Angewandte
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Controlled Release
Intracellular Delivery of Functional Native Antibodies under Hypoxic
Conditions by Using a Biodegradable Silica Nanoquencher
Peiyan Yuan, Hailong Zhang, Linghui Qian, Xin Mao, Shubo Du, Changmin Yu, Bo Peng, and
Shao Q. Yao*
Abstract: Antibodies are important biopharmaceuticals, but
almost all existing antibody-based drugs are limited to target-
ing antigens located at the cell exterior because of the inability
of antibodies to enter the cell interior. Available methods for
intracellular delivery of antibodies have major shortcomings.
Herein, we report an approach to encapsulate native antibodies
in a biodegradable silica nanoquencher (BS-qNP), which
could undergo efficient cellular uptake and intracellular
degradation to release antibodies only under hypoxic condi-
tions. By coating the surface of BS-qNP with cell-penetrating
poly(disulfide)s (CPD), the delivered antibodies (or other
proteins) avoided endolysosomal trapping. Doping of the silica
coating with a fluorescent dye and a dark hole quencher further
endowed BS-qNP with hypoxia-responsive fluorescence turn-
on property. Our antibody delivery system thus provides the
first platform capable of stable encapsulation, efficient uptake,
on-demand antibody release, and imaging of release/cell state.
Proteins play vital roles in biology and medicine. Despite
obvious benefits of protein-based therapy,[1] intracellular
delivery of functional, native proteins remains a key bottle-
neck.[2] Amongst various therapeutic proteins, antibodies,
generally with a Y-shape structure and molecular weight
greater than 150 kDa, are a special type because of their
ability of binding to virtually any immunogenic targets with
exquisite specificity and high affinity. This makes them the
ideal “magic bullets” against many diseases, if they are able to
reach the site of action.[3, 4] In fact, antibodies constitute one of
the largest classes of therapeutic proteins.[3] Almost all of
them, however, target cell-surface antigens overexpressed in
malignant cells, thus avoiding the need for intracellular
delivery.[3, 5] The number of therapeutic antibodies might
have been much higher, had an effective means for intra-
cellular delivery been available, for this would provide
opportunities to target intracellular antigens that are consid-
ered less druggable.[4, 6] Attempts have been made to deliver
antibodies intracellularly by standard protein-delivery proto-
[*] Dr. P. Yuan, Dr. H. Zhang, Dr. L. Qian, X. Mao, S. Du, B. Peng,
Prof. Dr. S. Q. Yao
Department of Chemistry, National University of Singapore
3 Science Drive 3, Singapore 117543 (Singapore)
E-mail: chmyaosq@nus.edu.sg
Dr. C. Yu
College of Materials Science & Engineering
South China University of Technology
510640 Guangzhou (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201705578.
Angew. Chem. Int. Ed. 2017, 56, 12481 –12485 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemie
International Edition: DOI: 10.1002/anie.201705578
German Edition: DOI: 10.1002/ange.201705578
cols including those using peptide/protein fusion, viruses,
materials, and microinjection, but they suffer from low/
variable delivery efficiency, technical complexity, insufficient
carrier stability, endolysosomal trapping, and other short-
comings.[4, 7, 8] In most cases, chemical and genetic modifica-
tions are introduced in the antibody, which inevitably alters its
antigen-binding property. As large extracellular proteins that
are part of the bodyQs defense system, most antibodies could
tolerate relatively harsh external conditions (for example,
non-physiological pH and temperature, high salt/detergent
concentrations, and organic cosolvents) but readily degrade in
highly reducing intracellular environments upon prolonged
exposure to endogenous GSH (1–10 mm) and proteolytic
enzymes.[4] One way to counter such problems is the use of
intracellularly expressed antibodies (intrabodies) by trans-
fecting cells with genes expressing structurally optimized
unnatural antibodies normally obtained through painstaking
genetic engineering.[4, 9] If a commonly available antibody
could be directly taken with minimal chemical, genetic, or
other modifications, efficiently delivered into cells without
being trapped by endocytic vehicles but still stably shielded
from the cytosolic milieu, and released on-demand in its
functional, native form upon stimulation by disease signals,
then the full potential of antibody-based therapy could be
realized. Herein, by using Cetuximab (an FDA-approved
antibody drug for cancer treatments) as a model, we report
the first biodegradable silica nanoquencher (BS-qNP)-coated
protein system (Protein@BS-qNP; henceforth protein refers
to a protein or antibody, unless otherwise specified) capable
of intracellular delivery (via endocytosis-independent
uptake) and controlled release of the functional, native
Cetuximab in the cytosol of cancer cells only when they are
in hypoxia. Cetuximab normally targets cancer cell surface-
exposed epidermal growth factor receptor (EGFR, a receptor
tyrosine kinase) without the need for intracellular delivery.[10]
In our study, we found intracellularly released Cetuximab had
similar cellular effects on hypoxic, EGFR-expressing A431
cancer cells. As an added advantage of this novel protein
delivery system, the cell state and intracellular protein release
could be concurrently monitored by fluorescence imaging.
