Transferrin-Appended Nanocaplet for Transcellular siRNA Delivery into Deep Tissues

Article abstract of DOI:10.1021/jacs.8b12501

Transferrin (Tf) is known to induce transcytosis, which is a consecutive endocytosis/exocytosis event. We developed a Tf-appended nanocaplet (^TfNC?siRNA) for the purpose of realizing siRNA delivery into deep tissues and RNA interference (RNAi)

Full text of DOI:10.1021/jacs.8b12501

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Communication
Transferrin-Appended Nanocaplet for
Transcellular siRNA Delivery into Deep Tissues
Ai Kohata, P.K. Hashim, Kou Okuro, and Takuzo Aida
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b12501 • Publication Date (Web): 06 Feb 2019
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Transferrin-Appended Nanocaplet for Transcellular siRNA Delivery
into Deep Tissues
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†
†
,†
,†,‡
Ai Kohata, P. K. Hashim, Kou Okuro,* Takuzo Aida*
†Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
1
13-8656, Japan
‡
Riken Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Supporting Information Placeholder
deep tissues, the carrier is preferred to be as small as possible7
ABSTRACT: Transferrin (Tf) is known to induce transcyto-
and needs to be reductively cleavable in cytoplasm. Fur-
thermore, it should be active for transcytosis (consecutive
endocytosis/exocytosis events). Although a few carriers that
bear particular ligands for activating transcytosis such as
sis, which is a consecutive endocytosis/exocytosis event. We
developed a Tf-appended nanocaplet ( NC⊃siRNA) for the
purpose of realizing siRNA delivery into deep tissues and
RNA interference (RNAi) subsequently. For obtaining
Tf
8
9
Tf
Az
transferrin (Tf), Tf-receptor binding peptides, and RGD
NC⊃siRNA, a macromonomer ( Gu) bearing multiple
1
0
+
peptides have been reported, successful examples of siRNA
delivery feature the best delivery depth of only 20–40 µm,9
most likely due to their large dimensions. Namely, siRNA
delivery into tissues deeper than 40 µm still remains a big
challenge.
guanidinium (Gu ) ion units, azide (N
Trt)-protected thiol groups in the main chain, side chains,
and termini, respectively, was newly designed. Because of a
3
) groups, and trityl
a,10a
(
+
Az
multivalent Gu –phosphate salt-bridge interaction, Gu can
adhere to siRNA along its strand. When I
2
was added to a
Az
preincubated mixture of Gu and siRNA, oxidative polymer-
Herein, we report a siRNA-containing nanocaplet append-
Az
Tf
ization of Gu took place along the siRNA strand, affording
ed with Tf units ( NC⊃siRNA, Figure 1e), which can deeply
Az
NC⊃siRNA, the smallest siRNA-containing reactive
deliver siRNA into tissues at depth of up to nearly 70 µm,
nanocaplet so far reported. This conjugate was converted
unprecedentedly (Figure 2). Prior to the present work, we
Glue/BP
+
11
into
NC⊃siRNA by the click reaction with a Gu -
developed water-soluble molecular glues bearing multiple
+
appended bio-adhesive dendron (Glue) followed by a ben-
guanidinium (Gu ) ion units that can strongly adhere to pro-
1
2
13
14
zophenone derivative (BP). Then, Tf was covalently immo-
teins, nucleic acids, phospholipid membranes, and even
clay nanosheets15 through multiple salt-bridge interactions
with their oxyanionic functionalities. We found that a siR-
NA strand can template oxidative polymerization of a Gu -
Glue/BP
+
bilized onto
NC⊃siRNA by Gu -mediated adhesion
1
6
followed by photochemical reaction with BP. With the help
of Tf-induced transcytosis, NC⊃siRNA permeated deeply
+
Tf
+
appended telechelic macromonomer carrying four Gu units
into a cancer spheroid, a 3D-tissue model, at depth of up to
nearly 70 µm, unprecedentedly.
as well as thiol (SH) termini, affording a siRNA-containing
nanocaplet NC (NC⊃siRNA) with a hydrodynamic diame-
ter (D
h
) of <10 nm.1 NC is the smallest nanocaplet for
3a
siRNA so far reported. The disulfide (SS) bonds forming the
Tissue-permeable nanocarriers for small interfering RNA
17
NC part are readily cleaved off under reductive conditions.
