubation
most solid tumours. Traditionally, tumour removal during
time, the shape of the nucleus did not change significantly.
surgery relies on the experience of the surgeons to
These results further confirmed that DPMPB-FONs show low
differentiate tumour from normal tissue, which is not easily
cytotoxicity and can be effectively internalized into cancer cells
quantifiable and do not offer the sensitivity to identify the
and have the ability to track cancer nucleoli for a long time. To
[25]
tumour margins.
Hence, the way to objectively assess
investigate the light-up response of DPMPB-FONs in cell
nucleus, fluorescence spectra were explored when DNA was
added into the DPMPB-FONs in PBS buffer solution (pH = 7.4).
As seen from Figure S9a, the fluorescence intensity of DPMPB-
FONs rises significantly with increasing amount of DNA and
reaches a maximum at 8 µg/mL. The observed fluorescence
enhancement may owe to the electrostatic interactions
between the oppositely charged DPMPB-FONs and DNA,
resulting in blocking the free rotation of DPMPB molecules. [24]
Moreover, the fluorescence intensity at 500 nm and DNA
tumour margins during surgery would be of great value.
Optical image-guided surgery was proven to be significant
potential to objectively assess the tumour margin and guide
the surgeon to adequately resect the tumour.[26] The success
of illuminating cancer cells inspired us to further use our probe
in cancer tissue and normal tissue. The breast cancer tissue
and para-carcinoma tissue were removed from a 44-year-old
patient with breast cancer. As shown in Figure S11, the
fluorescence of para-carcinoma tissue is obviously weaker
than the cancer tissues when observed by using fluorescence
microscope. The results indicated that DPMPB-FONs were
capable of specific staining of the cancer tissue with high
contrast. It is noted that, the short excitation wavelengths
have shallow tissue penetration depths and their application is
limited. In comparison with one-photon imaging, two-photon
imaging can provide better three-dimensional spatial
localization, higher imaging resolution, and increased
penetration depth.[27] We then further investigated the utility
of DPMPB-FONs in deep breast cancer tissue and para-
carcinoma tissue. The fluorescence images were obtained by
two-photon confocal microscopy, and the images of breast
cancer and para-carcinoma tissues stained with DPMPB-FONs
were recorded at green emission channels. The breast cancer
and para-carcinoma tissues stained with DAPI were collected
at blue emission channels. As shown in Figure 3a, 3D
concentrations exhibited a good linearity relationship (R2
=
0.981) in the range of 0 to 8 µg/mL (Figure S9b). This result
demonstrated that the DPMPB-FONs can sensitively detect
DNA in water solution. The cytotoxicity of DPMPB-FONs on
GES-1 cells was also evaluated by CCK-8 assay. As shown in
Figure S10, the cell viability was tested after the incubation
with various concentrations of DPMPB-FONs (0-20 µM) for 48
h. The cell viability remains above 62% high at DPMPB-FONs
concentrations as high as 20 µM, indicating low cytotoxicity of
DPMPB-FONs in the test and suitable for the application in the
long-term tracing.
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