Using Congo red to report intracellular hydrogelation resulted from self-assembly of small molecules

Article abstract of DOI:10.1039/b712971h

This communication reports the use of Congo red to stain the nanofibers of self-assembled small molecules for assaying intracellular supramolecular hydrogels, which provides a convenient method to explore molecular self-assembly inside cells. The Royal So

Full text of DOI:10.1039/b712971h

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COMMUNICATION
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Using Congo red to report intracellular hydrogelation resulted from
self-assembly of small molecules{
Gaolin Liang,abc Keming Xu,b Lihua Li,a Ling Wang,a Yi Kuang,ab Zhimou Yanga and Bing Xu*abc
Received (in Cambridge, UK) 22nd August 2007, Accepted 12th September 2007
First published as an Advance Article on the web 19th September 2007
DOI: 10.1039/b712971h
This communication reports the use of Congo red to stain the
nanofibers of self-assembled small molecules for assaying
intracellular supramolecular hydrogels, which provides
a
convenient method to explore molecular self-assembly inside
cells.
Supramolecular hydrogels,1,2 formed by hydrogelators self-
assembling into three-dimensional networks of nanofibers to trap
water, have shown promise for biological applications such as drug
delivery,3 biosensing,4 tissue engineering,5 and wound healing.6
Though extracellular formation and applications of supramole-
cular hydrogels have achieved considerable development, intracel-
lular hydrogelation to create artificial nanostructures and their
potential applications are just beginning to be explored as a
nascent direction in supramolecular hydrogel research. Recently,
we have demonstrated that small molecular hydrogelators, after
being produced inside cells, could form nanofibers intracellularly
and result in hydrogelation to induce cell death or to inhibit cell
growth.7 To confirm these nanofibers of hydrogels had formed
intracellularly, we used transmission electron microscopy (TEM),
which required laborious sample preparation and sophisticated
instrumentation. Therefore, a quick, convenient assay would
greatly help the research on intracellular hydrogelation by
eliminating the need for expensive instruments. Here we report
the use of Congo red (1) to stain the nanofibers of intracellular
supramolecular hydrogels inside two types of mammalian cell and
one strain of bacterium. After being stained by Congo red, the
intracellular hydrogel resulting from the self-assembly of Nap–
Phe–Phe motif retains the Congo red to exhibit red fluorescence as
the specific signal (Scheme 1). This Congo red assay, as an effective
and easy method for determining intracellular hydrogelation,
should also assist the exploration of the intracellular self-assembly
of small molecules.
Scheme 1 Illustration of Congo red to stain nanofibers.
b-sheets9 that can bind to Congo red.10 Among many kinds of
supramolecular hydrogels developed, a considerable proportion of
them are peptide derivatives.11,12 Similar to amyloid fibers, these
peptide-based supramolecular hydrogelators also self-assemble
into b-sheet-like nanostructures.13 Because Congo red has good
cell permeability and inhibits the formation of b-amyloid fibrils
inside cells,14 Congo red might be able to selectively stain this type
of nanofiber formed in supramolecular hydrogelation. Based on
this assumption, we chose to use Congo red to stain these
hydrogels for identifying intracellular hydrogelation. Scheme 2
gives the molecular structure of the sulfonated azo dye, Congo red
(1). Like its binding to amyloid fibers, the sulfonate groups of 1
interact with the hydrophilic part of the assembly of hydrogelators,
while the hydrophobic core of 1 binds to the hydrophobic part of
the nanofibers in the hydrogel. Being excited by a green light (510–
560 nm), 1 gives a red fluorescent emission peak at 616 nm that is
suitable for imaging the process inside cells (Fig. S1{).
We used Nap–D-Phe–D-Phe–tyrosine phosphate (2) and Nap–
Phe–Phe–NH(CH2)2OCO(CH2)2COOH (4) as the precursors for
Several diseases, including Alzheimer’s disease, spongiform
encephalopathies, familial amyloid polyneuropathy, and type II
diabetes, are associated with proteins that undergo abnormal
conformational changes and polymerize into amyloid fibers.8 A
distinctive feature of amyloid fibers is the predominant presence of
aDepartment of Chemistry, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China.
E-mail: chbingxu@ust.hk; Fax: +852 2358 1594; Tel: +852 2358 7351
bBioengineering Program, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China
cCenter for Cancer Research, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China
{ Electronic supplementary information (ESI) available: Synthesis, in vitro
conversions of 2 to 3, 4 to 5, CD spectra of Gel I and Gel II, UV and
fluorescence spectra of 1, MTT assay of 2 and 4. See DOI: 10.1039/
b712971h
Scheme 2 Molecular structures of Congo red (1), the precursors (2, and
4), and the hydrogelators (3, and 5).
4096 | Chem. Commun., 2007, 4096–4098
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enzymatic hydrogelation to test the binding of 1 to the self-
assembled nanofibers for the following reasons. (i) Enzymatic
hydrogelation is an effective route to create self-assembled
nanostructures of small molecules.11 (ii) Nap–Phe–Phe and
amyloid fibers share a common motif, Phe–Phe, that tends to
self-assemble into b-sheet or b-sheet-like structures that bind to
Congo red. (iii) Compounds 2 and 4 are the substrates of two
endogenous enzymes, phosphatase and esterase, respectively. The
