Galactose-decorated light-responsive hydrogelator precursors for selectively killing cancer cells

Article abstract of DOI:10.1039/c6cc05707a

A multi-functional gelator precursor with high photosensitivity is rationally designed, which can not only selectively target hepatocellular carcinoma (HCC) cells, but also form intracellular self-assembly triggered by light and subsequently inducing cell

Full text of DOI:10.1039/c6cc05707a

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Galactose-decorated light-responsive hydrogelator precursor for
selectively killing cancer cells
Wei Ji,a Guofeng Liu,a Fang Wang,a Zhu Zhu,b Chuanliang Feng,*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A multi-functional gelator precursor with high photosensitivity is
rationally designed, which can not only selectively target
hepatocellular carcinoma (HCC) cells, but also form intracellular
self-assembly triggered by light and subsequently inducing cell
death. The study develops a methodology to design biologic
targets of supramolecular self-assembly under external stimuli for
cancer research.
Intracellular self-assembly of small molecules is a hot topic for
its promising biomedical applications in tumor diagnostics1-2
and therapeutics.3 Recently, enzyme-responsive peptide-based
precursors have been frequently reported since they could
penetrate cell membranes and self-assemble into nanofibers
inside living cells,4-8 which could affect cellular functions and
further induce cell apoptosis due to the promiscuous
interactions between nanofibers and cytoskeletal proteins.9
However, the release of gelators from their precursors by
enzyme (an internal stimulus) is still difficult to be switched on
and off at will.10 Furthermore, these gelator precursors usually
passively attack cancer cells and could induce inefficient tumor
cell uptake. Whereas, this may be circumvented by grafting
specifically recognized ligands onto precursors, which can
allow the active target to cancer cells through the specific
interaction between ligands and receptors (over-expressed by
cancer cells).11 Thus, it is very necessary to develop an
innovative designing methodology to explore functional
gelator precursors that can not oly selectively target cancer
cells, but also precisely release gelators under external stimuli,
which is still a question remained to be answered so far.
Scheme 1. a) Molecular structure of precursor Gal-BHMC and its
conversion to gelator BHMC upon UV light irradiation. b) Schematic
demonstration of specific targeting of Gal-BHMC onto cancer cells and cell
death induced by intracellular self-assembly triggered by light.
Herein, a multi-functional gelator precursor (Gal-BHMC)
with high photosensitivity is rationally designed (Scheme 1a),
which can not only selectively target hepatocellular carcinoma
(HCC) cells, but also release gelators under light irradiation,
leading to the intracellular supramolecular self-assembly and
subsequently inducing cell death. Gal-BHMC contains three
a.State Key Lab of Metal Matrix Composites, School of Materials Science and
Engineering, Shanghai Jiao Tong University 800 Dongchuan Road, 200240,
Shanghai.
different functional groups:
a galactose unit, a photo-
b.Department of Nephrology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong
University 227 South Chongqing Road, 200011, Shanghai.
E-mail: clfeng@sjtu.edu.cn
responsive carbamate bond of (coumarin-4-yl)-methyl
derivative,12 and a fluorescent gelator (BHMC). The galactose
ensures Gal-BHMC to selectively bind with asialoglycoprotein
Electronic Supplementary Information (ESI) available: X-ray crystallographic data, receptor (ASGP-R) over-expressed on HCC cells membranes
because of their high affinity and rapid internalization rate,13-14
experimental procedures, supplemental figures, synthesis and characterization of
all new compounds. See DOI: 10.1039/x0xx00000x
which could be examined by the fluorescent emission of Gal-
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BHMC inside cells under light excitation. The carbamate bond Especially, the blue emission of hydrogel could be visualized
can be efficiently cleaved to precisely release BHMC gelators under UV light due to fluorescent property of coumarin
by UV light, since light offers the merits of relatively high derivative (Inverted vial in Figure 1b).16 The entangled
precision spatial, temporal, and dose control by varying nanofibrous structures with blue emission were further
irradiation parameters, such as wavelength, intensity, and observed by fluorescence optical microscopy (Figure 1b). In
time.16a After cleavage, BHMC can self-assemble into addition, the critical gelation concentration (CGC) and critical
nanofibers inside the cells and the nanofibers exhibit critical aggregation concentration (CAC) of BHMC are 8 mM and 120
damage on cellular function and in turn induce HCC cell death μM, respectively (Figure S23 and S24).
(Scheme 1b). The study develops a methodology to design
To investigate the self-assembly mechanism of BHMC, the
muti-functional nano-materials for the purpose of targeted colourless single crystal of BHMC was prepared by slow solvent
and efficient cancer therapy by releasing the assembled evaporation method from n-hexane/tetra-hydrofuran solution
gelators under external stimuli. Herein, UV light will be firstly and characterized by single crystal X-ray diffraction (SC-XRD).17
explored for confirming the feasibility of the methodology Detailed crystallographic data were summarized in Table S1A,
since it is readily achieved.
