Enzyme-MOF Nanoreactor Activates Nontoxic Paracetamol for Cancer Therapy

Article abstract of DOI:10.1002/anie.201801378

Prodrug activation, by exogenously administered enzymes, for cancer therapy is an approach to achieve better selectivity and less systemic toxicity than conventional chemotherapy. However, the short half-lives of the activating enzymes in the bloodstream has limited its success. Demonstrated here is that a tyrosinase-MOF nanoreactor activates the prodrug paracetamol in cancer cells in a long-lasting manner. By generating reactive oxygen species (ROS) and depleting glutathione (GSH), the product of the enzymatic conversion of paracetamol is toxic to drug-resistant cancer cells. Tyrosinase-MOF nanoreactors cause significant cell death in the presence of paracetamol for up to three days after being internalized by cells, while free enzymes totally lose activity in a few hours. Thus, enzyme-MOF nanocomposites are envisioned to be novel persistent platforms for various biomedical applications.

Full text of DOI:10.1002/anie.201801378

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Long-Persistent Enzyme-MOF Nanoreactor Activates Non-toxic
Paracetamol for Cancer Therapy
Authors: Xizhen Lian, Yanyan Huang, Yuanyuan Zhu, Yu Fang, Rui
Zhao, Elizabeth Joseph, Jialuo Li, Jean-Phillipe Pellois, and
Hongcai Zhou
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201801378
Angew. Chem. 10.1002/ange.201801378
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10.1002/anie.201801378
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Long-Persistent Enzyme-MOF Nanoreactor Activates Non-toxic
Paracetamol for Cancer Therapy
Xizhen Lian[a], Yanyan Huang[b], Yuanyuan Zhu[b], Yu Fang[a], Rui Zhao[b], Elizabeth Joseph[a], Jialuo
Li[a], Jean-Phillipe Pellois[a][c], and Hong-Cai Zhou*[a]
Abstract: Prodrug activation by exogenously administered enzymes
for cancer therapy is an approach to achieve better selectivity and less
systemic toxicity than conventional chemotherapy. However, the short
half-lives of the activating enzymes in bloodstream has limited its
success. Here, we demonstrate that a tyrosinase-MOF nanoreactor
activates the prodrug paracetamol in cancer cells in a long-lasting
manner. By generating reactive oxygen species (ROS) and depleting
glutathione (GSH), the product of enzymatic conversion of
paracetamol is toxic to drug-resistant cancer cells. Tyrosinase-MOF
nanoreactors cause significant cell death in the presence of
paracetamol for up to three days after being internalized by cells,
while free enzymes totally lose activity in a few hours. Thus, we
envision enzyme-MOF nanocomposites to be novel long-persistent
platforms for various biomedical applications.
potentially useful, the biggest drawback is that externally-
administered enzymes are highly vulnerable to degradation in the
bloodstream.[19-20] Thus the feasibility of this strategy remains
unclear and strategies that can improve the biocompatibility of
externally-administered enzymes are needed.
Herein we demonstrate that enzymatic nanoreactors based on
metal-organic frameworks (MOFs) can be potent prodrug
activators. MOFs are an emerging platform that demonstrates
potential for a number of biotechnological applications, including
sensing, imaging, drug delivery and enzyme encapsulation.[21-37]
Encapsulated enzymes demonstrate well-preserved catalytic
25-26]
activities.[22,
Encapsulated enzymes also show enhanced
stability under protein denaturation conditions, such as organic
solvents, extreme pH environments, or high temperatures.[21, 27, 38]
Moreover, MOFs efficiently protect encapsulated enzymes from
proteolytic degradation, presumably by preventing access of
Chemotherapeutics typically interfere with mitosis and
demonstrate maximum toxicity towards rapidly-dividing cells.[1]
However, chemotherapy drugs are poorly selective and often
result in severe adverse effects that include kidney damage, bone
marrow suppression, nerve damage, hair loss, low blood count,
and inflammation.[2-4] In addition, resistance to chemotherapy
occurs in certain cancer cell lines, thereby declining the efficiency
