Anchoring Drugs to a Zinc(II) Coordination Polymer Network: Exploiting Structural Rationale toward the Design of Metallogels for Drug-Delivery Applications

Article abstract of DOI:10.1021/acs.inorgchem.0c03550

A new series of coordination polymers (CPs) were synthesized and crystallographically characterized by single-crystal X-ray diffraction with the aim of developing drug-delivery systems via metallogel formation. Structural rationale was employed to design such coordination-polymer-based metallogels. As many as nine CPs were obtained by reacting two bis(pyridyl)urea ligands, namely, 1,3-dipyridin-3-ylurea (3U) and 1,3-dipyridin-4-ylurea (4U), and the sodium salt of various nonsteroidal antiinflammatory drugs, namely, ibuprofen (IBU), naproxen (NAP), fenoprofen (FEN), diclofenac (DIC), meclofenamic acid (MEC), mefenamic acid (MEF), and Zn(NO3)2. All of the CPs displayed 1D polymeric chains that were self-assembled through various hydrogen-bonding interactions involving the urea N-H and carboxylate O atoms and, in a few cases, lattice-occluded water molecules. The reacting components of the CPs produced five metallogels in dimethyl sulfoxide/water. The gels were characterized by rheology and transmission electron microscopy. Three selected metallogelators, namely, 3UMEFg, 3UNAPg, and 3UMECg, showed in vitro anticancer, cell imaging, and multidrug delivery for antibacterial applications, respectively. The shear-thinning properties of 3UMECg (rheoreversibility and injectability) make it a potential candidate for plausible topical application.

Full text of DOI:10.1021/acs.inorgchem.0c03550

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Article
Anchoring Drugs to a Zinc(II) Coordination Polymer Network:
Exploiting Structural Rationale toward the Design of Metallogels for
Drug-Delivery Applications
Protap Biswas and Parthasarathi Dastidar*
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ABSTRACT: A new series of coordination polymers (CPs) were synthesized and
crystallographically characterized by single-crystal X-ray diffraction with the aim of
developing drug-delivery systems via metallogel formation. Structural rationale was
employed to design such coordination-polymer-based metallogels. As many as nine CPs
were obtained by reacting two bis(pyridyl)urea ligands, namely, 1,3-dipyridin-3-ylurea
(3U) and 1,3-dipyridin-4-ylurea (4U), and the sodium salt of various nonsteroidal
antiinflammatory drugs, namely, ibuprofen (IBU), naproxen (NAP), fenoprofen (FEN),
diclofenac (DIC), meclofenamic acid (MEC), mefenamic acid (MEF), and Zn(NO3)2.
All of the CPs displayed 1D polymeric chains that were self-assembled through various
hydrogen-bonding interactions involving the urea N−H and carboxylate O atoms and, in
a few cases, lattice-occluded water molecules. The reacting components of the CPs
produced five metallogels in dimethyl sulfoxide/water. The gels were characterized by
rheology and transmission electron microscopy. Three selected metallogelators, namely,
3UMEFg, 3UNAPg, and 3UMECg, showed in vitro anticancer, cell imaging, and multidrug delivery for antibacterial applications,
respectively. The shear-thinning properties of 3UMECg (rheoreversibility and injectability) make it a potential candidate for
plausible topical application.
many reports on PCP-based DDSs.22−26 Various parameters
INTRODUCTION
■
such absorption, distribution, metabolism, excretion, and
Organic−inorganic hybrid systems (OIHSs), such as porous
toxicity (ADMET), which are important for developing
coordination polymers (PCPs) or metal−organic frameworks
DDSs, could be fine-tuned in such OIHSs.27,28 Nevertheless,
(MOFs), are emerging materials for various applications1
OIHSs as drug-delivery vehicles possess a number of
including drug-delivery systems (DDSs).2−10 Delivering an
disadvantages: loading of the API/drug into the pores requires
active pharmaceutical ingredient (API) or drug to the target
compatibility of the size and chemical environment of the
site is a challenging task. It requires the development of a
active ingredient with that of the pores of the PCPs or MOFs,
vehicle that protects the API/drug from being destroyed by the
and subsequent release of the API/drug in a controlled fashion
immune response of the subject. There have been a number of
is often found to be difficult. To overcome such challenges
various organic11−15 and inorganic16−20 systems developed as
associated with the use of vehicles for drug delivery, an
vehicles for drug delivery purposes. However, hybrid systems
alternative concept, namely, “self-delivery” (without involving
like OIHSs as drug-delivery vehicles offer quite a few
any vehicle), is being developed.29,30 Among the various
advantages over vehicles developed from purely organic or
approaches to achieving such goals, supramolecular gelati-
inorganic compounds; they (a) are relatively easily synthesized
on31−37 is found to be quite promising; in this approach, the
because of the less energy expensive metal−ligand coordina-
API/drug under study is converted to a supramolecular gelator
tion bond compared to the covalent bond, (b) are often highly
by following certain design strategies, and the resulting gel can
crystalline, allowing access to atomic-level structural informa-
then be administered to the target site via either a topical
tion by single-crystal X-ray diffraction (SXRD), crucial for fine-
tuning, (c) have tunable physicochemical properties arising
(noninvasive) or a subcutaneous (invasive) route.38−44,70−72
from virtually infinite combinations of ligands and metal salts,
and (d) have a relatively labile metal−ligand coordination
bond compared to the covalent bond, facilitating postapplica-
Received: December 4, 2020
Published: February 11, 2021
tion degradation. PCPs such as MIL-100 and MIL-101 were
́
exploited by Ferey et al. for the first time as vehicles for the
controlled release of nonsteroidal antiinflammatory drugs
(NSAIDs), namely, Ibuprofen.21 Since then, there have been
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Scheme 1. OIHS-Based Drug-Delivery Strategies
Because the gel is made from a modified API/drug, there is no
need for a vehicle; the API/drug-based gel itself is delivered to
the target site, hence the name “self-delivery”.
logels for drug-delivery applications reported herein were
hitherto unknown.
EXPERIMENTAL SECTION
In the context of developing OIHS-based DDSs, one can
make the API/drug part of the coordination framework in such
a manner that the resulting coordination compound produces
a metallogel, which can then be administered for such
biomedical applications. With the API/drug being stoichio-
metric, the difficulty of drug loading does not exist. On the
other hand, a strong and labile coordination bond may
facilitate the slow release of the API/drug from the gel
(Scheme 1). This approach, although quite appealing, is not
highly explored.45,46
Herein, we report a new series of metallogels derived from
rationally designed and crystallographically characterized
coordination polymers (CPs), which were synthesized by
reacting two bidentate bis(pyridyl)bis(urea) ligands, namely,
1,3-dipyridin-3-ylurea (3U), 1,3-dipyridin-4-ylurea (4U), and
sodium salts of various NSAIDs and Zn(NO3)2. As envisaged
(vide supra), some of the reactants of the CPs produced
metallogels (Scheme 2). Three selected metallogelators
(3UMEF, 3UNAP, and 3UMEC) were further evaluated for
drug-delivery applications. Most likely, the CP-based metal-
■
Materials and Methods. All of the chemicals and solvents were
commercially available and were used without further purification.
NMR spectra were recorded using a 400 MHz/500 MHz
spectrometer (Bruker Ultrasheild Plus-400/500). SXRD data were
collected in a Bruker APEX II diffractometer equipped with a CCD
area detector. Powder X-ray diffraction (PXRD) data were recorded
on a Bruker AXS D8 Advance (Cu Kα radiation, λ = 1.5406 Å)
powder X-ray diffractometer. Fourier transform infrared (FT-IR)
spectra were recorded on a PerkinElmer FT-IR spectrometer. UV−vis
spectroscopic measurements were carried out on a Hewlett-Packard
8453 diode-array spectrophotometer. The elemental compositions of
the purified compounds were confirmed by elemental analysis
(PerkinElmer Precisely, Series-II, CHNO/S Analyzer-2400). Trans-
mission electron microscopy (TEM) images were recorded using a
JEOL (JEM 2100F) instrument on 300 mesh copper TEM grid.
Matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry (MALDI-TOF MS) spectra were collected in a Bruker
instrument with a DHB matrix. Images were analyzed using ImageJ
software. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay plate scanning was conducted using a multiplate
ELISA reader (Varioskan Flash Elisa Reader, Thermo Fisher).
Fluorescence images were collected by laser scanning confocal
microscopy (LSCM; Carl Zeiss). Rheological experiments were
performed with an Anton Paar MCR 102 modular compact
rheometer.
Scheme 2. CPs and Metallogels Reported in This Study
The bis(pyridyl)urea ligands (3U and 4U) were synthesized
following the reported literature.73,74 The detailed synthetic
procedure is described in the Supporting Information.
Characterization Data of the Ligands. 3U. Yield: 900 mg,
1
66.25%. H NMR (500 MHz, DMSO-d6): δ 8.96 (s, 1H), 8.59 (s,
1H), 8.19 (d, J = 4.6 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.30 (dd, J =
8.2 and 4.7 Hz, 1H) (Figure S1). 13C NMR (125 MHz, DMSO-d6): δ
153.2, 143.6, 140.8, 136.6, 125.9, 124.1 (Figure S2). FT-IR (cm−1): ν
3045 (brs, urea N−H stretch), 1690 (s, urea CO stretch) (Figure
S3). MALDI-TOF MS (DHB matrix/MeOH). Calcd for [M + H]+:
̃
m/z 215.23. Found: m/z 215.25 (Figure S4).
1
4U. Yield: 960 mg, 69.75%. H NMR (500 MHz, DMSO-d6): δ
9.27 (1 H, s), 8.38 (2 H, d, J = 5.4 Hz), 7.44 (2 H, d, J = 5.6 Hz)
(Figure S5). 13C NMR (125 MHz, DMSO-d6): δ 151.7, 150.2, 145.9,
112.4 (Figure S6). FT-IR (cm−1): ν
̃
3004 (brs, urea N−H stretch),
1739 (s, urea CO stretch) (Figure S7). MALDI-TOF MS (DHB
matrix/MeOH). Calcd for [M + H]+: m/z 215.23. Found: m/z
215.26 (Figure S8).
Synthesis of CPs and Characterization Data. The CPs were
synthesized by a crystallization method. The sodium salt of the
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corresponding NSAID (1 equiv, 0.1 mmol) was dissolved in water (4
mL) and placed in crystallization test tube (15 cm height and 1 cm
diameter). 3U or 4U (0.5 equiv, 0.05 mmol) was dissolved in
methanol (MeOH; 4 mL) and poured slowly over the aqueous
solution of NSAIDs. Finally, a metal salt [Zn(NO3)2·6H2O, 0.5 equiv,
0.05 mmol] solution in ethanol (2 mL) was layered over the reaction
mixture. Typically, after 3 weeks, crystals had grown at the interface of
the water/MeOH layer. The crystals were collected by a paint brush,
washed with MeOH, and dried on filter paper under ambient
conditions.
PXRD. PXRD data were recorded using a Bruker AXS D8 Advance
(Cu Kα radiation, λ = 1.5406 Å) powder diffractometer equipped
with a superspeed LYNXEYE detector. Finely powdered samples
(∼20 mg) were placed over a glass slide to collect data. The
experiments were carried out at 40 kV and 40 mA with a scan speed of
0.2 s/step (step size = 0.02°) for a scan range of 5−35° (2θ). The
PXRD data for silver nanoparticles (AgNPs) with the gel were
collected with Rigaku Smartlab (40 kV, 110 mA, Cu Kα1 radiation, λ
= 1.5406 Å, step size = 0.010). Simulated PXRD patterns were
generated from the CIFs using Mercury 3.8 software.
TEM. In a typical experiment, the metallogel sample was smeared
on a carbon-coated copper grid (300 mesh) and dried at room
temperature for 24 h (except for 4UMEFg, which was dried in a
vacuum desiccator at room temperature), and the TEM images were
recorded at an accelerating voltage of 200 kV without any staining.
Rheology. Parallel-plate geometry (25 mm diameter with a 1 mm
gap) was employed to collect rheological data on freshly prepared
dimethyl sulfoxide (DMSO)/water (1:1, v/v) metallogels (10 wt %,
w/v). The frequency sweep data (G′ and G″ vs frequency ω, rad/s)
were collected at a fixed strain guided by the linear viscoelastic (LVE)
region obtained from an amplitude sweep (G′ and G″ vs strain). The
thixotropic data were collected using strains of 65% (higher than the
critical strain) and 1% with a relaxation time of 120 s for three
consecutive cycles.
Loading and Release of Sulfasalazine (SUL). The metallogel
3UMECg (1 mL, 10 wt %; DMSO/water, 1:1, v/v) was taken in a
vial. A solution of SUL (1 mL, 1 mg/mL; 1:1 DMSO/water) was
prepared and placed over the metallogel bed to diffuse the drug
(SUL) into the metallogel, and photographs were taken at various
time intervals. The loading efficiency was determined by UV−vis
using a supernatant (10 μL). After complete absorption of SUL, as
revealed from the UV−vis data, the supernatant was replaced with
phosphate-buffered saline (PBS; 1 mL, 7.2 pH) to monitor the release
of SUL, which could be qualitatively observed by the yellow
coloration of the supernatant and quantitatively determined by
UV−vis using 10 μL aliquots at various time intervals.
AgNP Generation in the Metallogel. A silver nitrate (AgNO3)
solution (1 mL, 1 mg/mL; 1:1 DMSO/water) was placed over
3UMECg (1 mL, 10 wt %) taken in a vial and kept at room
temperature under laboratory light for 24 h. A UV−vis spectrum of
the AgNP-containing metallogel was recorded by dispersing it in
DMSO.
Characterization Data of the CPs. 3UIBU. FT-IR (cm−1): ν
̃
3256, 3091, 2954, 1717, 1588, 1557, 1488, 1428, 1358, 1277, 1209,
1134, 1068, 912, 807, 696, 647, 542 (Figure S9). CHN anal. Calcd
(expt): C, 64.39 (64.26); H, 6.43 (6.78); N, 8.12 (8.21).
3UNAP. FT-IR (cm−1): ν
̃
3220, 2932, 1703, 1605, 1558, 1485,
1391, 1255, 1211, 1161, 1032, 926, 796, 750,700, 647, 521, 471
(Figure S10). CHN anal. Calcd (expt): C, 61.95 (61.68); H, 5.07
(4.96); N, 7.41 (7.41).
3UFEN. FT-IR (cm−1): ν
̃
3281, 3064, 1710, 1580, 1557, 1484,
1385, 1355, 1256, 1207, 1059, 929, 881, 763, 763, 692, 651, 484, 455
(Figure S11). CHN anal. Calcd (expt): C, 64.61 (64.85); H, 4.76
(4.47); N, 6.73 (6.67).
3UDIC. FT-IR (cm−1): ν
̃
3283, 3087, 1703, 1631, 1489, 1454,
1270, 1207, 745, 710, 692, 564, 534 (Figure S12). CHN anal. Calcd
(expt): C, 53.84 (53.85); H, 3.48 (3.10); N, 9.66 (9.10).
3UMEC. FT-IR (cm−1): ν
̃
3270, 3061, 1716, 1609, 1548, 1495,
1414, 1287, 1197, 1089, 1039, 806, 752, 695, 653, 604, 463 (Figure
S13). CHN anal. Calcd (Expt): C, 53.85 (53.55); H, 3.49 (3.48); N,
9.69 (9.69).
3UMEF. FT-IR (cm−1): ν
̃
3280, 3065, 1709, 1609, 1578, 1548,
1489, 1387, 1284, 1202, 1151, 1062, 783, 693, 653, 615, 470 (Figure
S14). CHN anal. Calcd (Expt): C, 64.61 (64.61); H, 4.76 (4.54); N,
6.76 (7.06).
4UIBU. FT-IR (cm−1): ν
̃
3262, 2957, 1739, 1585, 1505, 1435,
1335, 1288, 1190, 1066, 1025, 838, 734, 534 (Figure S15). CHN anal.
Calcd (Expt): C, 62.75 (62.82); H, 6.50 (6.55); N, 7.71 (7.88).
4UNAP. FT-IR (cm−1): ν
1293, 1179, 1067, 1031, 849, 830, 732, 664, 532, 478 (Figure S16).
CHN anal. Calcd (expt): C, 63.6 (63.98); H, 4.65 (4.69); N, 7.61
(7.50).
̃
3272, 2964, 1746, 1588, 1513, 1439,
4UFEN. FT-IR (cm−1): ν
̃
3278, 3078, 1744, 1576, 1490, 1437,
1395, 1249, 1191, 1066, 839, 762, 692, 603, 535 (Figure S17). CHN
anal. Calcd (expt): C, 64.61 (64.86); H, 4.76 (4.97); N, 7.73 (7.65).
SXRD. Intensity data of X-ray diffraction were collected on a
Bruker SMART APEX-II diffractometer (Mo Kα, λ = 0.7107 Å)
equipped with a CCD area detector. The data reduction, structure
solution, and refinement were carried out using the APEX-3 SMART
software package. All of the structures were solved by direct methods
and refined by full-matrix least squares based on F2 against all
reflections in the SHELXL-201447 suite of APEX-3. The final
refinement, disorder treatment, and CIF finalization were carried
out in OLEX2, version 1.2.8.48 3UMEC was found to be
nonmerohedrally twinned, with a twin fraction of 0.540(3) that was
solved by separating two domains in the reciprocal lattice; data scaling
was carried out using TWINABS program, and structure refinement
was carried out with the HKLF5 file in SHELXL in APEX-3. The
methyl C atom in the ibuprofen molecule of 3UIBU and the methyl C
atoms in both meclofenamate moieties in 3UMEC were disordered
over two positions with refined SOFs of 0.259(7)/0.741(7), 0.46(2)/
0.54(2), and 0.512(13)/0.488(13), respectively. The methoxy group
of 3UNAP was disordered over two positions with a refined SOF of
0.568(14)/0.432(14). The methyl and isopropyl groups of 4UIBU
were also disordered over two positions with a refined SOF of
0.354(10)/0.646(10). All of the non-H atoms were anisotropically
refined. The H atoms connected to the heteroatoms or hydrogen-
bonded H atoms were located on the difference Fourier maps and
refined; in the other cases, they were fixed whereever possible.
Crystallographic data for the structural analysis of compounds
reported herein have been deposited at the Cambridge Crystallo-
graphic Data Centre as CCDC 1878233−1878241.
MTT Assay. The cells (B16F10) were purchased from the
National Centre for Cell Science (Pune, India) and cultured in a
high-glucose Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal bovine serum and 1% penicillin/streptomycin
in a humidified incubator at 37 °C under a 5% CO2 atmosphere until
its confluence. The cells were then seeded in a 96-well plate at a
density of ∼104 cells/well. The culture plates were then incubated for
24 h in a humidified incubator at 37 °C under a 5% CO2 atmosphere.
Primary stock solutions of the CPs were prepared by dispersing the
corresponding CPs (20 mg) in cell-culture-grade DMSO (1 mL). The
stock solutions were further diluted with DMEM (40−160 μg/mL);
150 μL of DMEM containing the CPs was then added to the well and
incubated at 37 °C under a 5% CO2 atmosphere for 72 h. The culture
medium of each well plate was replaced with a MTT reagent (100 μL,
0.5 mg/mL, in DMEM) and incubated further for 3 h. The culture
medium was discarded and replenished with 100 μL of DMSO. The
well plate was then read under a multiplate ELISA reader (λ = 570
nm) to measure the intensity of formazan, which was attributed to the
live cell concentration. The percentage of live cells under treatment
with the CPs was calculated by considering the DMEM-treated cells
(control) to be 100%. All of the experiments were performed three
times.
Cell Migration. The cells were cultured and seeded in a 35 mm 6-
well plate and incubated until its confluence. A uniform scratch line
was drawn by a sterile pipet microtip in the middle of the well plate.
The CP 3UMEF (1 mL in DMEM at IC50 ∼ 80 μg/mL) was added
to the well plate. For control experiments, only DMEM, free drug
(MEFH), and 3U were added to the well plate. The amounts of
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MEFH and 3U added were equivalent to that present in 3UMEF at
its IC50 concentration. The well plate was then incubated for 24 h,
and during this incubation period, still images were captured under an
optical microscope (Olympus CKX31) at different time intervals in
order to measure the migration speed.
carboxylate O and pyridyl N atoms, respectively; while the
carboxylate of one of the IBU ligands displayed unidentate
coordination, the other one coordinated to the metal center
through a bidentate mode; the metal center Zn(II) showed
five-coordinated highly distorted trigonal-bipyramidal geome-
try wherein the equatorial positions were occupied by two
carboxylate O atoms from two IBU ligands and one pyridyl N
atom and the axial positions were coordinated by one pyridyl
N and one carboxylate O atoms; such five-coordinated
coordination geometry of Zn(II) was because of the bidentate
coordination mode of one of the carboxylates. The methyl
group in one of the IBU ligands was found to be disordered
over two positions (see the Experimental Section). Crystal
structure analysis revealed that the bis(pyridyl)urea ligand 3U
was highly nonplanar, displaying a dihedral angle (Φ) of ∼63°
involving the planes of the terminal pyridyl rings; overall,
3UIBU was an 1D zigzag CP formed because of the extended
coordination of the bidentate ligand 3U, and the drug
molecules IBU were found to be anchored to the Zn(II)
centers as envisaged. The 1D chains were found to self-
assemble into a 2D hydrogen-bonded network sustained by
N−H···O hydrogen-bonding interactions involving one of the
N atoms of the urea moiety and the IBU carboxylate O atom,
which was free from coordination. Such 2D hydrogen-bonded
networks were further packed in a parallel fashion sustained by
dispersion forces (Figure 1).
Cell Imaging. The cells were cultured in a 35 mm confocal dish
and incubated overnight in a humidified incubator at 37 °C under a
5% CO2 atmosphere. After 24 h, when the cells were adhered to the
dish, the old medium was discarded and the cells were washed with
PBS (pH 7.4). 3UNAP (2 mL in DMEM at IC50 = 80 μg/mL) was
added to the dish and incubated for 20 min at 37 °C under a 5% CO2
atmosphere. The medium was then sucked out, the dish was washed
several times with PBS (pH 7.4) to remove the unbound drugs, and
then the cells were further treated with 4% paraformaldehyde and
kept for 15 min in the dark for cell fixation. 4′,6-Diamidino-2-
phenylindole (DAPI) was used for staining the nucleus. For this
purpose, a DAPI/PBS solution (2 mL, 5 nM) was added to the cells,
followed by incubation for 5 min. Extracellular staining was removed
by washing the cells with PBS (pH 7.4). After fresh PBS (1 mL, pH
7.4) was added to the cells, images were recorded by using a Carl
Zeiss laser scanning confocal microscope.
Antibacterial Zone Assay. Klebsiella spp. was grown from single-
colony isolation in liquid broth media supplemented with peptone,
yeast extract, and NaCl. For zone assay, the bacteria (100 μL in broth
media) were spread uniformly on an agar-gel plate (1.5 wt %
containing the above-mentioned supplements). Grooves (1 cm) were
cut and filled with 100 μL of 3UMECg, AgNP@3UMECg, SUL@
3UMECg, and nothing (control). Then the plate was incubated at 37
°C for 24 h in an incubator, and a digital photograph was taken.
Crystal Structure of 3UFEN. Crystallized in the centrosym-
metric monoclinic space group P21/c, the asymmetric unit of
3UFEN comprised one 3U ligand, two FEN ligands, and one
Zn(II) metal center; both FEN molecules displayed a
RESULTS AND DISCUSSION
■
Design and Synthesis of the Metallogelators. Because
of the lack of adequate molecular-level information behind
gelation, designing a gelator, be it organic or metal−organic, is
a formidable task.49−52 Since the first report of metallogelation
displayed by calixarene-based CPs,53 there have been efforts to
bring in some rationale to design metallogelators; for example,
we and others have proposed that molecular systems that
encourage strong interactions with solvents through various
supramolecular interactions resulting in lattice-occluded
crystalline solids (LOCSs) are potential targets as gelators
because large amounts of solvent molecules are trapped in both
gels and LOCSs.54,55
Thus, to design CP-based metallogelators, we chose two
bidentate ligands having a urea backbone, namely, 1,3-
dipyridin-3-ylurea (3U) and 1,4-dipyridin-3-ylurea (4U), that
would form 1D CPs75−80 when reacted with Zn(NO3)2 and
various NSAIDs (ibuprofen or IBU, naproxen or NAP,
fenoprofen or FEN, diclofenac or DIC, meclofenamic acid or
MEC, and mefenamic acid or MEF) in their carboxylate form
(sodium salt); the drug molecules are expected to remain
anchored to the 1D coordination network, thereby making the
drug molecule a part of the coordination network as envisaged.
Possibilities of interchain assembly and entrapment of solvent
molecules because of the excellent hydrogen-bonding capa-
bility of the urea backbone make the OIHS suitable for
producing metallogels. As many as nine CPs were isolated as
single crystals from the reactions (see Scheme 2, Table 1, and
the Experimental Section). It may be noted that the reaction of
4U and Zn(NO2)2 with MEC, DIC, and MEF did not produce
any single crystals; instead, relatively poorly diffracting
microcrystals were obtained (Figure S46).
SXRD. Crystal Structure of 3UIBU. The space group of
3UIBU was determined to be the centrosymmetric monoclinic
P21/c. The asymmetric unit contained one Zn(II) metal center
coordinated by two IBU and one 3U ligands through
Figure 1. Crystal structure illustration of 3UIBU: (a) 1D CP chain
with the anchored drug molecule IBU and (b) packing of the
hydrogen-bonded 1D CP chains viewed down the c axis.
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unidentate coordination mode to Zn(II), and 3U remained
coordinated to the metal center by a pyridyl N atom. The
bis(pyridyl)urea ligand 3U displayed near-planar conformation
with dihedral angle Φ = ∼14°. Because of the extended
coordination involving the ligand 3U with Zn(II), a 1D zigzag
CP chain was formed wherein the drug component remained
anchored. The usual urea synthon was not observed in the
structure; instead, one of the urea N−H atoms was involved in
N−H···O interactions with the carboxylate O atom of the
neighboring chain, resulting in parallel packing of the chains
and producing a 2D hydrogen-bonded network that further
packed in a parallel fashion stabilized by dispersion forces
(Figure 2).
∼45 and 48° in two independent moieties, and the Zn(II)
atom showed distorted tetrahedron geometry in one moiety,
whereas it was a five-coordinated species in the other because
of the bidentate coordination mode of the carboxylate. Both of
the independent moieties formed 1D polymeric chains because
of the extended coordination involving the pyridyl N atoms of
the bidentate ligand 3U. Each crystallographically independent
1D chain self-assembled with itself via hydrogen-bonding
interactions of the type N−H···O involving the urea N−H and
carboxylate O atoms. The lattice-occluded water molecules
were located within the interstitial space via O−H···O
hydrogen bonding with the carboxylate O atoms. All of the
chains were packed in a parallel fashion (Figure 3).
Figure 2. Crystal structure illustration of 3UFEN: (a) 1D CP chain
with the anchored drug molecule FEN and (b) packing of the
hydrogen-bonded 1D CP chains viewed down the c axis.
Figure 3. Crystal structure illustration of 3UNAP: (a) two
crystallographically independent 1D CP chains (shown in different
colors) with the anchored drug molecule NAP and (b) packing of the
hydrogen-bonded dimer of the 1D CP chains with lattice-occluded
water molecules (shown in the space-filling model) viewed down the
b axis.
Crystal Structure of 3UNAP. The crystal structure belonged
to the noncentric monoclinic space group P21. The asymmetric
unit comprised two crystallographically independent moieties,
each one containing one Zn(II) atom, one 3U ligand, two
NAP ligands, and two molecules of lattice-occluded water. The
coordinating modes of the NAP units to the metal centers
were different in two moieties; in one moiety, the NAP
molecules were coordinated to the Zn(II) atom via a
unidendate mode, whereas in the other moiety, it displayed
both unidendate and bidentate coordination modes. The
methoxy group of NAP in one of the crystrallographically
independent moieties was found to be disordered over two
positions (see the Experimental Section). The bidentate ligand
3U displayed a significantly nonplanar conformation with Φ =
Crystal Structure of 3UDIC. The centrosymmetric mono-
clinic space group P21/c was assigned to 3UDIC. In the
asymmetric unit, one Zn(II) metal ion was found to be
coordinated by two DIC ligands via carboxylate Zn
coordination in a unidentate mode and one 3U ligand via a
pyridyl N atom; the contents of the asymmetric unit were in
general positions. Because of the extended coordination
involving the bis(pyridyl)urea ligand 3U and Zn(II), the
supramolecular architecture of 3UDIC may be best described
as a 1D zigzag polymer wherein the drug molecules DIC were
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anchored to the metal centers. The coordination geometry of
Zn(II) was distorted octahedral, wherein two sites were
occupied by the pyridyl N atoms of 3U and the other two
positions were coordinated by carboxylate O atoms of DIC.
The urea ligand displayed a highly nonplanar conformation
with Φ = ∼59°. 1D CP chains self-assembled to form 2D
hydrogen-bonded networks via N−H···O interactions involv-
ing the urea N−H and carboxylate O atoms of the interacting
chains. Such an assembly further packed in a parallel fashion
stabilized by dispersion forces (Figure 4).
were packed in a parallel fashion stabilized by dispersion forces
(Figure 5).
Figure 5. Crystal structure illustration of 3UMEC: (a) 1D CP chains
with the anchored drug molecule MEC and (b) packing of the
hydrogen-bonded dimer of the 1D CP chains viewed down the a axis.
Crystal Structure of 3UMEF. This compound crystallized in
the centrosymmetric triclinic P1 space group. The asymmetric
̅
unit contained one Zn(II) atom, one 3U ligand, and two MEF
molecules, all located on general positions. The bidendate
ligand 3U was involved in extended coordination with the
Zn(II) metal ions, resulting in 1D CP chains on which the
drug molecules were found to be anchored via unidentate
coordination Zn(II) through carboxylate O atoms. The
conformation of the ligand 3U was significantly nonplanar
with Φ = ∼65°, and the metal center displayed a distorted
tetrahedral coordination geometry. The 1D CP chains were
further self-assembled to form hydrogen-bonded dimers
through N−H···O interactions involving urea N−H and
carboxylate O atoms of the interacting chains; such dimer
chains were further packed in a parallel fashion sustained by
dispersion forces (Figure 6).
Figure 4. Crystal structure illustration of 3UDIC: (a) 1D CP chains
with the anchored drug molecule DIC and (b) packing of the
hydrogen-bonded dimer of the 1D CP chains viewed down the c axis.
Crystal Structure of 4UIBU. The crystal structure of 4UIBU
was solved in the centrosymmetric monoclinic space group
P21/c. One Zn(II) ion, one 4U ligand, two IBU ligands, and a
lattice-occluded water molecule were located in the asym-
metric unit. Both the methyl and one of the isopropyl groups
of one of the IBU moieties were disordered over two positions
(see the Experimental Section). The characteristic dihedral
angle Φ of 4U was ∼24°, indicating a nonplanar conformation,
and the Zn(II) ion showed distorted tetrahedral coordination
geometry, wherein two sites were occupied by the pyridyl N
atoms of two 4U ligands and the other two positions were
coordinated by unidentate coordination of the carboxylate O
atom of two IBU ligands. As a result, a 1D CP chain was
formed on which the drug molecules were coordinated to the
metal center. The 1D chains formed hydrogen-bonding
interactions involving one of the urea N−H and carboxylate
O atoms of the neighboring chain, and the interstitial space of
the crystal lattice was occupied by the solvate water molecules
Crystal Structure of 3UMEC. The refined model of 3UMEC
belonged to the centrosymmetric triclinic space group P1. The
̅
asymmetric unit contained one Zn(II) atom coordinated by
one 3U ligand by one pyridyl N and two MEC atoms via
carboxylate O atoms displaying unidentate coordination, all
located on general positions. The dichloromethylbenzene rings
of both of the crystallographically independent drug moieties
were rotationally disordered because of C−N bond rotation
(see the Experimental Section). While the metal center showed
distorted tetrahedral geometry, the bidentate bis(pyridyl)urea
ligand displayed a nonplanar conformation with Φ = ∼58°.
The 1D CP chain formed because of the extended
coordination involving 3U further self-assembled with another
neighboring chain sustained by N−H···O hydrogen bonding
involving the urea N−H and carboxylate O atoms of the
interacting chains, resulting in hydrogen-bonded dimers that
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showed a reasonably planar conformation (Φ = ∼6 and ∼5°),
and the metal center displayed distorted tetrahedral geometry
in both moieties. Because of the extended coordination of the
pyridylurea ligand, each crystallographically independent
moiety formed a 1D CP chain that interacted with another
symmetry-equivalent 1D chain by N−H···O hydrogen bonding
involving the urea N−H and carboxylate O atoms to form a
dimer assembly; such an assembly of chains further packed in a
parallel fashion sustained by dispersion forces (Figure 8).
Figure 6. Crystal structure illustration of 3UMEF: (a) 1D CP chains
with the anchored drug molecule MEF and (b) packing of the
hydrogen-bonded dimer of the 1D CP chains viewed down the b axis.
sustained by N−H···O and O−H···O interactions involving the
urea N−H and carboxylate O atoms (Figure 7).
Crystal Structure of 4UNAP. The crystal structure belonged
to the noncentric monoclinic space group P21. The asymmetric
unit contained two crystallographically independent moieties,
each having one Zn(II) atom, one 4U ligand, and two NAP
ligands. The NAP carboxylates displayed unidentate coordi-
nation to the metal center in both moieties. The urea ligand
Figure 8. Crystal structure illustration of 4UNAP: (a) two
crystallographically independent 1D CP chains (shown in different
colors) with the anchored drug molecule NAP and (b) packing of the
hydrogen-bonded dimer of the 1D CP chains viewed down the ac
plane.
Crystal Structure of 4UFEN. The SXRD-refined model
showed that 4UFEN crystallized in the centrosymmetric
monoclinic space group P21/n. The asymmetric unit contained
one Zn(II) ion, one 4U ligand, two IBU ligands, and one
lattice-occluded water molecule. The bis(pyridyl)urea ligand
4U (Φ = ∼25°) displayed an extended coordination mode
with the Zn(II) metal centers, resulting in a 1D CP chain
wherein the drug molecules remained anchored to the chain
via unidentate coordination involving the carboxylates to
Zn(II) metal centers. The polymer chains formed a hydrogen-
bonded dimer involving one of the N−H bonds of the urea
moiety and the carboxylate O atom of the interacting chains;
the solvate water molecule occupied the interstitial space
sustained by N−H···O and O−H···O hydrogen bonding
involving the urea N−H and carboxylate O atom. Such a
dimer assembly of the polymer chain further packed in a
parallel fashion stabilized by dispersion forces (Figure 9).
The crystalline phase purity of the bulk samples of the CPs
was found to be excellent to reasonably good, as revealed by
Figure 7. Crystal structure illustration of 4UIBU: (a) 1D CP chains
with the anchored drug molecule IBU and hydrogen-bonded water
molecules (in the space-filling model) and (b) overall packing of the
hydrogen-bonded 1D CP chains with lattice-occluded water
molecules (shown in the green space-filling model) viewed down
the a axis.
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solution of Zn(NO3)2 was added to a DMSO solution
containing the urea ligand and NSAID, instantaneous gelation
occurred at room temperature, as confirmed by an “inverted
vial” test wherein the gel was able to sustain its own weight
against gravity (Scheme 2 and Table S11). All of the gels were
thermoirreversible, indicating the CP nature of the gelator,
stable for a few days, and precipitated out with aging. To
confirm that they were really gels, we carried out dynamic
rheology of the gels. Gel, being a non-Newtonian liquid, shows
a viscoelastic response in dynamic rheology; a plot of G′
(storage modulus) and G″ (viscous modulus) versus angular
velocity ω (rad/s) under a constant strain chosen from the
LVE region obtained from amplitude sweep experiments
would show that G′ is much larger than G″ and largely
frequency-invariant at low ω, i.e., longer time scale.56 The nice
viscoelastic response in all cases indicated that all of these
materials that passed through “inverted vial” tests were indeed
gels (Figure 10). The gels had reasonable mechanical strength,
with tan δ ranging from 0.09 to 0.15 (Table S11).
Figure 9. Crystal structure illustration of 4UFEN: (a) 1D CP chains
with the anchored drug molecule FEN and hydrogen-bonded water
molecules (in the space-filling model) and (b) overall packing of the
hydrogen-bonded 1D CP chains with lattice-occluded water
molecules (shown in the green space-filling model) viewed down
the a axis.
comparing the PXRD patterns of the bulk sample and the
corresponding simulated PXRD patterns obtained from the
SXRD data (Figures S27−S35) except for 3UIBU; the major
mismatch of the PXRD patterns in this case might be due to
the formation of a polymorph during crystallization of the CP.
The foregoing discussions on the crystal structures of the
isolated OIHSs clearly highlighted a few key features that were
expected to be useful for developing metallogel-based DDSs:
(a) drug molecules were part of the OIHSs, thereby getting rid
of the difficulties involved in drug loading; (b) all of the crystal
structures were 1D CPs having hydrogen-bonding-capable
moieties [the urea functionality of the bis(pyridyl)urea ligands
3U and 4U and carboxylate O atoms of the drug molecules]
that helped self-assemble the 1D chains and, in three cases
(3UNAP, 4UIBU, and 4UFEN), interacted strongly with
lattice-occluded solvates (water), key features for gelation
(vide supra). Besides, the conformational flexibility of the
bis(pyridyl)urea ligands, as revealed from the wide range of
dihedral angles Φ = 6−65° of the terminal pyridyl rings
(Scheme 1), was expected to contribute toward effective
interactions in the presence of polar solvents facilitating
gelation.
Figure 10. Viscoelastic (gel-like) response of the metallogels in
dynamic rheology.
Metallogelation. SXRD analyses revealed that, in all CPs,
the ratio of the reactants, i.e., metal/ligand/NSAID, was 1:1:2.
We, therefore, decided to carry out metallogelation studies
with this reactant ratio for all nine CPs. Interestingly, as many
as five metallogels (3UNAPg, 3UMECg, 3UMEFg, 4UNAPg,
and 4UFENg) were obtained and the reaction of Zn(NO3)2,
4U, and MEF under identical conditions also produced
metallogel 4UMEFg. It may be noted that, under identical
conditions, 3U produced a gelatinous precipitate and colloidal
solutions when treated with IBU, DIC, and FEN, respectively,
whereas 4U generated a precipitate, a gelatinous precipitate,
and a colloidal solution when treated with IBU, MEC, and
DIC, respectively. In a typical experiment, when an aqueous
It is interesting to note that, in all five cases for which we
have single-crystal structures, the PXRD patterns of the dried
gel (xerogel) showed significant correspondence with that
obtained from the bulk sample, with the SXRD data
(simulated) indicating that the gel network essentially
represented that observed in the single-crystal structures
(Figures S37−S41). However, one cannot rule out the
possibility of having different crystalline phases of the xerogel
in minor amounts because of the fact that a complete match of
the PXRD patterns was not observed; evaporation of the
solvent during xerogel formation might be the driving force for
the formation of different crystalline phases in minor amounts.
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Thus, the rationale employed herein to design metallogelators
seemed to have worked to a significant extent.
The morphology of the gel networks was studied by TEM.
Figure 11 displayed networks of entangled fibers for 3UNAPg,
scratch assay was employed to evaluate the anticancer property
of the metallogelator 3UMEF. While the orchestrated
migration of cells in a particular direction is important for
any multicellular organism for its development and main-
tenance, cancer cells display uncontrolled migration, leading to
invasion and metastasis.63 Thus, the ability to inhibit cancer
cell migration by a potential anticancer agent is estimated by
determining the speed with which the cancer cells migrate
under in vitro conditions in the presence of the agent; the
slower the speed, the better the anticancer property of the
agent.64 Scratch assay was performed on B16F10 cells using
only media (control), the ligand 3U, the NSAID component,
i.e., mefenamic acid (MEFH), and the metallogelator 3UMEF.
The cell migration speed was lowest (4.9 μM/h) for 3UMEF,
establishing the beneficiary effect of the metallogelator as an
anticancer agent compared to that of its individual components
(Figure 12). These data clearly suggested that the metal-
logelator 3UMEF must have been internalized by the cancer
cells.
Cell Imaging. In order to probe the cell permeability of the
metallogelators, we selected 3UNAP for cell imaging studies
because the naproxen moiety of 3UNAP does have moderate
green emission in fluorescence when excited in the blue region.
LSCM of B16F10 cells in the presence of 3UNAP and a
nucleus staining dye DAPI clearly confirmed that 3UNAP was
successfully internalized, as is evident from the green and blue
fluorescence due to 3UNAP and DAPI in the cytosol and
nucleus, respectively (Figure 13).
Conventional Drug Delivery: Exploring Antibacterial and
Multidrug-Delivery Applications. To explore the possibility of
using one of the metallogels for antibacterial and multidrug-
delivery applications following conventional a drug-delivery
route, we chose to work with 3UMECg because of its stability
and bright-white color. We allowed a AgNO3 solution to soak
into the 3UMECg gel bed and exposed it to laboratory light for
24 h. The appearance of a dark-violet color in the gel bed
indicated the formation of AgNPs.81 The TEM image of
AgNP@3UMECg revealed that the nanoparticles were
adhered to the gel network and the sizes of the AgNPs were
Figure 11. Metallogel network morphology under TEM. Insets:
Inverted vial pictures of the corresponding gels.
4UNAPg, 4UFENg, and 4UMEFg typically observed for gels.
However, 3UMECg and 3UMEFg showed discrete short
fibers, which were also not so uncommon for gels.57−59
Biological Studies. Anticancer Property of the Metal-
logelators. Because the NSAIDs were now part of the
coordination network and many of them did possess anticancer
properties,60,61 we explored the effect of metallogelators on
cancer cells. For this purpose, we carried out MTT assay62 on
the murine tumor melanoma cell line (B16F10) in the
presence of crystallographically characterized metallogelators.
The B16F10 cell line was chosen because it is used as a model
for human skin cancer, and therefore the corresponding
metallogel could be applied topically. MTT assay revealed that
3UMEF possessed the best ability to kill the cancer cells with
IC50 = 80 μg/mL (Figure S42). We, therefore, decided to carry
out further experiments with 3UMEF. Cell migration assay or
Figure 12. Scratch assay: B16F10 cell migration under various conditions.
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network structure of the xerogel of 3UMECg (Figure 14a).
The antibacterial property of AgNPs is well documented.67−69
Therefore, AgNP@3UMECg can be used for antibacterial
application (vide infra).
To load another drug into the gel matrix for plausible
multidrug-delivery application, we soaked the 3UMECg gel
bed with a solution containing a NSAID, namely, sulfasalazine
(SUL). The bright-yellow color of SUL helped us to visualize
its intake by the gel bed. The loading efficiency of the drug
within the gel matrix was found to be ∼51% (Figures S43 and
S44). Steady release of the drug from the SUL@3UMECg gel
bed to PBS was also monitored by UV−vis; a gradual increase
in the intensity of λ = ∼366 nm with time indicated the release
of SUL into the bulk solvent. The percentage of release of SUL
from SUL@3UMECg was ∼73% (Figures 14b and S45). The
morphology of the gel network of SUL@3UMECg observed
under TEM was similar to that found for 3UMECg, indicating
that adsorption of the drug did not have any effect on the
supramolecular architecture of the metallogel. It was
interesting to note that both of the metallogels 3UMECg
and SUL@3UMECg could be spread on a Petri dish and the
characteristic color of SUL in SUL@3UMECg (yellow and
green) was visible under visible and UV light, respectively
(Figure 14b).
We demonstrated that the cargos in both AgNP@3UMECg
and SUL@3UMECg could be delivered in vitro. For this
purpose, we cut four grooves in an agar plate inoculated with a
Gram-negative bacterium, namely, Klebsiella spp. The grooves
were filled with nothing (control), 3UMECg, SUL@3UMECg,
and AgNP@3UMECg and incubated at 37 °C for 24 h.
Evidence of an inhibited zone (15 2 mm) around the groove
containing AgNP@3UMECg confirmed the delivery of
antibacterial AgNPs, whereas no such inhibited zones around
the grooves containing nothing and 3UMECg were observed.
The bright-yellow color due to the diffusion of SUL into agar
gel around the groove containing SUL@3UMECg suggested
its successful delivery under in vitro conditions. Finally, the
sheer-thinning property of 3UMECg required for topical
application was demonstrated by rheoreversibility as well as by
Figure 13. LSCM images of B16F10 cells under various conditions
(scale bar = 20 μm).
∼15 nm. Selected-area electron diffraction of the TEM image
showed nice diffraction spots, indicating the crystalline nature
of the AgNPs. Energy-dispersive X-ray spectroscopy also
revealed the presence of Ag in the TEM image. The UV−vis
spectrum of the AgNPs in DMSO also showed a characteristic
surface plasmon peak at ∼458 nm. These data clearly
demonstrated that AgNPs indeed formed within 3UMECg;
it may be mentioned that the formation of metal nanoparticles
without the assistance of an external reducing agent might have
been facilitated by the urea moiety of 3U in the metallogel
because urea as well as amide moieties are known to act as
reducing agents for such a purpose.65,66 Sometimes solvents
such as N,N-dimethylformamide could also act as reducing
agents in synthesizing AgNPs.82 Interestingly, the PXRD
pattern of AgNP@3UMECg matched quite well with that of
simulated, bulk, and xerogel of 3UMECg, suggesting that the
formation of AgNPs within the metallogel did not alter the
Figure 14. (a) Synthesis and characterization AgNPs inside 3UMECg. (b) Loading and release of the drug SUL using the 3UMECg gel matrix. (c)
Zone inhibition and sheer thinning of 3UMECg.
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Author Contributions
All authors have given approval to the final version of the
drawing of the gel into a syringe and then syringing it out into
a vial without phase separation (Figure 14c and video).
manuscript.
CONCLUSION
■
Notes
Thus, we have successfully demonstrated that structural
rationale could indeed be employed to synthesize a new series
of OIHSs that not only incorporated the drug molecules
(NSAIDs) in the coordination network, thereby getting rid of
the need for loading of the drug into a vehicle, but also
provided a series of metallogels. While the metallogelator
3UMEF showed anticancer behavior against the B16F10 cell
line (MTT and cell migration assay), 3UNAP was successfully
internalized by the cancer cells (LSCM with DAPI), thereby
suggesting the possibility of exploring the metallogel 3UMEFg
as a topical gel for self-drug-delivery application for the
treatment of skin cancer. The successful synthesis of AgNPs
within the metallogel 3UMECg and its in vitro delivery to
inhibit the growth of Gram-negative bacterium Klebsiella spp.
and the loading and delivery of another NSAID, namely, SUL,
using 3UMECg as a gel matrix suggested utility of the
metallogels for the development of conventional DDSs with a
potential for multidrug delivery. Although DMSO is known to
facilitate skin penetration of drugs including NSAIDs in topical
application,83 the amount of DMSO required to form the
metallogels studied herein may have an adverse toxic effect.
Therefore, the present data mainly support the proof of
concept and should be considered as a model for in vitro drug
release. Research toward the development of such metallogels
from pure water or aqueous solvent systems containing safe
amounts of DMSO is required for real-life application.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
P.D. thanks DST (CRG/2019/000790) for financial support.
A SXRD facility was availed at the Department of Organic
Chemistry, IACS, supported by the CEIB program of DBT
(BT/01/CEIB/11/V/13). P.B. acknowledges IACS for a
fellowship. P.B. thanks Dr. Hemanta K. Datta for help
pertaining to the antibacterial experiments.
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AUTHOR INFORMATION
Corresponding Author
Parthasarathi Dastidar − School of Chemical Sciences, Indian
Association for the Cultivation of Science, Kolkata 700032,
India; orcid.org/0000-0002-1011-2386; Email: ocpd@
iacs.res.in
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Rudzinski, W. E. Biodegradable polymeric nanoparticles as drug
delivery devices. J. Controlled Release 2001, 70, 1−20.
(12) Cao, Z.; Tong, R.; Mishra, A.; Xu, W.; Wong, G. C. L.; Cheng,
J.; Lu, Y. Reversible Cell-Specific Drug Delivery with Aptamer-
Functionalized Liposomes. Angew. Chem., Int. Ed. 2009, 48, 6494−
6498.
Author
Protap Biswas − School of Chemical Sciences, Indian
Association for the Cultivation of Science, Kolkata 700032,
India
Complete contact information is available at:
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