DOTA-Branched Organic Frameworks as Giant and Potent Metal Chelators

Article abstract of DOI:10.1021/jacs.9b09269

Multinuclear complexes as metallo-agents for clinical use have caught extensive attention. In this paper, using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as both a functioning unit and a constructing junction, we build a series of DOTA-branched organic frameworks with multiple chelating holes by organizing DOTA layer by layer. These giant chelators are well characterized, which reveals their nanosized and soft structures. Further experiments demonstrate that they could efficiently hold abundant metal ions with much higher kinetic stabilities than the conventional small DOTA chelator. Their corresponding polynuclear complexes containing Gd³⁺, Tb³⁺, or both show superior imaging properties, excellent feasibility for peripheral modification, and unusual kinetic stability. This work can be easily extended to the fabrication of diverse homomultinuclear complexes and core/shell heteromultinuclear complexes with multifunctional properties. We expect that this new type of giant molecules and the ligand-branching strategy would open up a new avenue for the design and construction of next-generation polymetallic agents with high performance and stabilities for biomedical applications.

Full text of DOI:10.1021/jacs.9b09269

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Article
DOTA-Branched Organic Frameworks as Giant and Potent Metal Chelators
Chengjie Sun, Hongyu Lin, Xuanqing Gong, Zhaoxuan Yang, Yan Mo, Xiaoyuan Chen, and Jinhao Gao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b09269 • Publication Date (Web): 11 Dec 2019
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DOTA-Branched Organic Frameworks as Giant and Potent Metal
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Chengjie Sun, Hongyu Lin,†,# Xuanqing Gong, Zhaoxuan Yang, Yan Mo, Xiaoyuan Chen, and
Jinhao Gao*
†
,#
†
†
†
‡
,†
†State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Laboratory of Spectrochemical Analysis
Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology,
&
College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China
‡Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering,
National Institutes of Health, Bethesda, MD 20892, United States
ABSTRACT: Multinuclear complexes as metallo-agents for clinical use have caught extensive attention. In this paper, using
DOTA as both a functioning unit and a constructing junction, we build a series of DOTA-branched organic frameworks (DBOF)
with multiple chelating holes by organizing DOTA layer by layer. These giant chelators are well characterized, which reveals
their nano-sized and soft structures. Further experiments demonstrate that they could efficiently hold abundant metal ions
with much higher kinetic stabilities than the conventional small DOTA chelator. Their corresponding polynuclear complexes
3
+
3+
containing Gd , Tb , or both show superior imaging properties, excellent feasibility for peripheral modification, and unusual
kinetic stability. This work can be easily extended to the fabrication of diverse homo-multinuclear complexes and core/shell
hetero-multinuclear complexes with multifunctional properties. We expect that this new type of giant molecules and the
ligand-branching strategy would open up a new avenue for the design and construction of next-generation polymetallic
agents with high performance and stabilities for biomedical applications.
ions with relatively high toxicity.1 Attention was then
INTRODUCTION
turned
to
1,4,7,10-tetraazacyclododecane-1,4,7,10-
Metal ion-based agents play an indispensable role in
tetraacetic acid (DOTA) and its derivatives (DOTAs) of
better thermodynamic and kinetic stabilities.
3
+
2+
11-15
biomedicine. Paramagnetic metal ions including Gd , Mn ,
In fact,
3
+
and Fe reside in the core of contrast agents for magnetic
gadolinium complexes of DOTAs (Gd-DOTA, Gd-HP-DO3A,
and Gd-DO3A-butrol) have been very successful as MRI
1
resonance imaging (MRI). Radioactive metal isotopes, such
9
0
3+ 111 3+ 177
3+
64
2+
1
68
as Y , In , Lu , and Cu , have been widely used in
clinic for single-photon emission computerized
tomography (SPECT)/positron-emission tomography
contrast agents in clinic. Recently, Ga-DOTA-TATE
®
177
®
(NETSPOT ) and Lu-DOTA-TATE (LUTATHERA ) have
also been approved by the FDA as radiopharmaceuticals for
PET and radiation therapy, respectively.
2
16
(
PET) and radiation therapy. In addition, some metal ions
own unique therapeutic effects for certain diseases, such as
Meanwhile, chelators with multiple holding sites for metal
2
+
3
3+
Pt in cisplatin for cancers and Bi for Helicobacter pylori-
12, 17-22
ions have caught extensive attention.