of treatment.[5-7] One strategy to approach these problems is to
employ prodrugs, compounds that are innocuous until
39]
proteases to the protein substrate.[28,
Notably, the porous
nature of MOFs allows for facile substrate diffusion and product
release, a feature typically superior to other well-established
carriers.[40] In this work, we selected PCN-333(Al) as the enzyme
carrier owing to its high enzyme encapsulation capacity, facile
modification with fluorophores, and chemical robustness in
cellular environment.[26] To achieve tumor specific prodrug
activation, tyrosinase (TYR), which is uniquely localized in
melanocytes and melanoma cells, is chosen as the activating
enzyme.[41] Paracetamol (APAP), the active ingredient of Tylenol,
is used as the nontoxic prodrug.[42] Both in cellulo and in vivo
experiments establish that APAP along with enzyme-MOF
nanoreactor induced significant cytotoxicity in drug-resistant
cancer cells and led to tumor regression. Mechanistically,
cytotoxicity arises from 4-acetamido-o-benzoquinone (AOBQ),
the enzymatic conversion product of APAP, and from subsequent
metabolized to give cytotoxic products in
a
tumor
microenvironment.[8-11] However, this approach involves a new
challenge, the identification of activation mechanisms specific to
cancer cells. To date, enzymes overexpressed in tumor cells have
been used to activate prodrug.[10, 12] Nevertheless, because these
targeting enzymes may also present in normal cells, albeit at
lower levels, many of these prodrug therapies retained poor
[13-14]
selectivity and demonstrated minimal success.
To
circumvent this, an alternative strategy involves delivering
exogenous “activating” enzymes into cancer cells. Tumor specific
accumulation of the enzyme “activator” is achieved by conjugation
to tumor directing antibodies or immunoliposomes.[15-18] While
[a]
X. Lian, Dr. Y. Fang, E. Joseph, J. Li, Prof. J.-P. Pellois, Prof. H.-C.
Zhou
Department of Chemistry
Texas A&M University
College Station, Texas 77843-3255, United States
E-mail: zhou@chem.tamu.edu
[b]
[c]
Dr. Y. Huang, Y. Zhu, Prof. R. Zhao
Beijing National Laboratory for Molecular Sciences
CAS Key Laboratory of Analytical Chemistry for Living Biosystems,
Institute of Chemistry, Chinese Academy of Sciences
Beijing, 100190, China
Prof. J.-P. Pellois
Department of Biochemistry and Biophysics
Texas A&M University
College Station, TX 77843-2128, United States
Scheme 1. Nontoxic prodrug APAP oxidation is catalyzed by tyrosinase,
yielding cytotoxic AOBQ, which induces ROS generation and GSH
depletion.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201801378
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COMMUNICATION
reactive oxygen species (ROS) generation and glutathione (GSH)
depletion.
cavity disappeared, consistent with the cavity being occupied by
TYR (Figure S5). Thermal gravimetric analysis (TGA) also
indicated the high loading of TYR in NPCN-333 (Figure S6).
TYR catalyzes the oxidation of APAP to an o-quinone compound,
4-acetamido-o-benzoquinone (AOBQ), which absorbs at 450 nm.
The enzymatic activities of free TYR and TYR@NPCN-333
nanoreactor in 50 mM Tris-HCl buffer (pH 6.8) at ambient
temperature, was monitored by UV-vis spectrometry.
TYR@NPCN-333 nanoreactor followed Michaelis-Menten
kinetics and showed a kcat value of 0.377 s-1 and a Km value of
1.119 mM (Figure S7). In comparison, the free enzyme displayed
a kcat of 0.405 s-1 and a Km of 1.338 mM (Figure S8). The catalytic
efficiencies, kcat/Km, were 0.302 s-1mM-1 for free TYR vs. 0.337 s-
1mM-1 for TYR@NPCN-333.
Resistance of enzyme-MOF nanoreactor towards degradation is
critical for practical applications in living biosystems. To elucidate
whether NPCN-333 could protect encapsulated TYR, free TYR
and TYR@NPCN-333 were incubated with trypsin solution for 2
hours at 37oC. Then, the enzyme was released by dissolving
TYR@NPCN-333 with HCl and the supernatant was subjected to
SDS-PAGE analysis. As shown in Figure S9, free TYR was
readily digested by trypsin as illustrated by the presence of small
molecular weight bands. In contrast, bands corresponding to
intact TYR were predominantly present for TYR@NPCN-333.
