Redox supramolecular self-assemblies nonlinearly enhance fluorescence to identify cancer cells

Article abstract of DOI:10.1039/c8cc02648c

Based on the nonlinear fluorescence enhancement, our H₂O₂ induced supramolecular self-assembly reveals a H₂O₂ threshold among multiple cancer and normal cells. Oxidative elimination restores an intramolecular hy

Full text of DOI:10.1039/c8cc02648c

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DOI: 10.1039/C8CC02648C
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Redox Supramolecular Self-Assemblies Nonlinearly Enhance
Fluorescence to Identify Cancer Cells
Zhentao Huang,1 Qingxin Yao,1 Jiali Chen1 and Yuan Gao1,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Based on the nonlinear fluorescence enhancement, our H2O2
induced supramolecular self-assembly reveals a H2O2 threshold
among multiple cancer and normal cells. Oxidative elimination
restores an intramolecular hydrogen bond which can planarize the
molecule to generate a fluorophore. The planarization enhances
the intermolecular - stacking to promote self-assembly.
Reactive oxygen species (ROS), mainly produced in
mitochondria, are the normal products in oxygen
metabolism.1,2 ROS studies have revealed its dual biological
properties: as signaling agents and as harmful molecules.3-5 At
the basal level with the strict regulation by redox homeostasis,
ROS participate in a wide range of biological processes.6
However, the dysfunction in ROS regulation, usually caused by
excessive and sustained inflammation, are suspectable of the
generation and exacerbation of a range of diseases including
epilepsy,7 Alzheimer's8 and cancer.9,10 In such a disease state,
one common feature is the ROS over-production to exceed
certain threshold.11,12 Therefore, a serials of molecular ROS
probes with various modalities including optical,13,14 13C MRI,15
and PET16 have been developed to observe the fluctuation of
ROS.17 For example, a typical hydrogen peroxide (the major
ROS) fluorescent probe restores its fluorescence signal after
removing the aryl boronate caging group via H2O2 mediated
oxidation.18 The swift reaction allows the real time read-out of
the elevated H2O2 level.19,20 It is worthy to mention that the
stoichiometric reaction gives the signal increase proportional to
the H2O2 (or RNS) concentration. As the detection limit is always
a holy grail for H2O2 probes, continuous efforts focus on the
amplification of the H2O2 sensitivity, e.g. by degrading polymer
backbone via single oxidation.21
Scheme 1. The illustration of ROS induced intracellular supramolecular self-assembly
in cancer cells.
Nevertheless, since ROS intrinsically exists in all living bio-
entities at a basal level, the ROS threshold is the real marker in
the pathophysiologic diagnosis. Then, pushing the detection
limit of ROS to an extremely low concentration is less relevant
to the concern in healthcare. Instead, the capability to report
the ROS threshold is critical for the judgment of normal or
pathologic regimes, which has been seldom explored. A recent
study has utilized a post-assembled nanoprobe with AIE
luminogens to report extracellular RNS (ONOO−) at the
inflammation detection window with a 20-fold enhancement in
fluorescence.22 However, the reason for the “silence” before
the onset ONOO− concentration remains unclear. Here we
developed a redox self-assembly system as a nonlinear reporter
for H2O2 (the major ROS) with a 260 times enhancement in
fluorescence emission. Our molecular design has enabled
fluorescent silence at basal H2O2 level but a burst of increase in
fluorescence intensity upon certain threshold, which is
dependent on the critical assembly concentration (CAC). In
addition, this redox self-assembly is applicable to profile the
native intracellular redox status, thus precisely distinguish
malignant cells from normal cells which is of urgent need to
reveal the heterogeneity in cancer diagnosis.
1CAS Center for Excellence in Nanoscience, CAS Key Laboratory of Biomedical
Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and
Technology Beijing 100190, China
E-mail: gaoy@nanoctr.cn
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C8CC02648C
Figure 1. (A) The synthesis of BQA-GGFF and the generation of BQH-GGFF upon
oxidation by H2O2. Comparison of (B) 1H NMR and (C) UV spectra of BQA-GGFF (black)
and the corresponding oxidation products (red) after addition of H2O2.
Following the bio-inspiration strategy, such as enzymatic self-
assembly,23-36 we designed the redox supramolecular assembly
(ROSA) using H2O2 as the trigger to conduct the oxidation
reaction and promote the intermolecular
- stacking. The
synthesized assembling molecule precursor composed of a
H2O2 activatable fluorogenic BQA and a tetra peptide GGFF.