They could form
4
related gastrointestinal disorders. However, most of these
polymetallic complexes with appropriate metal ions,
realizing synchronous delivery of multiple metal ions
functional metal ions are either toxic or not stable in the
5
body. A simple way to overcome these restrictions is to
(
either the same or different), which would significantly
form safe and stable complexes with chelators for better
biocompatibility and bioavailability.
increase the efficiency and sensitivity of the agents, or
achieve multifunctionalities.
1, 23-26
Besides, synergistic
Many efforts have been dedicated to the development of the
effects could be achieved through the cooperation among
different metal centers within the same molecule. For
example, multinuclear platinum complex BBR3464 has
been shown to be superior over cisplatin and entered Phase
6
-10
chelators
for
these
functional
metal
ions.
Diethylenetriamine pentaacetic acid (DTPA) and its
derivatives (DTPAs) was attractive during the early days
and frequently utilized for chelating these diagnostic or
therapeutic metal ions. Unfortunately, the correlation
between the use of Gd-DTPAs and the onset of nephrogenic
systemic fibrosis (NSF) resulted in a “black box warning”
from the U. S. Food and Drug Administration (FDA) and the
cessation of their clinical use for chelating functional metal
2
7-29
II clinical trials.
In addition, this cooperation could also
lead to new functions that cannot be accomplished by a
single metal site or a simple mixture of metal complexes,
which is demonstrated by the functioning of many
30
metalloenzymes and artificial enzymes developed based
21, 31-33
on them.
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Figure 1. Structure of DOTA-branched organic frameworks. (a) Schematic illustration of the structure of DOTA-branched
organic frameworks (DBOF), which is essentially different from traditional DOTA-modified dendrimers. (b) DO3AtBu-NH
could be used as a protected building block to construct DBOF divergently. F = functional group (-NH ), P = protective group
, EDC·HCl, HOBT, DIPEA,
2
2
(-tBu), C = coupling group (-COOH). (c) Experimental fabrication of DBOF. i) Coupling: DO3AtBu-NH
2
DMF; ii) Deprotection: TFA, RT. * indicates the binding site for -NH- or -OH group.
Therefore, this type of chelators is appealing for the
development of metallo-agents with higher efficiency,
increased sensitivity, better biosafety and potential
multifunctionality.
that this new giant molecular structure is completely
different from the simple conjugates of traditional
38-39
dendrimers and ligands in previous works.
To achieve
the fabrication of such giant chelators, we developed a
ligand-branching strategy that utilizes a small ligand as
both the functioning unit and the constructing junction. In
this work, DOTA was chosen as a ligand example to
demonstrate the strategy explicitly. These DOTA-based
giant chelators, named DOTA-branched organic
frameworks (DBOF), could be assembled divergently from
a central molecule derived from DOTA with repeating
attachments of 2-aminoethyl-monoamide-DOTA-tris(t-Bu
To further exploit the potential of polymetallic complexes
for biomedical applications, we embarked on the search for
unique chelating structures. Inspired by the distinctive
structures of dendrimers and metal-organic frameworks
3
4-37
(MOFs) that have been widely studied and utilized,
we
herein designed a new type of frameworks (“giant
chelators”) with multiple chelating holes both in the interior
and on the periphery (Figure 1a), which are single-
molecular chelating structures with small-molecular
ligands as the building blocks of the backbone. It is noted
2
ester) (DO3AtBu-NH ) molecules as
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Figure 2. Characterization of DBOF giant chelators. (a) MALDI-TOF mass spectra of G1~G4. (b) GFC traces of G1~G4. (c) DLS
intensity distributions of G2~G4. (d) Typical TEM images of G4. (e) An image of G4 by atomic force microscopy. (f) Schematic
illustration showing the deformation of DBOF on a plate.