Although a variety of protein-delivery systems, some of which
also possess stimulus-responsive features, have been
reported,[2, 11] our system is the first reported case for intra-
cellular delivery of native proteins with key attributes
including stable encapsulation, efficient delivery, imaging,
therapeutics, and cell fate-dependent targeting.
We chose hypoxia (an oxygen-deprived condition) as the
disease model for controlled release of intracellularly deliv-
ered proteins because it is found in the microenvironment of
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most solid tumors. Tumor hypoxia induces various biological
phenomena including enhanced EGFR-mediated signaling,
upregulated tumor gene expression, and resistance to chemo-
therapy.[12] In recent years, the development of hypoxia-
dependent prodrugs and hypoxia-sensing chemical probes
have drawn significant research interests.[13] An hypoxia-
mediated, tumor-targeting small molecule prodrug of EGFR
was recently reported, which showed promising anticancer
activity in A431 cells upon hypoxia activation.[13a] To our
knowledge, a prodrug-like EGFR-targeting antibody has not
been reported. To prove the feasibility of our novel antibody
delivery system (Scheme 1 and the Supporting Information,
Tables S1 and S2), it was first systematically optimized with
two model proteins, BSA and RNase A (BSA@BS-qNP and
RNaseA@BS-NP, respectively; Figure 1), then with Cetux-
imab (Cetuximab@BS-qNP; Figure 2).
As shown in Scheme 1 (and the Supporting Information,
Figures S1–S4), the preparation of our delivery system started
with encapsulation of the native protein in a biodegradable
silica nanocapsule, which was previously used to deliver well-
behaved small proteins and spontaneously release them
intracellularly under the cellQs natural reducing environ-
ment.[14] Whether or not similar nanocapsules could encap-
sulate antibodies, which are much larger, was however not
known. Noting the good stability of most antibodies in
external conditions, we hypothesized that various chemicals
used in the reverse nanoemulsion procedure to prepare
nanocapsules, though likely not suitable for delicate proteins,
would be well-tolerated by antibodies.[5, 15] Noting the already
superior physical rigidity and chemical stability of the
organosilica shells in these nanocapsules compared to other
protein carriers (for example, liposomes and hydrogels[2, 11]),
we further minimized the exposure of the encapsulated
protein to intracellular environments by rendering the nano-
capsules hypoxia-responsive with the addition of bisorthosi-
licate-containing azo monomer 1 during shell formation, thus
providing a release module in the nanocapsule (Protein@BS-
NP). Under hypoxic conditions, the azo moiety in BS-NP
Scheme 1. Scheme showing the preparation of CPD-protein@BS-qNP and its endocytosis-independent cell uptake (step I), endogenous GSH-
assisted CPD depolymerization (step II), and hypoxia-triggered intracellular protein release with fluorescence turn-on imaging (step III). The
imaging module and release module are highlighted.
12482 www.angewandte.org T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angewandte
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would be reduced by flavoproteins (that is, cytochrome P450
reductase), resulting in shell disintegration and subsequent
release of the encapsulated protein. To install an imaging
module, which would enable sensing of cell hypoxia and
protein release, we further doped Protein@BS-NP with
a fluorescent dye (FL-APS 2) and a black hole quencher
(BHQ1-APS 4), giving the corresponding nanoquencher
(Protein@BS-qNP) with a green turn-on property (Fig-
ure S2). If a different fluorescent color is preferred, RB-
APS 3 and BHQ2-APS 5 could be used instead. The use of
silica nanoquenchers for the simultaneous imaging of intra-
cellular protein release and cell state (hypoxia) is unprece-
dented.[16] A major issue plaguing most existing intracellular
protein delivery systems is the endolysosomal trapping of the
payload, although in some cases this could be ameliorated by
clever designs.[2, 11] This problem was eliminated in our system
by coating the Protein@BS-qNP surface with cell-penetrating
poly(disulfide)s (giving CPD-Protein@BS-qNP). CPD was
previously shown to facilitate efficient endocytosis-independ-
ent delivery of a variety of payloads with minimum cytotox-
icity.[16a, 17, 18] The coating of CPD on Protein@BS-qNP was
carried out by first modifying the nanoquencher surface with
N3-APS, followed by successive bioorthogonal reactions with
TCO-PEG12-DBCO and Tz-CPD (Figures S3 and S4).[16a] As
shown in Scheme 1, upon incubation with mammalian cells,
CPD-Protein@BS-qNP would be taken up via thiol-mediated
pathways and directly enter the cytosol (step I), where its
CPD coating would be rapidly depolymerized (< 5 min[17, 18])
by endogenous GSH (step II). The resulting nanoquencher
would remain intact (thus shielding the encapsulated protein
from the intracellular environment and minimizing degrada-
tion). Subsequently, when cells became hypoxic, both the
imaging module and release module in the nanoquencher
would be activated, leading to successful payload release and
simultaneous imaging of the cell state (step III). It should be
highlighted that, unlike our previously reported CPD-assisted
protein delivery method in which chemical and/or genetic
modifications were made to the payload itself in order to
Angew. Chem. Int. Ed. 2017, 56, 12481 –12485
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achieve CPD conjugation,[17a] the current nanoquencher-
based system requires no changes to the encapsulated
native protein/antibody, all modifications were performed
on the capsule coating instead.
As shown in Figure 1 A and the Supporting Information,
Figure S5, upon careful optimization of the amount of 1 and
other components in the reported reverse nanoemulsion
procedure,[14] we were able to obtain uniform, well-dispersed
nanocapsules (Protein@BS-NP) and nanoquenchers (Pro-
tein@BS-qNP) with an average diameter of circa 50 nm.
Further surface modifications leading to CPD-Protein@BS-
qNP were successfully confirmed by measurements of the
zeta potential (Supporting Information, Figure S6). As con-
trols, non-biodegradable nanocapsules (NonBS-NP), as well
as the corresponding non-biodegradable nanoquenchers,
were prepared by repeating the same nanoemulsion proce-
dure with the omission of 1 (Supporting Information, Fig-
ure S7 and Tables S1 and S3). To test the fluorescence turn-on
feature of the nanoquenchers, additional controls were
prepared and named FLProtein@BS-NP or FLProtein@BS-
qNP, by directly encapsulating an FITC-labeled protein in the
nanocapsule without or with quencher 4, respectively. In some
cases, a rhodamine dye was used instead and the correspond-
ing nanocapsules were named RBProtein@BS-NP or
of 82 % and 84 % were obtained with capsules doped with 2/4
and 3/5, respectively (Figure S6).
To study the hypoxia-responsive biodegradability of these
newly developed nanocapsules, BSA@BS-NP was incubated
with rat liver microsomes and NADPH at 37 8C (an in vitro
condition that mimics hypoxia[13b]). As shown in Figure 1 A–D
with SEM/TEM measurements, complete disintegration of
nanocapsules was observed in 24–48 h. Even more rapid
degradation was observed with nanocapsules treated with
sodium dithionite (a strong reducing agent; Supporting
Information, Figure S8). In stark contrast, no significant
morphological changes were observed with BSA@NonBS-
NP even after prolonged sodium dithionite treatment (48 h).
These results indicate that azo bond cleavage in 1 was directly
responsible for the degradation of these biodegradable nano-
capsules. Next, to directly monitor protein release, FITC-
labeled BSA (FLBSA; Supporting Information, Figure S9)
was encapsulated and the resulting FLBSA@BS-NP, together
with BSA@BP-qNP, were treated with microsomes/
NADPH(Figure 1 E and the Supporting Information, Fig-
ure S10); an obvious time-dependent fluorescence increase in
the collected supernatants was detected but not in the
supernatants of similarly treated non-degradable
FLBSA@NonBS-NP. To further confirm that the degradability

RBProtein@BS-qNP (the latter was doped with quencher 5).
FITC/BHQ1 and rhodamine/BHQ2 pairs were chosen in the
imaging module because of their overlapping fluorescence
emission/quenching spectra, and in our nanoquenchers (Pro-
tein@BS-qNP), excellent fluorescence quenching efficiencies
of our nanocapsules and the protein release were indeed
caused by hypoxia, lysates of cells incubated with
CPD-FLBSA@BS-NP followed by induction under hypoxic
or normoxic conditions were analyzed by SDS-PAGE/in-gel
fluorescence scanning (Figure 1 F); a 66-kDa fluorescent

Figure 1. A) SEM image of BSA@BS-NP. TEM images of BSA@BS-NP B) before and after incubation with rat liver microsomes/NADPH for
C) 24 h and D) 48 h. E) Time-dependent fluorescence spectra (lex = 488 nm) of BSA@BS-qNP (0.01 mg mL@1 in PBS) incubated with rat liver
microsomes/NADPH at 37 8C, and with the corresponding time-dependent plot (right; red line) of fluorescence intensity (at lem = 515 nm).
Similar protein-release profiles with FLBSA@BS-NP (black line) and FLBSA@NonBS-NP (green line) were plotted for comparison. F) SDS-PAGE/in-
gel fluorescence scanning of lysates from A549 cells treated with different nanocapsules/conditions. G) 3D projections showing Z-stack images
(step size = 0.163 mm) of A549 cells incubated with CPD-FLBSA@BS-NP (10 mg mL@1; 2 h), followed by staining with LysoTracker and Hoechst
dyes. H) Flow cytometric analyses (FACS) with a minimum of 10 000 cells were used for fluorescence quantification of A549 cells incubated with
CPD-FLBSA@BS-NP (10 mg mL@1; 2 h incubation) upon pretreatment with different inhibitors/conditions (10 mg mL@1 chlorpromazine, 50 nm
wortmannin, 50 mm methyl-b-cyclodextrin (M-b-CD)), 4.8 mm 5,5’-dithioobis-2-nitrobenzoic acid (DTNB), or at 4 8C) for 30 min prior to incubation
with CPD-FLBSA@BS-NP. See Figure S13 for further details. I) CLSM images of A549 cells treated with CPD-FLBSA@BS-qNP or CPD-BSA@BS-qNP
(10 mg mL@1, 6 h) followed by 24-h and 48-h incubation under hypoxic or normoxic conditions. Blue = Hoechst. Scale bar = 20 mm. J) % Viability
(determined by XTT assay) of A549 cells treated with different nanocapsules (50 mg mL@1; equivalent to ca. 2 mg RNase A·mL@1, 6-h incubation)
followed by 48-h incubation under hypoxic or normoxic conditions. Error bars were obtained from triplicate experiments. Inset: TEM image of
RNaseA@BS-NP; scale bar = 50 nm.

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band corresponding to FLBSA was detected in the sample
from hypoxic cells treated with CPD-FLBSA@BS-NP but not
with CPD-FLBSA@NonBS-NP. No protein release was
detected in normoxic cells treated with CPD-FLBSA@BS-
NP either (lane 5).
Next, by live-cell imaging experiments, we confirmed the
CPD-assisted cellular uptake with no endolysosomal trapping
of CPD-coated nanocapsules by using CPD-FLBSA@BS-NP.
With encapsulated FLBSA and no quencher doping, these
nanocapsules were always fluorescent and stable in DMEM,
allowing the uptake process to be imaged by confocal laser
scanning microscopy (CLSM; Figure 1 G and the Supporting
Information, Figures S11 and S12); unlike non-CPD-coated
nanocapsules, which showed little uptake after 4 h, rapid
uptake of these nanocapsules (< 2 h) directly into the cytosol
of treated cells was observed with no apparent endolysosomal
trapping, and the uptake profile was insensitive to common
endocytosis inhibitors (Figure 1 H and the Supporting Infor-
mation, Figure S13). As previously observed with other CPD-
assisted delivery systems,[16a, 17, 18] reduced temperature (4 8C)
or 5,5’-dithiobis-2-nitrobenzoic acid (DTNB, a thiol blocker)
led to significant inhibition in nanocapsule uptake. To directly
image hypoxia-responsive protein release following intra-
cellular nanocapsule degradation, we used CPD-FLBSA@BS-
qNP and CPD-BSA@BS-qNP, which were initially non-
fluorescent (Supporting Information, Figures S14 and S15).
In live cells treated with these nanocapsules, we observed the
development of strong intracellular fluorescence signals with
cells under hypoxia (Figures 1 I, S14, and S15). The intra-
cellularly released FLBSA, successfully detected in the pro-
teome lysate (Figure 1 F), was further shown by CLSM to be
free of endolysosomal trapping (Supporting Information,
Figure S16). Hypoxic cells treated with CPD-BSA@BS-qNP
(nanoquenchers encapsulated with native BSA) showed
similar cytosolic fluorescence increase, with profiles similar
to those obtained from CPD-FLBSA@BS-qNP experiments
(Figure 1 I, bottom). In contrast, CPD-BSA@NonBS-qNP
showed negligible fluorescence, indicating no capsule degra-
dation (Supporting Information, Figure S17). Our newly
developed protein delivery system was further used to
image the intracellular protein release/cell state under
a different fluorescence channel, thus offering the prospect
of multiplexity in future applications (Supporting Informa-
tion, Figure S18). Finally, to ensure the intracellularly deliv-
ered native proteins retain their biological activity, nano-
capsules encapsulated with RNase A were prepared (CPD-
RNaseA@BS-NP). RNase A is a ribonuclease known to have
cytotoxic activities.[14] Upon successful nanocapsule formation
and in vitro release, it was found to still possess circa 84.8 % of
its original enzymatic activity (Supporting Information, Fig-
ure S19). Significant cell death was detected only in hypoxic
cells treated with CPD-RNaseA@BS-NP (Figures 1 J and the
Supporting Information, Figure S20), indicating our biode-
gradable nanocapsules were stable upon being taken up in
cells and the encapsulated protein remained active for
extended time periods until stimulus activation.
Having successfully demonstrated our hypoxia-respon-
sive, imaging-enabled delivery system for rapid, endocytosis-
independent intracellular release of functional, native pro-
teins, we next used them to deliver native therapeutic
antibodies. Cetuximab was chosen as the model because of
its well-established cellular profiles in EGFR-expressing
A431 cancer cells.[19] The Cetuximab-encapsulated biode-
gradable nanocapsules (Cetuximab@BS-NP), which is essen-
tially an hypoxia-responsive prodrug-like antibody, would be
cellularly active only upon successful uptake/intracellular
release in hypoxic A431 cells. As shown in Figure 2 A,
following optimized protocols, we obtained Cetuximab-
encapsulated nanocapules (Cetuximab@BS-NP) with an
average diameter of circa 50 nm. Both in vitro release and
live-cell imaging experiments with FLCetuximab@BS-NP and
CPD-Cetuximab@BS-qNP confirmed the successful release

Figure 2. A) TEM results of Cetuximab@BS-NP. B) Time-dependent fluorescence spectra (lex = 488 nm) of FLCetuximab@BS-NP (10 mg mL@1)
incubated at 37 8C with microsomes/NADPH. Inset: The corresponding time-dependent plots (1, FLCetuximab@BS-NP; 2, Cetuximab@BS-qNP; 3,
FLCetuximab@NonBS-NP) of fluorescence intensity (at lem = 515 nm). C) SDS-PAGE/in-gel fluorescence scanning of lysates from A431 cells
incubated with FLCetuximab@BS-NP (50 mg mL@1) under hypoxic and normoxic conditions. D) CLSM with 3D projections showing Z-stack images
of A431 cells treated with CPD-Cetuximab@BS-qNP (10 mg mL@1; 6 h) followed by incubation under normoxic and hypoxic conditions for different
lengths of time (24 and 48 h). Blue = Hoechst, green = FITC. Scale bar = 20 mm. E) WB analysis of EGFR signaling pathway in A431 cells treated
with Dacomitinb (70 nm) or Cetuximab (10 mg mL@1) for 4 h (see Figure S29 for results of longer treatments). F) % Viability (by XTT) of A431 cells
treated with Dacomitinib (70 nm) or Cetuximab (10 mg mL@1) for different lengths of time. G) WB analysis of EGFR signaling pathway in A431 cells
treated with different nanocasules. H) % Viability (by XTT) of different cells treated with different nanocapsules (50 mg mL@1, equivalent to
2.4 mg mL@1 of Cetuximab, 6-h incubation) followed by incubation under normoxic or hypoxic conditions (for 48 h). Error bars were obtained from
triplicate experiments.

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Communications
of encapsulated antibodies (Figures 2 B–D and the Support-
ing Information, Figure S23) and the in vitro-released
FLCetuximab still possessed most of its original bioactivity
(ca. 91.3 %; see the Supporting Information, Figure S24)
when compared to the original FLCetuximab. In a series of
cell-based experiments, CPD-Cetuximab@BS-NP together
with other control nanocapsules/samples were incubated with
A431 cells (Supporting Information, Figures S25–S28). Daco-
mitinib (a small molecule EGFR inhibitor) and Cetuximab
(directly added to cell media or delivered to A431 cells by
liposome transfection[17a]) were employed as positive controls.
Total EGFR and pEGFR (for example, EGFR phosphory-
lated at Y1068) expressions were closely monitored by western
blot analysis (Figure 2 E); as previously reported,[13a, 19] both
Dacomitinib and Cetuximab inhibited pEGFR expression
leading to time-dependent cell death (Figures 2 F and the
Supporting Information, Figure S29). Similar effects were
observed with liposome-delivered Cetuximab. Next, A431
cells were treated with CPD-Cetuximab@BS-NP followed by
further incubation under normoxic or hypoxic conditions
(Figure 2 G/H, labeled with *); only hypoxic A431 cells
treated with these nanocapsules showed the expected
decrease in pEGFR expression as well as significant cell
death. Hypoxic cells treated with control nanocapsules
showed no effect, nor did normoxic cells treated with CPD-
Cetuximab@BS-NP. A549 and CHO cells, two cell lines that
have weak and no endogenous EGFR expression, respec-
tively, showed insignificant cell-killing effect under both
hypoxic and normoxic conditions (Figure 2 H). Collectively,
our results thus reaffirmed what was discovered earlier,
namely, that these nanocapsules were able to protect the
encapsulated Cetuximab for extended time periods inside
live-cell environments and release the functional, native
antibody upon activation by hypoxia.
In conclusion, key qualities of our newly developed,
nanocapsule-based system capable of intracellular delivery of
prodrug-like antibodies might provide great opportunities in
future protein-based therapy for targeting of other intra-
cellular antigens/targets.
Acknowledgements
Funding was provided by National Medical Research Council
(CBRG/0038/2013) and GSK-EDB Trust Fund, and NNSF of
China (51403065).
Conflict of interest
The authors declare no conflict of interest.
Keywords: antibodies · bioimaging · drug delivery · hypoxia ·
nanoparticles
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Manuscript received: May 31, 2017
Revised manuscript received: July 24, 2107
Accepted manuscript online: August 17, 2017
Version of record online: September 7, 2017
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