transformations of 2 and 4 (the precursors) to 3 and 5 (the
hydrogelators) are representative processes of enzymatic intracel-
lular hydrogelation of small molecules.
colorless after the same washing process (Fig. S4C{). A confocal
laser scanning micrograph showed the fiber-like features in the
Congo red stained Gel I (Fig. 2B), confirming that Congo red was
able to bind the nanofiber in the hydrogel.
After validating Congo red as the dye for Nap–Phe–Phe based
supramolecular hydrogel specifically, we tested the staining of self-
assembled nanofibers of hydrogels inside cells. We used HeLa cells
to investigate the intracellular gelation of 2 because HeLa cells
could over-express phosphatase.15 Presumably, the over-expressed
phosphatase can dephosphorylate the up-taken hydrogel precursor
(2) to accumulate enough hydrophobic hydrogelator (3), thus
leading to self-assembled nanofibers and forming the hydrogel
intracellularly. As shown in Fig. 3, Congo red cannot stain the cells
when HeLa cells were treated with 2 for 24 h and at a
concentration (250 mM) below the IC50 (410 mM) (Fig. 3E), likely
due to there being too few hydrogelators to induce intracellular
hydrogelation to result in cell death or inhibit cell growth. When
the concentration of 2 (1 mM) exceeded the IC50, the Congo red-
stained HeLa cells showed strong red fluorescence (Fig. 3F).
Apparently, most of the red fluorescence localized at the region of
the nucleus (Fig. S6{), suggesting that the nanofibers might form
around the nucleus. Similar to the staining of HeLa cells treated
with 2, Congo red cannot stain the HepG2 cells treated with 4 for
24 h and at a concentration (160 mM) below the IC50 (341 mM)
(Fig. 3K). When the concentration of 4 (640 mM) exceeded the
IC50, the Congo red-stained HepG2 cells also showed strong red
fluorescence (Fig. 3L).
We first tested the binding of Congo red to the self-assembled
nanofiber of the hydrogel extracellularly. After the addition of
alkaline phosphatase to the PBS buffer solution containing 2
(0.6 wt%), a transparent hydrogel forms within minutes (Fig. 1A,
inset). A TEM image of the cryo-dried Gel I shows that the
nanofibers in Gel I have a length of over 10 mm, a width of about
10–20 nm, and an average pore size of the network of about
100 nm. Similarly, the addition of esterase to the solution
containing 4 (0.5 wt%) at 37 uC results in Gel II, whose nanofibers
have a length of over 10 mm, a width of about 10–20 nm, and an
average pore size of the network of about 200 nm (Fig. S2{). The
diameters of the nanofibers in Gel I and Gel II are within the
same range as those of b-amyloid fibers formed in vitro and in vivo
(5–17 nm) that can be stained by Congo red.10 After the Congo red
stock was added to Gel I at a calculated final concentration of
10 mM, the transparent colorless Gel I was slowly stained by
Congo red solution and turned a bright red color (Fig. 1B, inset).
TEM revealed that there were black particles adhering to the
nanofibers of Gel I after Congo red staining (Fig. 1B). A high
resolution TEM image of Congo red stained Gel I and its EDX
analysis revealed that the dark particles attached to the nanofibers
and dispersed in the hydrogel matrix were Congo red (because
they contain sulfur element) (Fig. S3{). In addition, circular
dichroism (CD) spectra of the hydrogels showed a positive peak at
195 nm and a negative peak center at 225 nm (exciton splitting of
the peptide p–p* transition) for Gel I and a positive peak at 198 nm
and a negative peak at 214 nm for Gel II, indicating that most of 3
and 5 took a b-sheet-like arrangement in the gel phase (Fig. S1{).
To prove the specificity of the Congo red staining, we used a
polymeric hydrogel made of cross-linked polyacrylamide as the
control. Without Congo red staining, both the supramolecular
hydrogels and the polyacrylamide hydrogel were colorless. After
Congo red staining, a transparent hydrogel with bright red color
was obtained (Fig. S4B{). The Congo red remained in the
supramolecular hydrogel after being washed with PBS buffer three
times. On the other hand, the polyacrylamide hydrogel returned to
We also used 1 to stain the cells that were treated with 1% of
Triton X-100 as the necrosis model. Both the necrotic models of
HeLa cells and HepG2 cells showed no red fluorescence after the
staining of Congo red (Fig. 3D and 3J). This result excludes the
possibility that necrosis of cells causes the staining of Congo red
and confirms that the strong red fluorescence in Fig. 3F and 3L is
the result of the staining of nanofibers in the supramolecular
hydrogels by Congo red.
After confirming that Congo red selectively assays intracellular
hydrogelation in mammalian cell lines, we also used Congo red to
stain phosphatase overexpressed E. coli after they were incubated
with 2 for 24 h. Congo red could only stain phosphatase
overexpressed E. coli that were incubated with 2 (Fig. 3R),
agreeing with the formation of the hydrogel of 3. Because of the
short life cycle, some of the E. coli had not built up enough
concentration of 3 to form an intracellular hydrogel, and they
show little fluorescence as the result of the staining of Congo red.
Nevertheless, the observed fluorescence clearly suggests that
Fig. 1 TEM images of (A) Gel I (inset: optical image) formed by 3 via
enzymatic gelation of 2 in PBS buffer (conc. 5 0.6 wt%, pH 5 7.4); and
(B) Congo red stained Gel I (inset: optical image).
Fig. 2 (A) Phase contrast micrograph of Congo red stained Gel I; and
(B) confocal laser scanning micrograph of Congo red stained Gel I.
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Chem. Commun., 2007, 4096–4098 | 4097