spectroscopy (Figure S1-S17). Matrix-assisted laser
protected junctions (Figure 1b). According to this strategy,
desorption ionization time of flight mass spectrometry
we successfully synthesized DBOF with a series of
(MALDI-TOF-MS) results reveal the distributions of the
generations. As expected, DBOF could efficiently chelate
molecular masses of G2~G4 (Figure 2a), which is probably
metal ions to form stable giant polymetallic complexes (M-
due to structural imperfections and/or molecular
DBOF). These M-DBOF complexes (e.g., Gd-DBOF and Tb-
fragmentation by high laser power. The structural
DBOF) show superior imaging properties than their
imperfections may arise from the strong steric congestion
corresponding M-DOTA complexes. Besides, DBOF could be
on the periphery of the macromolecules, and the
easily functionalized on the periphery (e.g., PEGylation)
fragmentation is inevitable since high laser power is
because of abundant free carboxylic acid groups. More
4
0
necessary to ionize these macromolecules. Therefore, the
number of DOTA structures in each layer was estimated
using the central molecular masses of the distributions. The
numbers of the peripheral groups and DOTA chelating holes
of G1~G4 are summarized in Table S1. The gel filtration
chromatography (GFC) results (Figure 2b) suggest that the
size of the DBOF grows with the increase of generation as
expected, whereas the size distribution in each generation
remains narrow. The hydrodynamic diameters of the
frameworks were 4.1nm (PdI: 0.117), 6.9 nm (PdI: 0.200),
and 9.4 nm (PdI: 0.185) for G2, G3, and G4, respectively
interestingly, DBOF can chelate with different metal ions to
form hetero-multinuclear complexes with multifunctional
properties. Furthermore, kinetic studies reveal that M-
DBOF complexes are much more kinetically stable than M-
DOTA complexes, which is extremely critical for in vivo
applications. These DBOF giant chelators hold great
potential as potent carriers for metal ions and would be
enlightening for the development of next-generation
metallo-agents for biomedical applications.
RESULTS AND DISCUSSION
Synthesis and characterizations of DBOF. Using
(
Figure 2c). No credible result was obtained for G1,
indicating that G1 is too small to be measured by dynamic
light scattering (DLS) analysis. To further visualize these
giant chelators, G4 was characterized by transmission
electron microscopy (TEM) (Figure 2d) and atomic force
microscopy (AFM) (Figure 2e). TEM shows that G4 was
uniformly circular with an average diameter of 11.6 nm,
while AFM images shows that G4 was elliptic on the mica
plate with an average height of 1.29 nm and an average
grain size of 15.70 nm. This oblate morphology revealed by
TEM and AFM, which is different from the spherical
morphology as indicated by DLS, is probably due to the
symmetrical dendritic structure of the giant chelators that
is soft, open, and deformable. This also accounts for the
significantly larger particle size measured by AFM and TEM
than the hydrodynamic diameter (Figure 2f). The observed
particle size by AFM was slightly larger than that by TEM,
which could be ascribed to the interaction between the tip
DO3AtBu-NH
2
as a protected branching junction, the
strategy of which has not been reported before, DBOF could
be rationally constructed based on an iterative strategy
(Figure 1b). Through the efficient EDC-HOBt coupling (step
i) and TFA deprotection (step ii) reactions, the DOTA-
derived chelating holes could be packed layer by layer,
maintaining the growth of DBOF. We chose a DOTA
derivative that has eight carboxylic acid groups (Figure 1c,
the structure in blue, and Scheme S1, Compound 5) as the
initial core to make the product after first construction
cycle, DBOF Generation 1 (denoted as G1), large enough for
dialysis, since purification by column chromatography
could lead to considerable yield loss. Based on this strategy,
we accomplished the synthesis of four generations, G1, G2,
G3, and G4, without tedious column chromatography as
detailed in Supporting Information.
41
The experimental chemical structures of G1~G4 are shown
in Figure 1c, which were characterized by mass
spectrometry and nuclear magnetic resonance (NMR)
of AFM and G4. These results demonstrate that the size of
the chelators reaches nanometers after a few synthesis
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cycles. This would confer certain properties that are unique
to nanomaterials
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Figure 3. Characterization and properties of Gd-DBOF complexes. (a) DLS intensity distributions (top) and GFC (bottom)
profiles of Gd-DBOF. (b) Typical TEM images of Gd-G4. (c) Comparison of r values between Gd-DBOF and Gd-DOTA at 1.5 T.
d) Nuclear magnetic relaxation dispersion (NMRD) profiles of Gd-DBOF at 25 C. (e) Changes on R of 1 M HCl solutions
1
(
2
containing the complexes at 25 C over time. (f) The lifetimes of Gd-DBOF in 1 M HCl were significantly longer than that of
Gd-DOTA. N.A., no available.
on the frameworks, such as high cellular uptake, long
unimolecular micelles. Even that metal complexes could be
chemically conjugated to the end groups of the core of the
micelle, such conjugation is difficult to control and would
restrict surface modifications and further affect the in vivo
retention time, and controllable in vivo behaviors, which are
25
highly desired for biomedical applications.