BQA was a quinazolinone derivative37-39 capped with a typical
aryl boronate immolative linker which readily restored the
critical phenyl to form intramolecular hydrogen bond upon
oxidation. The formation of that intramolecular hydrogen bond
was strictly correlated the generation of the fluorophore BQH
which made the molecule planar. The planarization facilitated
Figure 2. (A) H2O2 mediated oxidation and the formation of the fluorophore upon
self-assembly. (B) The determination of the critical assembly and hydrogelation
concentration of BQH-GGFF in water at pH 7.4. TEM image of the assemblies of BQH-
GGFF at the concentrations of (C) 2 mM and (D) 10 mM. (E) The fluorescence
development (emission at 490 nm) during the titration of H2O2 (0.01-3 eq.) into 10
mM of BQA-GGFF. (F) The fluorescence intensities (emission at 490 nm) of a series
of concentrations (0.1-10 mM) of BQA-GGFF reacted to 3 eq. of H2O2.
the intermolecular -
stacking to promote self-assembly.40,41
Interestingly, in conjugation with GGFF, the formation of
fluorophore BQH was not straightforward but dependent on
the CAC. In soluble state (below CAC) the formation of
fluorophore BQH was suppressed which implied the disrupted
intramolecular hydrogen bond. While above CAC, the self-
assembly process made the formation of the fluorophore
(indication of the hydrogen bond) more favorable than its non-
fluorescent form in their equilibrium. Therefore, we observed
the simultaneous self-assembly and fluorescence turn-on.
Although the oxidation reaction was still stoichiometric and
linear, the synergistic self-assembly and fluorophore formation
resulted in the nonlinear fluorescence enhancement (Scheme
1). Further incubation of BQA-GGFF with various mammalian
cells revealed the selective formation of highly fluorescent
assemblies inside malignant cells (Hep G2, MCF-7, PANC-1 and
HeLa) instead of corresponding normal cells (L-O2, MCF-10A
and HUVEC). Inductively, our study demonstrated a redox self-
assembly strategy to construct intracellular fluorescent
assemblies which was dependent on oxygen metabolism level.
As the CAC can be easily modulated by the short peptide
sequence, the strategy is expected to be applicable in other
diseases with ROS threshold feature. In addition, without usual
targeting moiety such as triphenylphosphine,42 the assemblies
could also target mitochondria which is likely due to the
reaction (oxidation) based kinetic control.43
exchange reaction yielded the product of BQA-peptide
conjugate after HPLC purification. (Figure 1A) Then we tested
the hydrogelation capability of BQA-peptides upon oxidation by
H2O2. After screening a series of short peptides including FF, FG,
GF, GG, GFF, FFG, GGFF, BQA-GGFF could perform the solution-
hydrogel transition after oxidation under physiological pH and
will be investigated in further studies. (Table S1)
The oxidation of arylboronic acid on BQA-GGFF initiated a 1,
6-elimination to unmask the phenol group and convert BQA to
BQH. After the addition of H2O2 into the solution of BQA-GGFF,
two characteristic peaks of hydrogens (labelled 1 and 2) shifted
from 5.29 ppm and 7.21 ppm to 5.14 ppm and 6.72 ppm (1’ and
2’), respectively. (Figure 1B and Figure S1) Further in the UV
spectra, the BQA-GGFF showed a characteristic peak at 304 nm
while after oxidation the BQH-GGFF gave a red-shifted
absorption peak at 356 nm. (Figure 1C) Due to the molecular
structure change, the retention time of BQA-GGFF also differed
from that of BQH-GGFF in HPLC traces. (Figure S2)
The self-assembly and hydrogelation depended on both CAC
and the intramolecular hydrogen bond. The intramolecular
hydrogen bond would planarize the whole molecule, which
promotes the intermolecular interaction and assembly. The
generation of BQH with phenol group provided the possibility
We started the study with the synthesis of the BQA moiety
based on the classic synthesis of quinazolinone derivative
(Scheme S1).44 A sequential NHS activation and ester-amide
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for the formation of intramolecular hydrogen bond but it only
occurred when the concentration exceeded the CAC with the
indication of fluorescence turn-on. (Figure 2A) As shown in
Figure 2B, the solution of BQA-GGFF at the concentration as
high as 10 mM remained non-fluorescent. When adding 3 eq. of
H2O2 into the solution of BQA-GGFF at different concentrations,
the fluorescence significantly increased. Specifically, the
solution remained non-fluorescent at the concentration below
1 mM but a brightly fluorescent hydrogel formed at the
concentration of 10 mM with stable fluorescence emission
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DOI: 10.1039/C8CC02648C
Figure 4. BQA-GGFF distinguishes cancer cells from normal cells. Fluorescent images
of various cells incubated with 500 M of BQA-GGFF. Scale bar: 20 m.
against variant temperature or pH. (Figure S3-4)
A
concentration below 1 mM, BQH-GGFF was completely soluble
in water without the formation of intramolecular hydrogen
bond and thus remained non-fluorescent. Once the
concentration was above the CAC, the self-assembly would
occur with the formation of intramolecular hydrogen bond,
resulting in the simultaneous fluorescence turn-on. (Figure S10)
After correlating fluorescence and self-assembly in vitro, we
further verified if it can serve to nonlinearly detect ROS in
complex biological environment such as in cancer cells.45 In
screening the concentration and incubation time, we found that
concentration in-between (e.g. 3.7 mM) gave a fluorescent but
weak hydrogel. To verify the micro morphology of the
nanostructures, TEM images revealed the existence of
abundant nanofibers in both weak and stable hydrogel samples,
with the diameter at 11.80.5 nm and 9.50.6 nm, respectively.