S1B, and S1C. BHMC crystallizes in the monoclinic space group
P21/n with two molecules in the asymmetric unit (Z’=2),
containing 8 independent molecules in one unit cell (Z=8)
(Figure S25). The crystal structure and packing diagram of
BHMC suggested that the main driving forces of the self-
assembly were from two types of intermolecular hydrogen
bonds (O-H…O) (Figure 1c). Two O atoms of lactone in
coumarin ring were found to form hydrogen bonds with a
hydrogen of benzyl alcohol (O4-H4A...O2, O4-H4A...O3, O8-
H8A...O6, O8-H8A...O7), which have the bond length of 2.54 Å
(H4A...O2), 2.21
Å Å Å
(H4A...O3), 2.45 (H8A...O6), 2.14
(H8A...O7), respectively. In addition, two intramolecular
hydrogen bonds of C15-H15A...O4 and C32-H32A…O8 could be
also formed to further increase the stability of self-assembly
with the bond length of 2.34 Å (H15A...O4), 2.33 Å (H32A…O8),
respectively. Structure analysis proved that there were no π-π
stacking (Cg-Cg Distances
＞4.5 Å, Table S1D) between the
hydrogelators. Followed by the frequently occurred hydrogen
bonds between BHMC, they could self-assemble into three
dimensional (3D) crystal networks. Moreover, powder X-ray
diffraction (PXRD) was further investigated and it proved that
PXRD patterns were essentially superimposable for both single
crystal and xerogel states of BHMC (Figure S26), implying the
same self-assembly mechanism for two states.18
Fig. 1 a) SEM and b) fluorescent image of nanofibrous structures of freeze
dried hydrogel BHMC, scale bars represent 50 and 100 μm, respectively.
Insets are photographs of BHMC hydrogels before a) and after b) UV
irradiation. c) Packing mode of BHMC stabilized by hydrogen bonds in
single crystal state. (Gray C, black H, red O).
The precursor Gal-BHMC and hydrogelator BHMC were
synthesized in several steps starting from 1,3-benzenediol
(Scheme S1), respectively. The chemical structures of all the
new compounds were confirmed by 1H NMR, 13C NMR, and
HRMS (Figure S1-S21). To prove the self-assembly ability of
BHMC, the powder of BHMC was initially dissolved in dimethyl
sulfoxide (DMSO) and water was then added to trigger the
gelation (Inverted vial in Figure 1a) with final DMSO
concentration of 8% (a well-known method to make
hydrogels).15 To gain insight into the morphologies of hydrogel
BHMC, the freeze dried thin layer of xerogels was studied by
Fig. 2 a) HPLC and b) UV-Vis absorption traces for the photolysis of Gal-
BHMC (2 x 10-5 M) in PBS solution. TEM images of the cryo-dried Gal-BHMC
solution (500 μM) c) before and d) after UV irradiation for 30 s. Scale bar:
100 nm. Insert is the conversion of precursor Gal-BHMC to gelator BHMC
after UV cleavage.
scanning electron microscopy (SEM) and Transmission Electron
Microscopy (TEM). The well-defined nanofibers were observed
with the thickness of ~10-200 nm and the length in the range
of hundreds of micrometers (Figure 1a and Figure S22).
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The cleavage of precursors Gal-BHMC into hydrogelators Hela cells owing to the lack of ASGP-R. These results
BHMC in PBS solution was explored under UV light (Scheme demonstrated that cellular uptake of Gal-BHMC was through
S2). HPLC and UV-Vis absorption were used to assess the ASGP-R-mediated endocytosis in HepG2 cells.19
photolytic rate under mercury lamp irradiation at ~320 nm.
This wavelength ascribes to the characteristic absorption band
of coumarin rings from Gal-BHMC (Figure S27).12b By HPLC, the
photocleavage of Gal-BHMC was precisely dependent on
irradiation time and intensity, the photolytic processes were
effectively progressing with irradiation (Figure 2a and S28).
The photolytic consumption of Gal-BHMC followed a single
exponential decay and the photolytic amount could reach
~90% after 20 s irradiation (100 mW/cm2). In good agreement
with HPLC analysis, UV-Vis absorption intensity of Gal-BHMC
continually decreased at the wavelength of 324 nm with
irradiation time from 0 to 30 s (Figure 2b). Here, there were no
detectable side products during the photolysis for Gal-BHMC,
implying the efficient and controllable cleavage of Gal-
BHMC precursors under UV light irradiation.
After the cleavage of Gal-BHMC into BHMC hydrogelators,
the ability to self-assemble into nanofibers for the cleaved
BHMC was studied by Transmission Electron Microscopy
(TEM). No any nanofibers were detected for Gal-BHMC (500
μM) before UV light irradiation (Figure 2c). While, the formed
nanofibers were observed after irradiating Gal-BHMC for 30 s
by UV light (Figure 2d). The size of the nanofibers was ~10-20
nm and length was hundreds nanometers, suggesting the
successful cleavage of the precursor and self-assembly for the
cleaved BHMC. The stability of Gal-BHMC in PBS solution was
checked in the absence of light by detecting the remaining rate
of the precursor after 24 h incubation at 37 °C (Table S2). The
high remaining rate (97.38%) confirmed that Gal-BHMC was
sufficiently stable from hydrolysis and suitable for cell-related
study in vitro.