Gd-DBOF complexes. We firstly employed these
3
8, 42
3+
behaviors of the micelle.
In contrast, the periphery of M-
frameworks to chelate Gd
and investigated their
DBOF is open to various modifications and its in vivo
behaviors are almost free of interference from hydrophilic
metal complexes.
characteristics in metal chelation and their performance in
magnetic resonance imaging (MRI). MALDI-TOF-MS spectra
of Gd-G1 (Figure S18) demonstrate that nine metal ions
were quantitatively coordinated by one G1 molecule, which
is consistent with the number of DOTA chelating holes in
one G1 molecule. The metal contents (Table S2) of Gd-
DBOF complexes approached the metal content of the small
Gd-DOTA complex, which are higher than those of most
nanocarriers for metal ions, suggesting a considerable
efficiency of DBOF in carrying metal ions.
3
+
As Gd is widely used in clinical MRI, we further studied the
MRI-related properties of Gd-DBOF. The ionic longitudinal
relaxivities (r
3.6-fold, 4.0-fold, and 4.5-fold higher than that of Gd-DOTA
(Figure 3c), respectively. The high r of Gd-DBOF should be
ascribed to the longer rotational correlation times of the
giant complexes, which was evidenced by the peak at 10-
00 MHz in the nuclear magnetic relaxation dispersion
1
NMRD) profiles (Figure 3d). The T -weighted phantom
images of Gd-DBOF were significantly brighter than that of
Gd-DOTA at a given Gd concentration (Figure S20), which
1
) of Gd-G1~G4 at 1.5 T were about 2.4-fold,
1
1
(
DLS profiles (Figure 3a, top panel) reveal that the
hydrodynamic diameters of Gd-G2~G4 were 4.8 nm (PdI:
4
3
0
.139), 6.4 nm (PdI: 0.075), and 9.5 nm (PdI: 0.094),
3+
respectively. Notably, only little change in either
hydrodynamic diameter or size distribution was observed
during chelation, indicating that metal ions were embedded
into the ligands as expected. GFC profiles (Figure 3a, bottom
panel) of Gd-G1~G4 and TEM images (Figure 3b) of Gd-G4
indicate that these giant chelates were spherical and
monodispersed, which is inherited from DBOF. The average
particle size of Gd-G4 in TEM was 10.2 nm, which is slightly
smaller than that of G4 (11.6 nm), indicating that the
structure of Gd-G4 is more rigid than G4. These results
indicate that DBOF acts like a umimolecular micelle
customized for carrying hydrophilic metal complexes,
which is hard to be accomplished by traditional
umimolecular micelles since hydrophilic metal complexes
are difficult to be physically enwrapped into the core of
further demonstrates that Gd-DBOF could act as high-
performance contrast agents for T -weighted MRI.
1
We further explored the chelating stability of Gd-DBOF by
a classic relaxometric method. It is well-known that small
M-DOTAs complexes are highly kinetically stable, which
44
4
5-47
could hardly be achieved by other forms of chelation.
Surprisingly, under highly acidic conditions (1 M HCl), much
slower changes of proton transverse relaxation rates (R
were observed with Gd-DBOF than with Gd-DOTA (Figure
3e). Gd-DOTA was completely decomposed within 50 h,
while the decompositions for Gd-G2~G3 required more
than 1000 h. Unfortunately, the decomposition of Gd-G1
could not be evaluated by this method since the relaxivity of
2
)
3
+
Gd-G1 is similar to that of free Gd ion under acidic
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solutions. The exponential decay model was used to
quantitatively evaluate the lifetimes of these complexes in
the acidic solutions (detailed in the Supporting Information,
Table S3), which are shown in Figure 3f. All Gd-DBOF
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Figure 4. Tb-DBOF complexes for cell imaging. (a) DLS intensity distributions (top) and GFC (bottom) profiles of Tb-DBOF.
(b) Typical TEM images of Tb-G4. (c) Emission spectra of Tb-DBOF and Tb-DOTA (left) and an optical photograph of the
solution containing Tb-G2 with exciting light (right). (d) Confocal microscopy images of HeLa cells after incubation with Tb-
®
G2 (top) and Lysotracker Red (middle), the merged picture (bottom) reveals a significant degree of co-localization for both
®
probes. (e) Comparison of photostabilities of Tb-G2 and Lysotracker Green in live HeLa cells.