Hence, in cytoplasm that contains glutathione (GSH) abun-
(
siRNA) are essential for the realization of RNA interference
1
(RNAi)-based gene therapy for diseases that develop in
dantly (Figure 1d), NC⊃siRNA is possibly disrupted to lib-
deep tissues at depth of >40 µm from the vascular endotheli-
1
3a
erate siRNA that causes RNAi. We envisioned that its Tf-
2
um. For instance, metastatic cancers often spread over tis-
Tf
appended version ( NC⊃siRNA) may deliver siRNA into
3
sues at depth of up to ~300 µm from blood vessels. For the
deep tissues.18 In the present study, we synthesized a siRNA-
tissue permeation of nanocarriers, paracellular and transcellu-
Az
containing reactive nanocaplet NC⊃siRNA (Figure 1e) by
4
lar pathways are considered. However, paracellular siRNA
Az
oxidative polymerization of Gu, an azide (N
3
Az
)-appended
5
delivery is unlikely since intercellular gaps (5–10 nm) are
6
telechelic macromonomer (Figure 1a). Then, NC⊃siRNA
was allowed to react with a Gu -appended bioadhesive den-
dron (Glue-alkyne) followed by a benzophenone (BP) deriv-
not large enough for siRNA (~5 nm) to pass through. For
+
the purpose of achieving transcellular delivery of siRNA into
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Az
+
Figure 1. Molecular structures of (a) Gu, an azide (N
3
)-appended telechelic macromonomer bearing guanidinium (Gu ) ions and
+
trityl (Trt)-protected thiol (STrt) termini, (b) Glue-alkyne bearing Gu -appended dendron (Glue) and an alkyne group at the focal
core, and (c) BP-alkyne (alkyne-appended benzophenone). (d) Iodine (I
2
)-mediated disulfide formation between Trt-protected
thiols and reductive cleavage of a disulfide bond into thiols by glutathione (GSH). (e) Synthesis of a siRNA-containing nanocaplet
Tf
Az
appended with transferrin (Tf) units ( NC⊃siRNA): Gu is oxidatively polymerized using siRNA as a template to yield a siRNA-
Az
containing N
3
-appended nanocaplet ( NC⊃siRNA). Then, Glue-alkyne and BP-alkyne are allowed to react successively with
Az
NC⊃siRNA (click reactions) to yield Glue/BPNC⊃siRNA. At the initial stage, Tf is noncovalently attached to the surface of
Glue/BP
+
NC⊃siRNA through a multivalent salt-bridge interaction with the dendritic Gu pendants and then immobilized covalently by a
photochemical reaction with BPs.
ative (BP-alkyne) to obtain Glue/BPNC⊃siRNA (Figure 1e).
Subsequently, this conjugate was incubated with Tf, and the
mixture was exposed to UV light for covalent immobilization
of the attached Tf units by reacting with the BP units, afford-
ing NC⊃siRNA (Figure 1e). As highlighted in this com-
munication, NC⊃siRNA deeply permeated into a cancer
spheroid, a 3D tissue model, at depth of up to nearly 70 µm.
19
TOF mass spectrometry (Figure S5) after the reductive
Glue/BP
depolymerization of
(DTT).
NC⊃siRNA using dithiothreitol
Tf
NC⊃siRNA was successfully obtained by 10-min UV ir-
Tf
Glue/BP
radiation (λ = 310 nm) of a mixture of
NC⊃siRNA
Tf
([siRNA] = 0.2 µM) and Tf (1.1 µM; Figure 1e). Dynamic
Tf
light scattering (DLS) analysis indicated that NC⊃siRNA
has a D value of 15.8 ± 5.0 nm (Figure 3c, blue), which is
This unprecedented achievement possibly takes advantage of
h
Tf
the small dimensional aspect of NC⊃siRNA and its
reasonable considering that the
D
h
values of
transcytosis activity mediated by the surface Tf units.