(Figure 2C-D) While in the 1 mM solution sample, we noticed
the absence of any significant nanostructure. (Figure S5) A
further rheological test confirmed the formation of a stable
hydrogel. The storage modulus was around 3 kilopascal and
both the storage and loss modules were frequency
independent. (Figure S6-7) We determined the CAC by titrating
H2O2 into the solution of 10 mM of BQA-GGFF. With the
excitation at 406nm, the solution emitted at 490nm and the
intensity rose along the addition of H2O2. (Figure S8)
Interestingly, the plot of the emission intensity vs. the amount
of H2O2 was obviously nonlinear, showing a burst increase
between 0.1-0.2 eq. and a plateau with 1 eq. of H2O2, reaching
a 260-times enhancement in emission. The burst increase
indicated the CAC between 1-2 mM for BQH-GGFF. (Figure 2E)
Alternatively, the trend of the emission intensities of a series of
BQA-GGFF solutions at concentrations ranging from 0.1 to 10
mM after the addition of 3 eq. of H2O2. (Figure S9) The plot of
intensity vs. BQA-GGFF concentration further indicated the CAC
around 1.2 mM, which was consistent with previous
determination. (Figure 2F) Such a nonlinear enhancement was
attributed to the occurrence of self-assembly. At low
the 500 M BQA-GGFF was non-toxic (Figure S11) and sufficient
to form fluorescent assemblies inside HeLa cells. (Figure 3A &
S12) The optimized incubation time was 8 h since the brightness
faded thereafter. (Figure S13) In contrast, fluorescent
assemblies were absent in HeLa cells incubated with BQA-GG
which cannot self-assemble at physiological condition. (Table S1
& Figure S14) As the over-production of ROS was due to the
dysfunction of mitochondria,9 we expect that the formation of
fluorescent assemblies are related to mitochondria. The co-
staining showed the strong overlap (92.6%) between BQH-GGFF
(green) and MitoTracker (red). (Figure 3C) Based on a standard
fractionation procedure,46 in the mito fraction we could observe
the nanofibers with the diameter of 9.10.9nm which was
similar to in vitro one, while the nanofibers was absent in
HUVEC cells. (Figure S15)
Further, we verified that the ROS-induced supramolecular
self-assembly will generally form in a broad range of cancer cells
instead of normal cells. (Figure 4) For the group of malignant
cancer cells including Hep G2, MCF-7, and PANC-1, there were
significant amount of fluorescent assemblies formed inside
cells. The corresponding normal cells such as L-O2, MCF-10A,
and HUVECs cells were largely dark under confocal microscope.
We interpreted that the formation of fluorescent assemblies
related to the ROS level which was further verified via the ROS
attenuation. For a group of HeLa cells pre-treated with 5 mM of
ROS inhibitor N-Acetyl-cysteine (NAC), no fluorescent
assemblies formed. In contrast, the 3 M of ROS inducer 4-
hydroxyphenylretinamide (HPR) treated HUVECs acquired
intracellular fluorescent assemblies as many as other malignant
cancer cells. A further statistical analysis of the fluorescence
intensity in each individual cell by image J showed the
significant difference between “malignant” and “normal” cells.
The intensities for all malignant cells were above 4 while below
4 for all normal cells. (Figure S16) Therefore, the fluorescence
intensity could be an empirical reference for the judgment of
the malignance of individual cell.
Figure 3. The fluorescent images of a group of HeLa cells incubated with 500 M
BQA-GGFF and co-stained with Mito tracker. (A) The green channel of BQH-GGFF,
(B) The red channel of Mito tracker and (C) The merged channels. (D) A typical TEM
image showed the existence of nanofibrils in the mitochondria fraction among
cellular fractions.
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21 C. D. Lux, S. Joshi-Barr, T. Nguyen, E. Mahmoud, E. Schopf, N.
Fomina and A. Almutairi, J. Am. Chem. Soc., 201V2i,ew Article Online
134
In conclusion, we have demonstrated a redox supramolecular
self-assembly triggered by H2O2. By correlating the occurrence
of supramolecular self-assembly with the nonlinear
fluorescence enhancement, the CAC reflects the threshold of
H2O2. As this redox supramolecular self-assembly could
selectively construct assemblies inside multiple malignant cell
lines, this work reveals that the threshold, rather than an
absolutely low level of ROS is critical in distinguishing cancer
cells from normal ones. In addition to cancer, over-produced
ROS also behave critical roles in pathological conditions such as
inflammation and necrosis. We would expect that the redox
supramolecular self-assembly will be applicable for a vast range
of theranostic purposes with proper molecular design.
,
DOI: 10.1039/C8CC02648C
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