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fluorescence.2 To improve the specificity of this type of assay,
other chromophores or fluorophores that show definitive features
of aggregation16 or molecular imaging agents17 are worthy to be
explored.
BX acknowledges the financial support from RGC (Hong
Kong), and HIA (HKUST). GLL thanks Mr Wei Zhang for
helping with confocal imaging.
Notes and references
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Fig. 3 (A, B, C) Optical images (objective: 206) of HeLa cells stained by
Congo red: (A) Triton X-100 treated for 1 min before staining; (B) 250 mM
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images of (A, B, C). (G, H, I) Optical images (objective: 206) of HepG2
cells stained by Congo red: (G) Triton X-100 treated for 1 min before
staining; (H) 160 mM and (I) 640 mM of 4 treated for 24 h; (D, E, F)
corresponding fluorescence images of (G, H, I). (M, N, O) Optical images
(objective: 1006) of phosphatase overexpressed Escherichia coli stained by
Congo red: (M) Control; (N) 1.73 mM and (O) 3.46 mM of 2 treated for
24 h at 18 uC; (P, Q, R) corresponding fluorescence images of (M, N, O).
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In summary, we have successfully demonstrated that Congo red
serves as an assay to stain the peptide-based supramolecular
hydrogel extra- and intracellularly. Although only two types of
hydrogelator, two cancer cell lines and one strain of bacterium
were investigated in this work, it is conceivable that Congo red can
stain other supramolecular hydrogels intracellularly because
Hamachi et al. have shown the amphiphilic nanofibers in
supramolecular hydrogels can dramatically enhance the
4098 | Chem. Commun., 2007, 4096–4098
This journal is ß The Royal Society of Chemistry 2007