5
7
(Gd-G2~G4) showed significantly longer lifetimes than Gd-
DOTA. These results demonstrate that Gd-DBOF are much
more kinetically stable than Gd-DOTA, which is crucial for
between the D
4
state and the F
j
(j = 3~6) ground states
5
0
3+
(Figure 4c). Tb complexes are widely explored as
fluorescence probes for cell imaging. However, most of the
reported probes were small Tb complexes that are quickly
4
6, 48
biomedical applications.
5
1
excreted from cells. In our study, after incubated with
HeLa cells for 4 h, Tb-G2~G4 could stay in the cells while
Tb-DOTA and Tb-G1 were cleared (Figure S21a).
Kinetic stability is one of the critical factors governing the
design of metal-chelate-based agents, especially after the
3
+
discovery of the relevance between the utilization of Gd
4
9
chelates and NSF. Most of current approaches tackling the
problem of kinetic stability are focusing on the structure
optimization of small molecular ligands or simply attaching
multiple small molecular ligands to the periphery of a
certain polymeric backbone. For the first time, we
established a macromolecular approach to fabricate giant
chelators with multiple chelating ligands both in the
interior and on the periphery of dendritic frameworks.
Since these DBOF chelators are nanosized with dendritic
structure, it is feasible to impart certain properties desired
for biomedical applications, such as adjustable in vivo
behaviors and lesion targeting, through size manipulation
and post-construction functionalization.
Interestingly, Tb-G2~G4 were found to localize in
3
+
lysosomes. The degree of co-localization between the Tb
®
complexes and Lysotracker Red was evaluated by the
Pearson’s coefficient (Figure S21b), which is larger than 0.6
for each of Tb-G2~G4, indicating their accumulation in
lysosomes. Among them, Tb-G2 had the highest co-
localization degree (Figure 4d). It is well-known that
52
lanthanide-based fluorescences are highly photostable,
therefore, we examined the photostability of Tb-G2 in cells
®
with Lysotracker Green as a control. Upon continuous
®
irradiation, the fluorescence of Lysotracker Green faded
away within 20 min, while the fluorescence of Tb-G2
sustained even after 50 min (Figure 4e and Supplementary
movie 1), which indicates that Tb-G2 could stay in living
cells for a relatively long time. Tb-DBOF complexes are
promising potential fluorescence probes for long-time live
cell imaging and lysosome tracking.
Tb-DBOF complexes. To further demonstrate the
3
+
versatility of DBOF, we prepared their Tb complexes to
study the fluorescence imaging properties. The results of
DLS, GFC, and TEM characterizations of Tb-DBOF were
similar to those of Gd-DBOF (Figure 4a-b). The emission
Gd-G4-PEG complex. DBOF not only have chelating holes
in frameworks but also possess plentiful peripheral groups
(carboxylic acid groups) for further modifications (Table
3
+
fluorescence spectra of Tb-DBOF featured typical Tb -
based peaks, which originates from electron transitions
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S1). To showcase this advantage, we modified G4 with
methoxylPEG1000-amine (marked as G4-PEG), which is
widely used in biomedical applications.53 The successful
PEGylation of G4 was evidenced by MALDI-TOF-MS, DLS,
1
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Figure 5. The PEGylation on the periphery of DBOF and Gd-DBOF complexes. (a) DLS intensity distributions (top) and GFC
bottom) profiles of G4-PEG. (b) DLS intensity distributions (top) and GFC (bottom) profiles of Gd-G4-PEG. (c) Typical TEM
images of Gd-G4-PEG. (d) Changes on R of 1 M HCl solution containing Gd-G4-PEG at 25 C over time. (e) GFC profiles of FBS
before (top) and after (bottom) incubated with Gd-G4-PEG. (f) MTT assays of HeLa cells incubated with Gd-G4-PEG for 24 h.
(
2
Figure 6. Facile formation of core/shell hetero-multinuclear complexes with multifunctional properties. (a) Using Gd
2
O
3
as
3
+
3+
the source of Gd and TbCl
3
as the source of Tb , the Tb/Gd-G2 core/shell complex was synthesized based on the difference
in stability constant between the DOTA monoamide chelating holes in the shell and the DOTA tetraamide chelating holes in
the core. (b) Molar Gd:M ratio of the core/shell complexes. (c) Relaxivity measurements of the core/shell complexes at 0.5 T,
25 ˚C. (d) Emission spectra of Tb-G2, Gd/Tb-G2, and Tb/Gd-G2, λex = 254 nm. (e) Fluorescence images (top, λex = 254 nm)
and T MRI phantom images (bottom) of G2, Gd(shell)-G2, and Tb/Gd-G2 (0.5 T, 25 C, TR = 100 ms, TE = 0.004 ms).