Glue/BP
NC⊃siRNA and Tf are 6.1 ± 0.7 nm (Figure 3c, red)
and 7.1 ± 0.2 nm (Figure S6), respectively. Accordingly,
cryogenic transmission electron microscopy (cryo-TEM)
AzGu, Glue-alkyne, and BP-alkyne (Figures 1a–c) were syn-
thesized according to the procedures described in the Sup-
porting Information and characterized unambiguously using
a variety of analytical methods.19 NC⊃siRNA (Figure 1e)
19
Tf
allowed for visualizing NC⊃siRNA as spherical objects with
Az
a diameter of ~20 nm (Figure 3d). For the purpose of char-
acterizing its charge density by means of agarose gel electro-
phoresis, we carried out the above reaction (Figure 1e, right)
was prepared in the presence of siRNA by deprotection of the
Az
Trt groups of Gu using I
2
and subsequent oxidative
Rhd
polymerization of the deprotected macromonomer (Figure
NC⊃siRNA was obtained by the copper-catalyzed
successive ‘click’ reactions of Glue-alkyne (Figure 1b) and
BP-alkyne (Figure 1c) with the azide groups of NC⊃siRNA
(Figure 1e), where the ratio of Gu /BP/N
using rhodamine-attached fluorescent Tf ( Tf). As shown
in Figure 3a (right), NC⊃siRNA migrated toward a nega-
tive electrode side, whereas, in the absence (Figure 3a, left)
NC⊃siRNA, nega-
tively charged Tf migrated toward a positive electrode side.
Accordingly, NC⊃siRNA displayed a positive zeta-
Glue/BP
Tf
1
d).
Az
Glue/BP
and presence (Figure 3a, center) of
+
Rhd
3
in
Glue/BP
Tf
NC⊃siRNA was estimated as 23/5/22 by MALDI-
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Figure 3. (a) Agarose gel electrophoretic profiles (λext = 495
Rhd
nm) of rhodamine-attached fluorescent Tf ( Tf, 1.1 µM) be-
Glue/BP
fore and after being mixed with
NC⊃siRNA ([siRNA] =
0
.2 µM), followed by 10-min photoirradiation at 310 nm. (b)
Agarose gel electrophoretic profiles (λext = 495 nm) of siRNA
fluorescently labeled with Alexa Fluor 488 (siRNA , 0.5 µM)
and NC⊃siRNA ([siRNA ] = 0.5 µM) before and after
A488
Tf
A488
A488
Tf
Figure 2. Permeation of NC⊃siRNA into cells located in a
1
1
-h incubation at 25 °C in the presence of dithiothreitol (DTT,
0 mM). (c) Dynamic light scattering histograms in HEPES
Tf
deep tissue via Tf-mediated transcytosis. Once NC⊃siRNA
escapes from the endosomes in a cell, glutathione (GSH),
abundantly present in the cytoplasm, liberates siRNA to cause
Glue/BP
buffer (20 mM, pH 7.3) at 25 °C of
NC⊃siRNA ([siRNA]
Tf
= 2 µM, red), NC⊃siRNA ([siRNA] = 2 µM, blue) and
RNAi by reductive depolymerization of the nanocaplet part
BSA
Tf
NC⊃siRNA ([siRNA] = 2 µM, green). (d) A cryogenic
(
NC).
transmission electron microscopy (cryo-TEM) image of a
HEPES buffer (20 mM, pH 7.0) solution of NC⊃siRNA
Tf
([siRNA] = 3 µM). Inset: a magnified image of (d).
potential (ζ) of 5 ± 2 mV (Table S1).19 TfNC⊃siRNAA488
A488
incorporating Alexa Fluor 488-labeled siRNA (siRNA ; 1
µM) displayed the same electrophoretic tendency as
Tf
λext = 488 nm). As shown in Figure 4a, the cells became fluo-
rescent, indicating the incorporation of fluorescently labeled
NC⊃siRNA (Figure 3b, center). As described above, the
SS bonds in NC are readily cleaved off in a reductive envi-
A488
Tf
A488
siRNA
into the cells, whereas the cells incubated with
ronment. In fact, when NC⊃siRNA was incubated with
DTT (10 mM) for 1 h at 25 °C in HEPES buffer, the electro-
phoretic profile of the reaction mixture became broadened
toward a positive electrode side (Figure 3b, right), indicating
Az
A488
A488
NC⊃siRNA
(Figure 4c) or naked siRNA
(Figure
A488
4
d) ([siRNA ] = 50 nM) did not fluoresce. When the Tf
Tf
A488
units in NC⊃siRNA
were replaced with bovine serum
albumin (BSA; BSANC⊃siRNAA488), the fluorescence emis-
Tf
the occurrence of reductive breakdown of the NC part
1
9
sion from Hep3B cells was again negligibly weak (Figure 4b),
(
Figure S8).
BSA
Tf
although NC⊃siRNA has a comparable size (D = 18.2 ±
h
NC⊃siRNA enters living cells via endocytosis: Human
4
0.3 nm; Figure 3c, green) and surface charges (ζ = 3 ± 0.4
hepatocellular carcinoma Hep3B cells (1.0 × 10 cells/well)
1
9
Tf
mV; Table S1) to those of NC⊃siRNA (D
h
= 15.8 ± 5.0
were incubated for 4 h at 37 °C in a serum-free minimal es-
Tf
A488
nm and ζ = 5 ± 2 mV; Figure 3c, blue and Table S1, respec-
sential medium (MEM, 200 µL) containing NC⊃siRNA
1
9
A488
tively). It should be noted that at 4 °C, where endocytosis is
(
[siRNA ] = 50 nM). Then, the sample was rinsed with
2
0
known to be suppressed, Hep3B cells barely took up
Dulbecco’s phosphate buffered saline (D-PBS, 100 µL × 2),
and subjected to confocal laser scanning microscopy (CLSM;
Tf
A488
19
NC⊃siRNA (Figure S7). Hence, the cellular uptake of
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Figure 4. (a–d) Confocal laser scanning microscopy (CLSM; λext = 488 nm, λobs = 510–590 nm) images of Hep3B cells after a 4-h in-
Tf
A488
A488
BSA
A488
A488
cubation at 37 °C in MEM with (a) NC⊃siRNA
([siRNA ] = 50 nM), (b) NC⊃siRNA
([siRNA ] = 50 nM), (c)
Az
A488
A488
A488
A488
NC⊃siRNA ([siRNA ] = 50 nM), and (d) siRNA ([siRNA ] = 50 nM). Scale bars = 25 µm. (e) Schematic illustration
of the experimental setup for obtaining cross-sectional CLSM images of a Hep3B spheroid. (f–g) Cross-sectional CLSM (λext = 488
nm, λobs = 510–590 nm) images of Hep3B spheroids at depth of 10, 30, 50, and 70 µm from the surface after a 3-day incubation at
Tf
A488
A488
Az
A488
A488
3
7 °C in MEM (10% FBS) in the presence of (f) NC⊃siRNA ([siRNA ] = 100 nM), (g) NC⊃siRNA ([siRNA ] = 100
A488
nM), and (h) siRNA (100 nM). Scale bars = 100 µm.
Tf
19
Tf
A488
NC⊃siRNA occurs most likely via endocytosis, and this
cell-repellent plates, and incubated with NC⊃siRNA
Tf
A488
process is mediated by the Tf units in NC⊃siRNA.
([siRNA ] = 100 nM) in MEM (100 µL, 10% FBS) for 3
days at 37 °C. After being rinsed with D-PBS buffer (100 µL
× 2), the resultant spheroid was subjected to CLSM (λext
88 nm). Fortunately, we found that the spheroid fluoresced
The above observations prompted us to investigate wheth-
Tf
=
er NC⊃siRNA indeed activates transcytosis or not. For this
purpose, we prepared a 3D-cultured Hep3B spheroid with an
average diameter of ~500 µm (Figure 4e) by using 96-well
4
throughout its cross sections at depth of 10, 30, and 50 µm
(Figure 4f).
Notably, even at depth of 70 µm,
Tf
A488
NC⊃siRNA reached the central part of the cross section
(Figure 4f), indicating the deep permeation of
NC⊃siRNA . In sharp contrast, the spheroid barely flu-
Tf
A488
Az
A488
oresced when incubated with NC⊃siRNA
(Figure 4g)
A488
A488
and siRNA
(Figure 4h) ([siRNA ] = 100 nM), in con-
sistency with their poor cellular uptake behaviors (Figures 4c
and 4d, respectively). All these observations indicate that
Tf
NC⊃siRNA activates transcytosis.
TfNC⊃siRNA successfully induced RNAi to suppress the
expression of a target gene. Thus, mutant Hep3B cells stably
expressing luciferase (Hep3B-luc) were incubated for 72 h at
Tf
3
7 °C in MEM (10% FBS) containing NC⊃siRNA ([siR-
Figure 5. (a) Normalized luciferase activities of Hep3B-luc cells
using the PicaGene LT 2.0 luciferase assay kit. Here, Hep3B-luc
cells were incubated at 37 °C for 72 h in MEM (10% FBS) in the
NA] = 50 nM). Then, the sample was subjected to the lucif-
erase activity assay using PicaGene LT 2.0 as a luminescence
reagent (TOYO INK). As shown in Figure 5a, the cells incu-
mis
presence of siRNA (50 nM, black bar), siRNA (50 nM, gray
Tf
Tf
mis
bated with NC⊃siRNA exhibited a much lower luciferase
bar), NC⊃siRNA ([siRNA or siRNA ] = 50 nM; siRNA and
mis
siRNA , purple and light purple bars, respectively), and
activity (purple bar, 30%) than the reference cells incubated
with naked siRNA (black bar). Similarly, siRNA conjugated
with DharmaFECT 1 (DFECT), a commercially available
transfection agent, suppressed the luciferase activity as ex-
mis
DFECT⊃siRNA ([siRNA or siRNA ] = 50 nM; siRNA and
mis
siRNA , green and light green bars, respectively). (b) Normal-
ized cell viabilities of Hep3B cells using Cell Counting Kit-8.
Hep3B cells were incubated at 37 °C for 24 h in MEM (10%
pected (Figure 5a, green bar, 7%).
NC⊃siRNA , obtained with mismatched siRNA (siR-
NA ) that is incapable of inducing RNAi for the luciferase
As a control,
Tf
Tf
mis
FBS) in the presence of NC⊃siRNA ([siRNA] = 25, 50, 100,
mis
and 200 nM).
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Tf
in Development of siRNA Delivery Vehicles for Cancer Therapy. Adv. Drug
Delivery Rev. 2016, 104, 61.
gene, was used instead of NC⊃siRNA for the above exper-
iment. As shown in Figure 5a, the luciferase activity of
Hep3B-luc cells remained substantially unchanged (light
purple bar, 87%). Hence, the decrease in the luciferase activi-
ty observed in Figure 5a (purple bar) indicates the occur-
1
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(3) Nacev, A.; Kim, S. H.; Rodoriguez-Canales, J.; Tangrea, M. A.;
Shapiro, B.; Emmert-Buck, M. R. A Dynamic Magnetic Shift Method to
Increase Nanoparticle Concentration in Cancer Metastases: A Feasibility
Study Using Simulations on Autopsy Specimens. Int. J. Nanomed. 2011, 6,
2907.
(4) (a) Tuma, P. L.; Hubbard, A. L. Transcytosis: Crossing Cellular Bar-
riers. Physiol. Rev. 2003, 83, 871. (b) Zheng, M.; Tao, W.; Zou, Y.;
Farokhzad, O. C.; Shi, B. Nanotechnology-Based Strategies for siRNA Brain
Delivery for Disease Therapy. Trends Biotechnol. 2018, 36, 562.
Tf
rence of RNAi. Fortunately, NC⊃siRNA did not show any
appreciable cytotoxicity with its concentration up to 200 nM
(
Figure 5b).
In conclusion, we developed a transferrin (Tf)-appended
siRNA nanocaplet ( NC⊃siRNA) capable of delivering
siRNA into deep tissues at depth of up to ~70 µm. As
demonstrated with a cancer spheroid, NC⊃siRNA perme-
ates into deep areas of tissues via transcytosis (Figure 2).
NC⊃siRNA eventually transfers siRNA into cytoplasm and
causes RNAi and gene knockdown (Figure 2). Because brain
endothelial cells are known to express high level of Tf-
receptors, NC⊃siRNA has the potential for overcoming
the blood-brain barrier (BBB). Thus, an in vivo study on
siRNA delivery to brain tissues using NC⊃siRNA is a sub-
ject worthy of further investigation.
(5) (a) Friend, D. S.; Gilula, N. B. Variations in Tight and Gap Junctions
Tf
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2
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ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website.
(9) (a) Wei, L.; Guo, X.; Yang, T.; Yu, M.; Chen, D.; Wang, J. Brain Tu-
Synthesis of Gu, Glue-alkyne, and BP-alkyne; H NMR, 13C
Az
1
mor-Targeted Therapy by Systemic Delivery of siRNA with Transferrin
Receptor-Mediated Core-Shell Nanoparticles. Int. J. Pharm. 2016, 510, 394.
(b) Youn, P.; Chen, Y.; Furgeson, D. Y. A Myristoylated Cell-Penetrating
Peptide Bearing a Transferrin Receptor-Targeting Sequence for Neuro-
Targeted siRNA Delivery. Mol. Pharmaceutics 2014, 11, 486.
NMR, and MALDI-TOF-MS spectral data; and related experi-
mental procedures (PDF)
AUTHOR INFORMATION
(10) (a) Waite, C. L.; Roth, C. M. PAMAM-RGD Conjugates Enhance
Corresponding Authors
okuro@macro.t.u-tokyo.ac.jp; aida@macro.t.u-tokyo.ac.jp
siRNA Delivery Through a Multicellular Spheroid Model of Malignant
Glioma. Bioconjugate Chem. 2009, 20, 1908. (b) Jiang, X.; Xin, H.; Gu, J.;
Xu, X.; Xia, W.; Chen, S.; Xie, Y.; Chen, L.; Chen, Y.; Sha, X.; Fang, X. Solid
Tumor Penetration by Integrin-Mediated Pegylated Poly(trimethylene
carbonate) Nanoparticles Loaded with Paclitaxel. Biomaterials 2013, 34,
Notes
The authors declare no competing financial interest.
1
739. (c) Liu, X.; Lin, P.; Perrett, I.; Lin, J.; Liao, Y.; Chang, C. H.; Jiang, J.;
Wu, N.; Donahue, T.; Wainberg, Z.; Nel, A. E.; Meng, H. Tumor-
Penetrating Peptide Enhances Transcytosis of Silicasome-Based Chemo-
therapy for Pancreatic Cancer. J. Clin. Invest. 2017, 127, 2007.
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Early-Career Sci-
entists (18K14353 and 18K14270) to K.O. and P.K.H., respec-
tively, and partially supported by Grant-in-Aid for Scientific
Research (S) (18H05260) to T.A. A.K. thanks JSPS for the
world-leading innovative graduate study program for life science
and technology (WINGS-LST). We appreciate Prof. H. Cabral
and Dr. Y. Anraku (the University of Tokyo) for zeta-potential
measurements and fruitful discussions on transcytosis assays
using cancer spheroids.
(11) Mogaki, R.; Hashim, P. K.; Okuro, K.; Aida, T. Guanidinium-Based
‘‘Molecular Glues’’ for Modulation of Biomolecular Functions. Chem. Soc.
Rev. 2017, 46, 6480.
(12) (a) Okuro, K.; Kinbara, K.; Tsumoto, K.; Ishii, N.; Aida, T. Molecu-
lar Glues Carrying Multiple Guanidinium Ion Pendants via an Oligoether
Spacer: Stabilization of Microtubules against Depolymerization. J. Am.
Chem. Soc. 2009, 131, 1626. (b) Okuro, K.; Kinbara, K.; Takeda, K.; Inoue,
Y.; Ishijima, A.; Aida, T. Adhesion Effects of a Guanidinium Ion Appended
Dendritic “Molecular Glue” on the ATP-Driven Sliding Motion of Actomy-
osin. Angew. Chem., Int. Ed. 2010, 49, 3030. (c) Uchida, N.; Okuro, K.;
Niitani, Y.; Ling, X.; Ariga, T.; Tomishige, M.; Aida, T. Photoclickable
Dendritic Molecular Glue: Noncovalent-to-Covalent Photochemical Trans-
formation of Protein Hybrids. J. Am. Chem. Soc. 2013, 135, 4684. (d) Gar-
zoni, M.; Okuro, K.; Ishii, N.; Aida, T.; Pavan, G. M. Structure and Shape
Effects of Molecular Glue on Supramolecular Tubulin Assemblies. ACS
Nano 2014, 8, 904. (e) Mogaki, R.; Okuro, K.; Aida, T. Molecular Glues for
Manipulating Enzymes: Trypsin Inhibition by Benzamidine-Conjugated
Molecular Glues. Chem. Sci. 2015, 6, 2802. (f) Okuro, K.; Sasaki, M.; Aida,
T. Boronic Acid-Appended Molecular Glues for ATP-Responsive Activity
Modulation of Enzymes. J. Am. Chem. Soc. 2016, 138, 5527. (g) Mogaki, R.;
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