Additionally, the enzymatic activity of TYR@NPCN-333 was
almost unchanged after trypsin treatment (Figure S10). Moreover,
in order to study whether MOF protect encapsulated enzymes in
an acidic medium, free TYR and TYR@NPCN-333 were
incubated in pH 5 buffer, and enzymatic activities were measured
at 2 h and 12 h. While a 2 h incubation only resulted in a 15%
activity loss on free enzyme, 75% of the original activity was lost
PCN-333 is composed of trimeric-oxo cluster and a planar
triangular ligand, 4,4′,4″-s-triazine-2,4,6-triyl-tribenzoic acid
(TATB), which self-assembles into a supertetrahedron (STH) with
metal clusters on the corner and TATB on the face (Figure 1a). A
1.5 nm cavity is present in the STH. Two types of mesoporous
cavities with diameters of 4.2 and 5.5 nm are composed of STHs
through vertex sharing (Figure 1b). PCN-333 nanoparticles
(NPCN-333) were prepared by solvothermal reaction of AlCl3 and
TATB in DMF at 95oC. The particle size determined by
transmission electron microscopy (TEM) was around 100 nm
(Figure 2a). A fluorescent version of NPCN-333 was prepared via
a ligand metathesis reaction with a BTB ligand functionalized with
a green fluorescent BODIPY fluorophore (Figure S1). The well-
maintained crystallinity of NPCN-333 after enzyme loading and
after soaking in cell culture medium Roswell Park Memorial
Institute 1640 (RPMI 1640) for up to 7 days was confirmed by the
TEM images (Figure 2b, c) and by the nearly unchanged PXRD
pattern (Figure 2d). The monodispersity of NPCN-333 immersed
in RPMI 1640 over an extended period of time was verified by the
particle size distribution diagram derived from dynamic light
scattering (DLS) data collected on day 0 and day 7 (Figure 2e).
Figure 1. Structure of PCN-333. (a) NPCN-333 is composed of aluminum
trimeric cluster and TATB ligand, which self-assembles into
supertetrahedrons as the secondary building block of NPCN-333. (b) Two
types of mesoporous cavities in NPCN-333: 4.2 nm dodecahedral cage
(light purple) and 5.5 nm hexacaidecahedral cage (light green). Based on
the size of tyrosinase, it can only be accommodated in the 5.5 nm cage.
One molecule of TYR contains a single 43 kDa polypeptide with
dimension of 5.5 × 5.5 × 5.6 nm3.[43] Therefore TYR may be
accommodated in the 5.5 nm cavity, but presumably not in the 1.5
and 4.2 nm cavity. After incubating TYR with NPCN-333 slurry for
20 minutes, the enzyme loading capacity was estimated to be
0.80 g/g according to BCA method, which is in consistent with the
theoretical value (1.08 g/g). The distribution of TYR in the material
was studied by N2 isotherm at 77K. The pore size distribution
diagram of TYR encapsulated NPCN-333 (TYR@NPCN-333)
clearly showed the presence of a microporous cavity around 1.5
nm and a mesoporous cavity at around 4 nm. Notably, the 5 nm
Figure 2. Structural characterization of NPCN-333. TEM images of NPCN-
333 (a), TYR@NPCN-333 (b), and TYR@NPCN-333 after soaking in
RPMI-1640 for 7 days (c). Scale bar: 200 nm. (d) Powder X-ray diffraction
(PXRD) patterns of pristine NPCN-333 (black), TYR@NPCN-333 (red),
TYR@NPCN-333 after trypsin treatment (blue), and TYR@NPCN-333
after soaking in RPMI 1640 media for 7 days (magenta). (e) Particle size
distribution measured by dynamic light scattering (DLS): (from front to
back) pristine NPCN-333, TYR@NPCN-333, TYR@NPCN-333 after
trypsin treatment, and TYR@NPCN-333 after soaking in RPMI 1640 media
for 0 and 7 days.
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10.1002/anie.201801378
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after 12 h incubation. In contrast, TYR@NPCN-333 maintained
over 75% activity after 12 h incubation at pH 5 (Figure S11).
lysosomes (Figure S12).[47] This indicates that the nanoreactor
accumulated within endocytic organelles. Next, the cytotoxicity
induced by TYR@NPCN-333 was quantified by the MTT assay.
The prodrug, or the enzyme-MOF nanoreactor, did not display
any noticeable cytotoxicity (Figure 3a). In contrast, cells that were
pretreated with TYR@NPCN-333 for 6 h, and then subsequently
exposed to increasing concentrations of APAP (0.1 to 2 mM)
displayed a correlated decrease in viability. Besides, similar
cytotoxicity was observed in other cancer cell lines, including non-
small lung carcinoma cell H1299 and cervical carcinoma cell
HeLa (Figure S13 and S14). In complementary assays, the
cytotoxicity of the TYR@NPCN-333 – APAP combination was
assessed by flow cytometry. After different treatments, cells were
stained with annexin V-FITC, a fluorescent marker of surface
exposed phosphatidyl serine, and with propidium iodide (PI), a
DNA stain that detects the permeabilization of the plasma
membrane. As observed previously, neither APAP nor
TYR@NPCN-333 caused an increase in annexin V or PI staining,
indicating that these conditions are not toxic to cells (Figure 3b).
However, cells pretreated with TYR@NPCN-333 and exposed to
APAP displayed dual labeling, which is indicative of cell death.
Because activating enzymes must be administered before
treatment with a prodrug, it is important to ensure that their activity
can be maintained over a prolonged period of time.[48] To study
how the enzyme-MOF nanoreactor performs in this respect,
SKOV3-TR cells pretreated with TYR@NPCN-333 for 6 h were
cultured in fresh media for several days. MTT assay revealed that
a 2 mM APAP treatment on day 3 could lead to 50% cell
proliferation inhibition (Figure S15). On the contrary, free TYR
showed slight inhibition effect (<20%) on cells after APAP
treatment on day 0. However, the activity of free TYR totally
disappeared from day 1 (Figure S15).
Next, we sought to characterize how TYR@NPCN-333 – APAP
induces toxicity. When used as the sole therapeutic, neither
TYR@NPCN-333 nor APAP display cytotoxicity. We
hypothesized that the cytotoxicity observed was a result of the in
situ generation of AOBQ as an enzymatic product. Based on the
property of p-quinone compounds, we suspected that AOBQ
would undergo single electron redox reactions and induce the
generation of reactive oxygen species (ROS) in cells.[49-52] Real-
time monitoring of superoxide ( O2− ) concentration using a
superoxide detection reagent in SKOV3-TR cells upon different
treatments was performed to test this hypothesis. The intensity of
red fluorescence is proportional to the concentration of
intracellular O−2 . Cells treated with TYR@NPCN-333 – APAP
displayed elevated levels of O−2 whereas the O2− level in cells
treated with either TYR@NPCN-333 or APAP remained
unchanged (Figure S16).
Besides inducing ROS generation, o-quinone compounds are
notorious for consuming glutathione (GSH) in living cells.[53-54]
Therefore we sought to measure the GSH concentration in cells
upon TYR@NPCN-333 – APAP treatments. GSH reacts with
DNTB (5,5’-dithio-bis-2-(nitrobenzoic acid) and produces a yellow
colored compound (5-thio-2-nitrobenzoic acid), which can be
quantitatively measured by UV-vis spectroscopy at 405 nm.[55]
Because GSH is the most abundant non-protein thiol in eukaryotic
cells, this measurement yields a good estimation on intracellular
GSH concentration.[56] GSH level remained unchanged in cells
treated with either TYR@NPCN-333 or APAP. However, when
Figure 3. Cytotoxicity of TYR@NPCN-333 – APAP. (a) Cell proliferation of
SKOV3-TR cells upon different treatments measured by MTT assay. The
experiment was performed in quintuplicate in 96 well plates (n=5), mean ±
s.d., *** represents p≤0.001, multiple t test. (b) Cell viability of SKOV3-TR
cells stained with annexin V-FITC/propidium iodide (PI) and measured by
flow cytometry after different treatments. Cells with (+) or without (-) 6 h
TYR@NPCN-333 pretreatment was incubated with 0—2 mM APAP for 2 h.
The experiment was performed three times and representative results from
one test are displayed.
In vitro results indicated that TYR@NPCN-333 is enzymatically
active and relatively resistant to perturbations. Cell-based
experiments were therefore conducted to examine whether
TYR@NPCN-333 could activate APAP in cancer cells. To test the
performance of TYR@NPCN-333, we chose SKOV3-TR, an
ovarian adenocarcinoma cell that is resistant to cisplatin, tumor
necrosis factor, diphtheria toxin, adriamycin, and taxane.[44-46]
First, the cellular internalization of the enzyme-MOF nanoreactor
was established using confocal microscopy. A fluorescent particle,
TYR@FNPCN-333, was used to monitor cellular trafficking. After
incubation with cells, TYR@FNPCN-333 displayed an
intracellular punctate distribution around the nucleic area that
colocalizes with LysoTracker, a marker for late endosomes and
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10.1002/anie.201801378
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COMMUNICATION
cells were pretreated with TYR@NPCN-333 for 6 h, treatment
with APAP solution resulted in GSH depletion, which showed a
strong correlation with APAP concentration (Figure S17). Notably,
after 2 mM APAP treatment for only 2 h, intracellular GSH
concentration decreased to about 50% of its original level (cells
were not permeabilized at this time point, as shown by absence
of PI staining in Figure S18). Mass spectrometry analysis of the
cell lysate revealed the presence of a species with molecular
weight of 492, which is in consistent with that of APAP-GSH
conjugate (Figure S19).[53] In addition, to further confirm the role
of ROS generation and GSH depletion in AOBQ induced
cytotoxicity, we did two control experiments in which ascorbic acid
(AA) and GSH were supplemented to cell culture medium along
with TYR@NPCN-333 – APAP treatment (APAP concentration
was 500 µM). Compared with the negative control group, the cell
viability showed 20% and 30% increase for AA and GSH
supplement groups, respectively (Figure S20). Overall, these
results have established that TYR@NPCN-333 – APAP exhibits
cytotoxicity by inducing oxidative stress, as manifested by a
combination of ROS induction and GSH depletion.
To evaluate the antitumor efficacy of APAP in the presence of
MOF nanoreactor, in vivo experiments were performed on a HeLa
subcutaneous xenograft model. Mice were treated with PBS,
TYR@NPCN-333 (1.5 mg/kg), APAP (200 mg/kg), TYR@NPCN-
333 (1.5 mg/kg) and APAP (200 mg/kg), respectively. Compared
with PBS control, tumor regression only occurred in the group
receiving TYR@NPCN-333 – APAP treatment (tumor size
decreased from ~50 to ~20 mm3), while neither the nanoreactor
nor the prodrug alone induced any antitumor efficacy (Figure 4).
More importantly, in the TYR@NPCN-333 – APAP group, two out
of five tumors were completely eradicated by the end of the
experiment upon
a single treatment. Additionally, tumor
regression failed in mice treated with TYR@NPCN-333 – APAP
but supplemented with GSH (700 mg/kg), further confirming that
tumor regression is attributable to oxidative stress. Hematoxylin
and eosin (H&E) staining of tumor tissues showed that only the
group receiving TYR@NPCN-333 – APAP therapy demonstrated
cell apoptosis/necrosis (Figure S22, 23).
In summary, we demonstrate that the enzyme-MOF nanoreactor
can be a highly efficient activator for resistant cancer therapy
coupled with a prodrug. Encapsulated enzymes are well protected
by MOFs in proteolytic conditions and in acidic environment,
where free enzymes would quickly lose activity. The prodrug can
be efficiently activated by the enzyme-MOF nanoreactor,
generating a cytotoxic compound, which inhibits cell proliferation
and induces apoptosis/necrosis by promoting oxidative stress. In
vivo experiment shows a 2.5-time regression of tumor volume
after a single treatment. Overall, we envision that the enzyme-
MOF nanoreactor system as a new platform for vast biomedical
and biotechnological applications due to the ability to cater for
intrinsic diversities in structure and functionality.
Acknowledgements
This work was supported by the Welch Foundation through a
Robert A. Welch Chair in Chemistry to H.C.Z. (A-0030), by a grant
from the National Institutes of Health (to J.P.P., award number
R01GM110137), by a grant from Strategic Transformative
Research Program, College of Science, Texas A&M University to
X.L., by China Scholarship Council and by Youth Innovation
Promotion Association CAS.
Keywords: Metal-organic frameworks • Enzyme encapsulation •
Prodrug activation • Nanoparticles • Cancer therapy
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