1
GFC, and TEM. The result of MALDI-TOF-MS (Figure S19)
indicates that approximately 200 PEG chains were attached
to G4, which has about 330 peripheral carboxylic acid
groups. The hydrodynamic diameter increased from 9.4 nm
min), indicating a significant increase in particle size after
PEGylation. None of retention time or hydrodynamic
diameter changed during further chelation of G4-PEG with
3
+
Gd (marked as Gd-G4-PEG) (Figure 5b, hydrodynamic
(
(
5
PdI: 0.185) to 22.3 nm (PdI: 0.216) after PEGylation
Figure 5a, top panel). Concomitantly, GFC profiles (Figure
a, bottom panel) also reveal a significant change on
diameter: 19.5 nm, PdI: 0.159, retention time: 26.6 min),
3
+
suggesting that Gd were successfully implanted inside G4-
PEG. TEM reveals that Gd-G4-PEG inherited the high
monodispersity of G4 with a size of 15.2 nm (Figure 5c).
retention time of the product (G4: 30.7 min, G4-PEG: 26.5
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Journal of the American Chemical Society
-
1
-1
Relaxivity measurents show that the r
1
value (8.7 mM s )
1
Gd/Y-G2), while Gd-G2 has an average level of r value
1
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of Gd-G4-PEG at 0.5 T was smaller than that of Gd-G4 (15.0
mM s ). This decrease in r may be ascribed to the
1
decrease in the water exchange rate of Gd-G4-PEG, which is
caused by the amidation of the carboxylic acid groups in the
shell of G4 and the formation of the thick PEG shell. The
(12.1 mM s ) (Figure 6c) (see Supporting Information
S1.10 for details). The special properties found in these
complexes demonstrate the achievement of the core/shell
structure and reveal some of the distinct chelating
characteristics of these giant chelators. The quantum yields
(QYs) of the Tb complexes showed positive correlation with
-1 -1
-1
-1
exploration on kinetic stability further indicates that the R
2
3
+
change of Gd-G4-PEG (Figure 5d) was even slower than
those of Gd-DBOF in 1 M HCl solution. The lifetime of Gd-
G4-PEG was about 334 h, which is twenty-fold longer than
that of Gd-DOTA. No significant change in relaxivity or
hydrodynamic diameter was observed for Gd-G4-PEG and
Gd-G1~G4 during incubation in phosphate buffer saline (1
PBS) at 37 ˚C for 2 weeks (Figure S22), indicating that
these giant complexes are highly stable under physiological
conditions.
the number of Tb ions per molecule (Figure 6d and Table
S4), which could be attributed to the increased efficiency in
3
+
energy transfer between Tb
ions and organic
5
4
chromophores in the complexes. As expected, Tb/Gd-G2
and Gd/Tb-G2 are capable of acting as dual-modal probes
for both fluorescence imaging and contrast-enhanced MRI
(Figure 6e and S26), indicating the enormous potential of
DBOF as a platform for multimodal imaging. Further
investigations are ongoing in our laboratory.
0
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0

The extremely high kinetic stability of Gd-G4-PEG offers
great opportunities in fabrication of metallo-agents with
high biosafety. To this end, we further evaluated the
biocompatibility of Gd-G4-PEG. GFC analysis reveals few
interactions of Gd-G4-PEG with serum proteins (Figure 5e),
indicating no formation of protein corona on Gd-G4-PEG.
Furthermore, MTT assays (Figure 5f) show that Gd-G4-PEG
was of no apparent cytotoxicity even at up to 600 M with
respect to Gd . Time dependent MRI of several organs and
Gd biodistribution analysis (Figure S23) reveal that Gd-G4-
PEG may have a favorable in vivo behavior for further
applicaions in biomedical imaging. These preliminary
results strongly indicate the promising potential of surface-
engineered DBOF as metallo-agents with high biosafety and
advanced properties.
CONCLUSIONS
In conclusion, an innovative ligand-branching strategy has
been successfully utilized to construct a series of DOTA-
branched organic frameworks (DBOF) as giant chelators.
