A glucose-depleting silica nanosystem for increasing reactive oxygen species and scavenging glutathione in cancer therapy

Article abstract of DOI:10.1039/c9cc06043j

Cancer cells adapt to cellular oxidative stress by increasing glutathione (GSH). An organically modified silica nanosystem (ORMOSIL@GOx) was fabricated to achieve a lethal level of oxidative stress in cancer cells by depleting intracellular GSH while incr

Full text of DOI:10.1039/c9cc06043j

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A glucose-depleting silica nanosystem for
increasing reactive oxygen species and
scavenging glutathione in cancer therapy†
Cite this: Chem. Commun., 2019,
55, 13374
Received 3rd August 2019,
Accepted 11th October 2019
Wee Kong Ong, Deblin Jana and Yanli Zhao
*
DOI: 10.1039/c9cc06043j
rsc.li/chemcomm
Cancer cells adapt to cellular oxidative stress by increasing glutathione
(GSH). An organically modified silica nanosystem (ORMOSIL@GOx) was
fabricated to achieve a lethal level of oxidative stress in cancer cells by
depleting intracellular GSH while increasing reactive oxygen species.
The use of reactive oxygen species (ROS) for cancer therapy is
controversial because it has been reported to promote cancer
growth.1,2 On the other hand, more comprehensive studies
have proven that ROS can kill cancer cells by elevating oxidative
stress to a toxic level.3,4 This process can be achieved by increasing
ROS and reducing antioxidants.5 To date, this strategy usually
revolves around the use of polymers and prodrugs.6–8 Nevertheless,
challenges such as synthetic complexity, drug leakage, and
insufficient targeting hindered the progression toward clinical
translation.9 Moreover, most systems require external energy
sources or drug cargos, further limiting their effectiveness.10–12
Silica nanomaterials are endowed with simple synthesis, superior
stability, biocompatibility, and scalability, and accepted as ‘‘generally
recognized as safe’’ by the US FDA.13,14 The abundance of silanol
groups on the surface also facilitates post-modifications for
¨
Scheme 1 Schematic illustration of (a) fabrication of ORMOSIL and
ORMOSIL@GOx and (b) therapeutic mechanism via ROS generation,
ROS-triggered QM release, and glucose depletion. Cell image was taken
using Medira software (2012) on Wikimedia Commons.
additional functions.15 Protocols based on the Stober method (APTES-GOx). Each component introduced is critical in treating
enable precise control of the nanoparticle size.16 This control cancer. The bornate ester-protected quinone methide silane is
allows for enhanced permeation and retention (EPR) effect for the first silica precursor. It is cleaved to release p-quinomethane
passive targeting.17 These properties would compensate for the (QM) in response to hydrogen peroxide.20 The resulting QM
challenges mentioned above. However, the use of organically mod- would in turn react with GSH to form a GSH-QM adduct. This
ified silica (ORMOSIL) nanosystems for simultaneous glutathione process not only depletes the level of GSH, but also recovers the
(GSH) depletion and ROS generation has not been well studied.18
amine group of APTES. Although it is well known that cancer cells
Herein, we report the fabrication of an organically-modified have a higher level of hydrogen peroxide, it is insufficient to
silica nanosystem (ORMOSIL@GOx) in two steps (Scheme 1).19 release QM rapidly.21 As such, it is essential to include a
First, a bornate ester-protected quinone methide silane ligand component that can generate hydrogen peroxide. GOx is an
was co-condensed with tetraethyl orthosilicate (TEOS) in a enzyme that converts intracellular glucose in cancer cells into
templated sol–gel methodology, followed by a post-modification hydrogen peroxide.22,23 The carboxylic group on the enzyme can
with (3-aminopropyl)triethoxysilane-conjugated glucose oxidase react with APTES in the presence of N-hydroxysuccinimide to
form the second silica precursor, APTES-GOx (Scheme S1, ESI†).24
Division of Chemistry and Biological Chemistry, School of Physical and
After the incorporation onto the silica nanoparticle surface,
APTES-GOx not only produces additional hydrogen peroxide,
Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link,
Singapore 637371, Singapore. E-mail: zhaoyanli@ntu.edu.sg
but also starves cancer cells in terms of decreasing their glucose
supply.25 Equally importantly, hydrogen peroxide from APTES-GOx
† Electronic supplementary information (ESI) available: Synthesis and characterization
details. See DOI: 10.1039/c9cc06043j
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serves as a source of ROS. This increase in ROS and the decrease in deionized water, but would react with the hydrogen peroxide
GSH increase the oxidative stress level in cancer cells above the provided to generate QM.
apoptosis threshold.26 One may argue that it is sufficient to simply
Encouraged by the above results, the silane ligand was added
further increase ROS to kill cancer cells. But, cancer cells can adapt jointly with TEOS to form the primary organically modified
by generating GSH to counteract ROS.27 Hence, the released QM is silica nanoparticles (ORMOSIL).29 ORMOSIL was later modified
important in weakening the antioxidant ability of cancer cells.28 with APTES-GOx to form ORMOSIL@GOx: Bare silica nano-
Thus, in this paper, we present a proof-of-concept of using the particles (SiNP) were also fabricated as the unmodified control
responsive ORMOSIL@GOx for anticancer application. This to ORMOSIL. The fabrication of the respective nanoparticles
functional nanosystem provides simultaneous consumption of was characterized by powder X-ray diffraction (XRD), Brunauer–
glucose supply, increase of intracellular oxidative stress, and Emmett–Teller (BET) surface area, scanning electron microscopy
suppression of antioxidants.
(SEM), transmission electron microscopy (TEM), X-ray photo-
The bornate ester-protected quinone methide silane ligand electron spectroscopy (XPS), dynamic light scattering (DLS) and
was prepared in a two-step synthetic route by first reacting zeta potential. The XRD analysis reveals a broad peak between
4-(hydroxymethyl)phenylboronic acid with carbonyldiimida- 15 and 351 for all nanosystems (Fig. S3, ESI†), matching
zole, followed by a conjugation with APTES in dimethylamino- well with the reported peaks of amorphous silica.30 The BET
pyridine. The chemical structures of successive compounds isotherm patterns indicate the low porous properties of the
were well characterized by 1H and 13C NMR spectroscopy as-synthesized ORMOSIL and ORMOSIL@GOx (Fig. S4, ESI†).
(Fig. S1 and S2, ESI†). Before utilizing the ligand for the The TEM images show that ORMOSIL and ORMOSIL@GOx have
templated sol–gel synthesis, we investigated whether the ligand spherical structures of 45 ꢀ 3 and 75 ꢀ 2 nm in diameter,
can be specifically cleaved by hydrogen peroxide to release QM. respectively (Fig. S5 and S6, ESI†). Indeed, the SEM image of
1H NMR analysis was employed to study the release of QM in ORMOSIL@GOx also shows a spherical morphology with a
deionized water and 1 mM H2O2 aqueous solution, respectively. diameter of around 100 nm (Fig. S7, ESI†). The zoomed-in image
Five new NMR peaks corresponding to 4-hydroxybenzyl alcohol validates the low porous nature of ORMOSIL and ORMOSIL@GOx
were identified only in the H2O2 aqueous solution, including a (Fig. S8, ESI†). The lack of mesopores would hinder the access of
phenolic proton at 9.2 ppm, two aromatic doublets at 6.8 and hydrogen peroxide to the silane ligand and result in sustained
7 ppm, hydroxyl peak at 5.9 ppm and aliphatic singlet at release of QM. The DLS results present larger hydrodynamic
4.3 ppm (Fig. 1a). 4-Hydroxybenzyl alcohol was the hydrolyzed sizes of 155 ꢀ 25, 108 ꢀ 10 and 150 ꢀ 10 nm for SiNP, ORMOSIL
product of QM, which means that the preparation of this and ORMOSIL@GOx, respectively (Fig. S9, ESI†). Obvious zeta
silane ligand was successful. The silane ligand is dormant in potential fluctuation after each reaction step demonstrates the
integration of the silane ligand from ꢁ21.7 mV in SiNP to
+26 mV in ORMOSIL, effective removal of the CTAB template
in ORMOSIL from +26 mV to +7.79 mV, and the incorporation of
APTES-GOx from +7.79 mV to 18.2 mV (Fig. S10, ESI†). XPS
comparison between SiNP and ORMOSIL reveals an additional
boron 1s peak in the latter, further confirming the successful
incorporation of the silane ligand (Fig. S11, ESI†). A new UV-vis
absorbance peak corresponding to 4-(hydroxymethyl)phenyl-
boronic acid pinacol ester (4-HPBA) appears upon incorporation
of the silane ligand into ORMOSIL (Fig. S12, ESI†). 4-HPBA has
an intense absorbance peak at 225 nm because of its aromaticity.
However, this peak drastically decreases after covalent conjugation
with APTES-GOx, attributing to the effective surface loading of
APTES-GOx on ORMOSIL.
To further confirm the attachment of APTES-GOx on the
ORMOSIL surface, fluorescence labeling and thermogravi-
metric analysis (TGA) were conducted. In the former study,
GOx was initially labeled with fluorescein isothiocyanate (FITC)
before the conjugation with APTES.25 According to a given
formula, the number of labeled FITC per GOx was 4.2 units
(Fig. S13, ESI†). The FITC-labeled GOx was further reacted with
APTES and then ORMOSIL to form FITC-labeled ORMOSIL@GOx.
By matching the fluorescence of FITC-labeled ORMOSIL@GOx
to the calibration curve, the GOx loading was determined to be
9.3 wt% (Fig. S14, ESI†). Michaelis–Menten steady-state kinetics was
Fig. 1 (a) QM release in the presence and absence of H2O2 recorded by
1H NMR spectra. (b) TEM image of ORMOSIL@GOx. Scale bar: 100 nm.
(c) Michaelis–Menten kinetics of ORMOSIL@GOx. (d) Lineweaver–Burk
plotting of ORMOSIL@GOx. (e) TGA comparison of SiNP and ORMOSIL for
used to determine the catalytic performance of ORMOSIL@GOx
toward glucose conversion to hydrogen peroxide (Fig. 1c and d).
the determination of the silane ligand content. (f) Release of QM from
ORMOSIL@GOx (10 mg mLꢁ1) in 5 mg mLꢁ1 glucose. Mean ꢀ s.d., n = 3.
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The Michaelis–Menten constant (Km) and maximum velocity (Vmax
of ORMOSIL@GOx were calculated to be 44.7 mM and 12.8 ꢂ
10ꢁ8 M sꢁ1, respectively (Table S1, ESI†). The Km value is compar-
able to the commercially available GOx between 33 and 110 mM,
meaning that for 44.7 mM of glucose about half of the maximum
catalytic activity could be achieved.
)
It is also important to characterize the QM component
content of ORMOSIL@GOx. TGA comparison between SiNP
and ORMOSIL shows a weight difference of 11%, corresponding
to the additional weight from the silane ligand (Fig. 1e). From
this result, the loading efficiency of the silane ligand in ORMOSIL
was calculated to be 59% (Fig. S15, ESI†). Since each silane ligand
holds a unit of QM, the total QM content could similarly be
calculated to be 0.229 mmol gꢁ1 of ORMOSIL. The cumulative QM
release profile indicates that the release of QM starts after the 7 h
mark (Fig. 1f). This observation is attributed to the low porous
nature of ORMOSIL@GOx, which restricts the access of hydrogen
peroxide to the silane ligand. The release occurs rapidly until its
plateau after 50 h, giving approximately 60% total release. QM
released from the bornate ester-protected QM silane ligand is very
reactive, and it would eventually be hydrolyzed to produce 4-
hydroxybenzyl alcohol in aqueous medium. In a GSH-rich medium
such as the lysosome of cancer cells, 4-hydroxybenzyl alcohol could
produce the GSH-adduct via thiol substitution at the allylic alcohol
(Scheme 1). Liquid chromatography-mass spectrometry analysis of
ORMOSIL@GOx incubated in 1 mM glucose and 1 mM GSH over
24 h reveals a distinct peak at 412 m/z, attributing to the GSH-
adduct (Fig. S16, ESI†). This is direct evidence of its GSH
scavenging ability.
Fig. 2 In vitro cytotoxicity and catalytic performance. (a) Confocal images
of MDA-MB-231 cells after co-incubation with PBS, GOx, ORMOSIL and
ORMOSIL@GOx in 1 mg mLꢁ1 glucose Dulbecco’s modified Eagle medium
for 4 h and subsequently stained with the ROS fluorescent probe DCFH-DA.
Scale bar: 20 mm. (b) In vitro cytotoxic profile of GOx, ORMOSIL and
ORMOSIL@GOx at varying GOx concentrations. (c) IC50 comparison
between free GOx and ORMOSIL@GOx. (d) Live/dead cell apoptotic assay
of control, GOx, ORMOSIL, and ORMOSIL@GOx after 24 h incubation with
10 mU mLꢁ1 GOx equivalents.
In vitro studies were conducted to investigate the cytotoxicity
and cellular effectiveness of the nanosystems. The half maximal
inhibitory concentration (IC50) of SiNP is 2140 mg mLꢁ1, while equivalent. This is 7.6 and 1307 times better than that of free
the IC50 of ORMOSIL is 753 mg mLꢁ1 (Fig. S19, ESI†). This GOx and ORMOSIL in terms of anticancer efficiency, respectively
decrease is attributed to the incorporation of the silane ligand (Fig. 2c). The released QM makes antioxidant GSH to reach a
that increases the hydrophobicity of the nanosystem. At the sufficiently low concentration in order to enhance the cancer cell
same time, the released QM could disrupt the ROS homeostasis killing effect of ROS. Thus, simultaneous depletion of glucose
in cancer cells. Nonetheless, a high concentration of 200 mg mLꢁ1 nutrient and continued generation of hydrogen peroxide exert
only yielded 29% killing efficiency, indicating that QM alone is immense stress on cancer cells. The therapeutic outcome was
inadequate for effective cancer therapy (Fig. 2a and b). On the again confirmed by flow cytometry analysis at a GOx concen-
other hand, the cellular results reflect the exceptionally low tration of 10 mU mLꢁ1. 20.6% of ORMOSIL@GOx treated cancer
cytotoxicity of ORMOSIL relative to the control groups. Since cells reached the late apoptotic stage compared to 1.61% and
GOx serves as the main source of hydrogen peroxide and is 2.53% in the case of GOx and ORMOSIL, respectively (Fig. 2d).
subsequently conjugated to the surface of ORMOSIL, it is These experimental results highlight the synergy between
important to determine the cytotoxicity of GOx so that a healthy ORMOSIL and GOx in ORMOSIL@GOx for greatly inhibiting
amount of the GOx equivalent is used for cellular studies. The the cancer cell growth.
GOx cytotoxicity in the presence of 1 mg mLꢁ1 glucose was
Taking the reference to Scheme S2 (ESI†), ORMOSIL@GOx
concentration dependent. The IC50 of GOx in MDA-MB-231 initially catalyzes b-D-glucose into b-D-glucono-1,5-lactone and
breast cancer cells was determined to be 41 mU mLꢁ1 (Fig. S20, hydrogen peroxide. The produced hydrogen peroxide would
ESI†). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide raise the level of ROS in cancer cells. The subsequent QM
(MTT) assay was carried out using an equivalent GOx concentration release impedes the decrease of ROS scavenged by GSH. Thus,
of 10 mU mLꢁ1. At this range of GOx, both GOx and ORMOSIL the overall oxidative stress and ROS level would be substantially
showed high cell viability of more than 75%. Interestingly, the higher than under normal conditions. In order to confirm that
therapeutic efficiency greatly increased when both GOx and an ROS-mediated therapy occurs, an ROS fluorescent probe
ORMOSIL were integrated together as ORMOSIL@GOx. The 20,70-dichlorofluorescin diacetate (DCFH-DA) was used. MDA-MB-
IC50 of ORMOSIL@GOx in MDA-MB-231 breast cancer cells 231 cells showed the highest level of green fluorescence after
was determined to be 0.576 mg mLꢁ1 or 5.4 mU mLꢁ1 GOx incubation with ORMOSIL@GOx (7.5 mU mLꢁ1 GOx equivalent)
13376 | Chem. Commun., 2019, 55, 13374--13377
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quinone methide silane and APTES-GOx. The nanosystem could
be effectively internalized by cancer cells, converting intra-
cellular glucose to hydrogen peroxide for enhancing the ROS
level. Hydrogen peroxide in turn triggers the release of QM to
scavenge GSH to achieve synergistic anticancer treatment.
Importantly, in vitro experiments have well demonstrated this
cancer treatment strategy provided by ORMOSIL@GOx. Further-
more, this nanosystem does not rely on any external input or
drugs, which offers an easy platform for cancer therapy. Thus,
this research is expected to pave the way for developing next
generation approaches in cancer treatment.
This research was supported by the Singapore Agency for
Science, Technology and Research (A*STAR) AME IRG grant
(No. A1883c0005) and the Singapore National Research Founda-
tion Investigatorship (No. NRF-NRFI2018-03).
Fig. 3 (a) Confocal images of MDA-MB-231 cells incubated with FITC-
labeled GOx and ORMOSIL@GOx after 2 h. Cell nuclei were stained by
H33342. Scale bar: 20 mm. (b) Flow cytometry profiles of MDA-MB-231
cells incubated with FITC-labeled GOx and ORMOSIL@GOx after 2 h.
(c) Cellular GSH level in MDA-MB-231 cells after the incubation with PBS,
GOx, ORMOSIL and ORMOSIL@GOx at an equivalent GOx concentration
of 10 mU mLꢁ1 in the presence of 1 mg mLꢁ1 glucose for 24 h. Mean ꢀ s.d.,
n = 3. **p o 0.05, ***p o 0.005 (t-test).
Conflicts of interest
There are no conflicts to declare.
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