Fig. 3 a) Schematic demonstration of receptor-mediated endocytosis in
HepG2 cells. b) Fluorescent, brightfield, and merged images of HepG2 cells
with Gal-BHMC. Scale bar = 50 μm. c) Fluorescent intensity of HepG2 and
HeLa cells pre-treated with free galactose and then incubated with Gal-
BHMC. (d) The viability of HepG2 and HeLa cells detected by CCK-8 assay
under different conditions by Gal-BHMC, BHMC, and galactose,
respectively.
To evaluate the anticancer effects of intracellular self-
assembly triggered by light, the viability of both HepG2 and
HeLa cells was quantified by CCK-8 assay. UV light intensity of
40 mW/cm2 was used in order to minimize the phototoxicity
and the photolytic amount could reach over 70% after 30 s
irradiation (Figure S28). The cells were firstly irradiated for 30 s
and then incubated for 2 h with Gal-BHMC (500 μM), BHMC
(500 μM), and galactose (500 μM), respectively. There was no
significant cell death observed for both HepG2 and HeLa cells
(Figure 3d), which implied that Gal-BHMC, BHMC, and
galactose had no obvious toxicity to the cells. If the cells were
firstly incubated for 2 h with BHMC (500 μM) and then
irradiated for 30 s, which also showed little cytotoxicity for
cells. However, the significant cell death was observed for
HepG2 cells when they were firstly incubated with Gal-BHMC
(500 μM) and then irradiated by UV light. This can be
attributed to the conversion of Gal-BHMC into BHMC inside
cells triggered by UV light and BHMC could further self-
assemble into nanofibers, which may induced cell apoptosis
through the promiscuous interactions between nanofibers and
cytoskeletal proteins.9 As comparison, there was no significant
cell death observed for HeLa cells under the same
experimental condition.
For validating the efficiently targeted cancer cells of Gal-
BHMC (Figure 3a), HepG2 cells (a type of HCC cell lines) with
high ASGP-R expression level and Hela cells (as control) with
little expression of ASGP-R were selected. Due to the specific
interaction between galactose and asialoglycoprotein, the
selectively targeted HepG2 cells and effective endocytosis
inside cells was directly proved by fluorescent images under
UV light because of fluorescent emission property of Gal-
BHMC (Figure 3b). As control, scarce amount of fluorescent
signal was detected for Hela cells due to less expression of
ASGP-R (Figure S29). To further prove whether the endocytosis
process was predominantly mediated by ASGP-R and
galactoside interactions, HepG2 and Hela cells were pre-
incubated with free galactose (a well-known inhibitor of the
receptor) for 15 min by varying concentration from 0 to 200
mM and then incubated with Gal-BHMC (500 μM) for 2 h. Cell
viability experiment suggested that the concentration of free
galactose have little damage to cells (Figure S30). The distinct
decrease of fluorescence intensity was detected for HepG2
cells and the decreased intensity was proportional to the free
galactose concentration (Figure 3c and S31). All these ascribed
to the pre-block of ASGP-R by the free galactose and the
precursors had little chances to bind with ASGP-R. As
expected, the fluorescence intensity didn’t change so much for
To further explore the formation of intracellular self-
assembly of BHMC gelators, dead HepG2 cells were collected
and broken by ultrasonication. TEM image of the cell lysate
revealed the existence of entangled nanofibers (arrows in
Figure 4) with ~10 nm in width and hundreds nanometers in
length. The morphologies were very similar to the formed
nanofibers by the cleaved BHMC in aqueous solution after UV
irradiation (Figure 2d). It should be noted that the cell lysate of
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Fig. 4 TEM image of the lysate of dead HepG2 cells with the self-assembled
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In summary, a fluorescent and multi-functional gelator
precursor with high photosensitivity was rationally designed,
which can selectively target cancer cells through the receptor
mediated interaction between galactose and ASGP-R over-
expressed by cancer cells. Typically, the precursor can release
hydrogelators inside cells under photo-irradiation, leading to
intracellular self-assembly and subsequently inducing cell
death. Especially, coumarin derivatives have the property of
two-photon absorption, enabling to irradiate the precursors
with near infrared light (non-invasive wavelength), which is still
going on in our group. The study develops a methodology to
design multi-functional nano-materials for the purpose of
selective and efficient tumor therapeutics through precisely
releasing the assembled gelators under external stimuli. The
methodology can be further exploited in the design of similar
systems to target different cancer cells by exchanging the
galactose unit into other functional groups, which can
specifically recognize the receptors over-expressed at cell
membrane.
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We thank the NSFC (51573092, 51273111, 51173105), the
National Basic Research Program of China (973 Program
2012CB933803), the Shanghai Jiao Tong University
(SJTU)−University of Michigan (UM) Collaboraꢀve Research
Program, and the Program for Professor of Special
Appointment (Eastern Scholar) at the Shanghai Institutions of
Higher Learning.
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