These frameworks feature abundant interior and
peripheral organized DOTA-derived chelating holes that
could efficiently hold metal ions. The M-DBOF complexes
are uniformly nanosized with high metal contents, which
offer opportunities for the development of metallo-agents
with high efficiency and sensitivity. Furthermore, the high
kinetic stabilities of M-DBOF complexes also provide a
potential solution for the stability problem of current
metallo-agents. The versatility of DBOF in biomedical
3
+
3+
3+
imaging are exemplified by Gd or Tb homometallic
3+
3+
2
1
chelation,
peripheral
PEGlation,
and
Tb /Gd
M /M -G2 core/shell complex. Because of their multiplex
nature, we explored the possibility of DBOF for
heterometallic chelation to form hetero-multinuclear
heterometallic chelation. The applications of DBOF could be
easily extended to other metal-ion-based biomedical
imaging, such as Cu for PET and
attractively, heterometallic DBOF complexes present a
great opportunity for multimodal imaging. We believe that
DBOF and their analogs will be emerging as a promising
chelating platform for biomedicine, and the strategy behind
the fabrication of these frameworks, the ligand-branching
strategy, will be inspiring for the design and construction of
novel giant molecules.
6
4
99m
Tc for SPECT. More
complexes with multifunctional properties. Using Gd
2
O
3
as
the source of Gd ions and ethylenediaminetetraacetic acid
EDTA) to control the amount of Gd3+ ions, Gd(shell)-G2
3+
(
could be constructed (Figure 6a), as demonstrated by the
model reaction (Supporting Information S1.3, Figure S24,
3
+
and Figure S25). By further Tb chelation with Gd(shell)-
G2, the fabrication of a Tb/Gd-G2 core/shell complex could
be accomplished (Figure 6a). Based on this protocol,
Gd/Tb-G2, Y/Gd-G2, and Gd/Y-G2 core/shell complexes
could also be constructed. It is worth noting that during the
chelating process, even that the metal reagents were used
in large excess, the molar Gd:M ratios for Tb/Gd-G2,
Gd/Tb-G2, Y/Gd-G2, and Gd/Y-G2 were maintained at 2.6,
ASSOCIATED CONTENT
Supporting Information. The supporting information is
available free of charge via the Internet at http://pubs.acs.org.
Materials and details for synthesis and characterizations, NMR
and mass spectra, MR images and fluorescence pictures,
structure information (PDF).
0
.27, 3.2, and 0.40, respectively (Figure 6b), which is
consistent with the ratio between the number of peripheral
chelating holes and the number of interior chelating holes
in G2 (Table S1), indicating the achievement of the
core/shell structure. Based on these core/shell complexes,
we could further study the chelating property of DBOF
AUTHOR INFORMATION
Corresponding Author
*jhgao@xmu.edu.cn
3
+
selectively in the shell or in the core. Interestingly, the Tb
ions in the core have a larger q value (q = 1.43) than those
in the shell (q = 0.90), and Tb-G2 has a moderate q value (q
Author Contributions
#C.S. and H.L. contributed equally.
Notes
=
0.99) consistent with the average of the q values of both
the core and shell (Table S4). Furthermore, the complexes
with Gd in the shell (Tb/Gd-G2 and Y/Gd-G2) have a
The authors declare no competing interest.
3
+
-
1
-1
-1
-
much larger r
1
value (~13 mM s ) than that (8 ~ 9 mM s
ACKNOWLEDGMENT
1
3+
)
of the complexes with Gd in the core (Gd/Tb-G2 and
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Journal of the American Chemical Society
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We thank Prof. B. Mao, Prof. J. Yan, and Y. Peng for the help in
AFM test and data analysis. This work was supported by
National Natural Science Foundation of China (21771148,
(17) Mastarone, D. J.; Harrison, V. S.; Eckermann, A. L.; Parigi,
G.; Luchinat, C.; Meade, T. J., A modular system for the synthesis of
multiplexed magnetic resonance probes. J. Am. Chem. Soc. 2011,
33, 5329.
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1602186, 21521004, and 81430041), IRT_17R66, the Natural
(
18) Debroye, E.; Parac-Vogt, T. N., Towards polymetallic
Science Foundation of Fujian Province of China (2018J01011),
and Fundamental Research Funds for the Central Universities
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Table of contents (TOC)
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3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment