Supramolecular Hydrogel Derived from a C3-Symmetric Boronic Acid Derivative for Stimuli-Responsive Release of Insulin and Doxorubicin

Article abstract of DOI:10.1021/acs.langmuir.7b03326

A C₃-symmetric triazine based triboronic acid (HG1) was designed and synthesized. HG1 was found to give hydrogel in DMSO-water (1:9). The hydrogel was rheo-reversible and thermoreversible over a few cycles. Single-crystal X-ray diffraction (SXR

Full text of DOI:10.1021/acs.langmuir.7b03326

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Article
Supramolecular Hydrogel Derived from A C3-symmetric Boronic acid
Derivative for Stimuli Responsive Release of Insulin and Doxorubicin
Koushik Sarkar, and Parthasarathi Dastidar
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03326 • Publication Date (Web): 11 Dec 2017
Downloaded from http://pubs.acs.org on December 14, 2017
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Supramolecular Hydrogel Derived from A C3-symmetric Boronic ac-
id Derivative for Stimuli Responsive Release of Insulin and Doxoru-
bicin
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Koushik Sarkar, and Parthasarathi Dastidar*
Department of Organic Chemistry, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road,
Kolkataꢀ700032, India
KEYWORDS: Boronic acid, Hydrogel, Honeycomb network, Drug release, Cell imaging
ABSTRACT: A C3ꢀsymmetric triazine based triboronic acid (HG1) was designed and synthesized. HG1 was found to give
hydrogel in DMSOꢀwater (1:9). The hydrogel was rheoꢀreversible and thermoreversible over a few cycles. Single crystal Xꢀray
diffraction (SXRD) studies on the crystals of HG1 established the presence of honeycomb network in which solvent molecules
(DMSO and water) were occluded. SXRD data corroborated well with the hypothesis based on which HG1 was designed. Stimuli
responsive release (in vitro) of insulin and doxorubicin from the hydrogel was also achieved.
We20,21 and others27 reported crystal structures of hydrogelaꢀ
tors derived from low molecular weight gelators (LMWGs)
INTRODUCTION
Gelation is a phenomenon wherein a large amount of liquid
(solvents) is entrapped and immobilized within a 3D fibrous
network arising due to covalent crosslinking of monomers
(polymer gel)1,2 or anisotropic selfꢀassembly of small moleꢀ
cules sustained by various supramolecular interactions (suꢀ
pramolecular gel).3ꢀ9 The resulting solidꢀlike softꢀmaterials
popularly known as gels are becoming increasingly important
in material science because of their various potential applicaꢀ
tions.10ꢀ17 Supramolecular hydrogel18 is a special type of suꢀ
pramolecular gel wherein, instead of organic solvent as in
organogel, the gelling solvent is pure water or aqueous solꢀ
vent.19ꢀ22 Unlike organogel wherein strong and directional
hydrogen bonding is the main driving force for selfꢀassembly
of gelator molecules, such hydrogen bonding becomes weak in
aqueous media in hydrogel; consequently hydrophobic interacꢀ
tions that lack the strength and directionality become imꢀ
portant in aqueous media. Thus a perfect balance between
hydrophobic and hydrophilic interactions must be achieved in
order to obtain hydrogel. Water being benign and intrinsically
biogenic, supramolecular hydrogel has enormous potential in
biomedical applications;23,24 it is becoming an attractive alterꢀ
native of polymeric counterpart mainly because of the followꢀ
ing properties that are unattainable by polymer hydrogel – i)
rapid response to external stimuli, ii) inherent thermoreversiꢀ
bility and iii) rapid excretion of low molecular weight gelator
molecule from the system. However, designing a hydrogelator
a priori is nontrivial because the molecular level mechanism
of gelation phenomenon (including hydrogelation) is unclear.
Nevertheless, there are a few groups involved in designing
hydrogelators following systematic molecular approach; for
example, van Esch and coꢀworkers reported a rational design
of a hydrogelator based on 1,3,5ꢀtriamide cis,cisꢀcyclohexane
core;25 Bing Xu and coꢀworkers exploited both hydrophobic
(aromatic…aromatic interactions) and hydrogen bonding to
achieve the required selfꢀassembly in water resulting in hydroꢀ
gels.26
that showed lattice inclusion phenomenon.28 Intrigued by the
striking analogies that exist between supramolecular gels and
lattice occluded crystalline solids (i.e. in both the cases, a large
amount of solvents are entrapped within the corresponding
networks that are formed by the supramolecular selfꢀassembly
of the molecules), we29ꢀ31 and others32 made deliberate atꢀ
tempts to develop lattice inclusion compounds suitable for
gelation. Lattice inclusion materials may be designed by synꢀ
thesizing a molecule which either cannot pack in all directions
and therefore needs solvent molecules as guest for crystallizaꢀ
tion or it has sticky functionality that recognizes each other via
supramolecular interactions (for example hydrogen bonding)
resulting in porous architecture that requires solvent molecules
as guests to stabilize the crystal structure. The design strategy
for generating such porous architecture lies on the judicious
selection of small building units (small molecules) known as
molecular tectons33 that can recognize themselves via supraꢀ
molecular interactions such as Hꢀbonding in a more specific
and directional manner. Such supramolecular system, under
suitable conditions, may result in gel.
Scheme 1
Like carboxylic acid (ꢀCOOH), boronic acid (ꢀB(OH)2) also
forms hydrogen bonded dimers; in majority cases, the hydroxꢀ
yl groups display syn-anti conformation with respect to
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Scheme 2: An overview of the results described in the present work.
the location of the hydrogen atom (type I) whereas dimers
displaying anti-anti and syn-syn conformation (type II) are
also reported34,35 (Scheme 1). Thus boronic acid functionality
can act as sticky site to selfꢀassemble the molecules to form a
network structure. Cambridge Structural Database (CSD)
analyses reveal that boronic acid functionality has hitherto
been marginally exploited to generate designed network strucꢀ
ture (Supporting Information). For example, Wuest et al. inꢀ
stalled boronic acid functionality on a tetrahedral C and Si and
successfully generated 5ꢀfold interpenetrated diamondoid netꢀ
work sustained by boronic acid hydrogen bonding.36 It is unꢀ
derstandable that if boronic acid functionality is installed in
strategic positions like 1, 3 and 5 position of a C3 symmetric
molecule, it can drive the molecules to selfꢀassemble to form a
honeycomb network structure having large pores to occlude
solvent molecules, which under suitable conditions may result
in gel.
In this article, we disclosed a new hydrogelator derived
from a C3ꢀsymmetric triboronic acid derivative of triazine
(HG1) having boronic acid functionality installed in the straꢀ
tegic 1,3,5 positions; the hydrogel so derived displayed the
ability to entrap and pH responsive release of an antiꢀcancer
drug (doxorubicin or DOX), and also showed glucose responꢀ
sive release of hydrogelꢀentrapped insulin. The single crystal
structure of HG1 displayed hitherto unknown honeycomb
network involving boronic acid dimeric hydrogen bonding
interactions and corroborated well with the hypothesis based
on which the hydrogelator (HG1) was designed. The gelator
molecule being photoluminescent was also exploited to perꢀ
form cell imaging (Scheme 2). Boronic acid based supramoꢀ
lecular hydrogelators were scarcely reported.14, 37ꢀ39
EXPERIMENTAL SECTION
Materials and Physical measurements:
All the chemicals were commercially available and used
without further purification. Solvents were laboratory reagent
(LR) grade and were used without any further purification.
Mouse macrophage RAW 264.7 was purchased from the
American Type Culture Collection (ATCC). FTꢀIR spectra
were obtained from a FTꢀIR instrument (FTIRꢀ8300, Shimadꢀ
zu). The elemental compositions of the purified compounds
were confirmed by elemental analysis (Perkin Elmer Precisely,
SeriesꢀII, CHNO/S Analyserꢀ2400). TGA analyses were perꢀ
formed on a SDT Q Series 600 Universal VA.2E TA instruꢀ
ment. Xꢀray powder diffraction patterns were recorded on a
Bruker AXS D8 Advance Powder (CuKα1 radiation, λ=1.5406
Å) Xꢀray diffractometer. TEM images were recorded using a
JEOL instrument with 300 mesh copper TEM grid. Scanning
electron microscopy (SEM) was performed with a JEOL JMSꢀ
6700F microscope. Rheology studies were performed using an
SDT Q series AR 2000 advanced rheometer. UV−Vis spectroꢀ
scopic measurements were carried out on a VARIAN CARY
50 Bio UVꢀVisible Spectrophotometer. NMR spectra (both 1H
and 13C) were recorded using 500 or 400 MHz spectrometer
(Bruker Ultrasheild Plusꢀ500/400). Emission spectra were
recorded with a Horiba Jobin Yvon Fluoromaxꢀ4 spectrofluoꢀ
rometer. MTT assay were conducted using a multiplate ELISA
reader (Varioskan Flash Elisa Reader, Thermo Fisher). Fluoꢀ
rescence microscopy was done in a Nikon TiꢀU eclipse invertꢀ
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ed microscope. CD data were collected in a JASCO CD specꢀ
Rheological studies: Rheology studies were carried out with
an Anton Paar Modular Compact Rheometer MCR 102 on at
25 °C in parallelꢀplate geometry (25 mm diameter, 1 mm gap).
Single crystal X-ray diffraction: Single crystal Xꢀray data
for HG1 was collected using CuKα (λ = 1.54184 Ǻ) radiation
on a XtaLAB Synergy, Dualflex, Pilatus 200K fourꢀcircle difꢀ
fractometer equipped with CCD plate Pilatus 200K detector.
Data collection, data reduction, structure solution and refineꢀ
ment were carried out using the software package of CrysAꢀ
lisPro 1.171.39.9b (Rigaku Oxford Diffraction, 2015). The
crystal was nonꢀmerohedrally twinned (59:41). Single crystal
Xꢀray data for NG1 was collected using MoKα (λ = 0.7107 Ǻ)
radiation on a SMART APEXꢀ II diffractometer equipped with
CCD area detector. Data collection, data reduction, structure
solution and refinement were carried out using the software
package of SMART APEXꢀII. The structure was solved by
direct methods and refined in a routine manner. The solvent
dioxane molecule was found to be disordered over three posiꢀ
tions (SOFꢀ 0.35414, 0.30393 and 0.34195).
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trometer (modelꢀJ815). Viscosity was collected using a
Brookfield viscometer. ImageJ software was used to determine
the size of the fibers in TEM and SEM.
Synthesis of
the precursor ((1,3,5-triazine-2,4,6-
triyl)tris(benzene-4,1-diyl))triboronic acid (HG1): HG1
was synthesized by acid catalyzed trimerisation reaction with
4ꢀcyanophenylboronic acid. 1.47
g (10 mmol) of 4ꢀ
cyanophenylboronic acid was taken in a 100 ml round botꢀ
tomed flask and 10 ml of trifluoromethanesulfonic acid was
added under ice bath. The reaction mixture was rotated 1 hour
in ice bath and at room temperature for overnight. The mixture
was worked up by adding drop wise in ice cold water with
continuous stirring. The precipitate was collected by filtration
and washed with water thoroughly. Yield: 380 mg. elemental
analysis of the crystals of HG1: calcd (%) for
C23H32B3N3O11S: C 46.74, H 5.46, N 7.11; found: C 46.31, H
5.24, N 7.29; ESIꢀMS on crude: m/z: 479.339 [M+K]+; FTꢀIR
(KBr): 3724ꢀ2717, 1945, 1824, 1613, 1575, 1518, 1466ꢀ1205,
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1
1173, 1122, 1014, 841, 809, 720 cmꢀ1. H NMR (500 MHz,
In all the cases, nonꢀhydrogen atoms were treated anisotropꢀ
ically except for the disordered atoms. Whenever possible, the
hydrogen atoms were located on a difference Fourier map and
refined. In other cases, the hydrogen atoms were geometrically
fixed at their idealized positions.
DMSOꢀd6, 25°C): δ= 8.712ꢀ8.696 (d, J= 8 Hz, 6H), 8.350 (s,
6H), 8.055ꢀ8.039 (d, J= 8 Hz, 6H). 13C NMR (500 MHz,
DMSOꢀd6, 250 C): 171.33, 139.60, 136.76, 134.62, 127.66.
Synthesis of the precursor 4,4',4''-(1,3,5-triazine-2,4,6-
triyl)trianiline (NG1): NG1 was synthesized by acid cataꢀ
lyzed trimerisation reaction with 4ꢀcyanoaniline. 1.16 g (10
mmol) of 4ꢀcyanoaniline was taken in a 100 ml round botꢀ
tomed flask and 10 ml of trifluoromethanesulfonic acid was
added under ice bath. The reaction mixture was rotated 1 hour
in ice bath and at room temperature for overnight. The mixture
was worked up by adding drop wise in ice cold water with
continuous stirring. The precipitate was collected by filtration
and washed with water thoroughly. Yield: 330 mg. elemental
analysis of the crystals of NG1: calcd (%) for C25H26N6O2: C
67.86, H 5.92, N 18.99; found: C 67.51, H 5.64, N 19.29; ESIꢀ
MS: m/z: 355.6 [M+H]+; FTꢀIR (KBr): 3282, 2966, 2871,
1629, 1509, 1452, 1373, 1360, 1199, , 892, 705 cmꢀ1. 1H NMR
(400 MHz, DMSOꢀd6, 25°C): δ= 8.36ꢀ8.34 (d, J= 8 Hz, 6H),
6.70ꢀ6.68 (d, J= 8 Hz, 6H), 5.92 (s, 6H). 13C NMR (400MHz,
DMSOꢀd6, 250 C): 170.80, 154.33, 131.69, 124.01, 114.31.
Gelation experiment
Crystallographic data for the structural analysis of comꢀ
pounds reported herein have been deposited at the Cambridge
Crystallographic Data Centre, CCDC Nos. 1541748ꢀ1541749.
Copies of this information may be obtained free of charge
from The Director, CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK (fax: +44 1233 336 033; eꢀmail: deposꢀ
it@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk/deposit).
Powder X-ray diffraction: PXRD data were collected using
Bruker AXS D8 Advance Powder (CuKα1 radiation, λ =
1.5406 Å) Diffractometer equipped with super speed
LYNXEYE detector. The sample was prepared by making a
thin film of finely powdered sample (~30 mg) over a glass
slide. The experiment was carried out with a scan speed of 0.3
sec/step (step size = 0.02˚) for the scan range of 5ꢀ35˚ 2θ.
Insulin release study
Loading of insulin on the gel matrix:
14 ꢀL of human insulin (1.388 mgmLꢀ1 or 40 IUmLꢀ1) stock
solution was diluted by 346 ꢀL of PBS. In a separate vial 13.2
mg of HG1 has been taken and 40 ꢀl of biological grade
DMSO has been added and slightly heated and cooled to room
temperature to form a colloidal solution. 360 ꢀL insulin soluꢀ
tion in PBS, prepared earlier, has been added to the gelator and
kept for few minutes until the gel formation takes place.
Release of insulin from the gel matrix:
10 mg of the HG1 was taken in a vial and 40 ꢀL of DMSO
was added and heated. After that 360 ꢀL of water was added to
the hot solution and kept undisturbed. After ∼ 2ꢀ3 minutes gel
was formed. Tgel was measured by the dropping ball method at
various gelator concentrations. In this experiment, a glass ball
weighing 205.50 mg was placed on a 0.4 mL of hydrogel (at
their respective minimum gelator concentration, MGC) taken
in a test tube (15x100 mm). The test tube was then immersed
in an oil bath placed on a magnetic stirrer to ensure uniform
heating. The temperature was noted when the ball touched the
bottom of the test tube.
Glucose solutions of different concentrations (5, 10, and 20
mM) were prepared in PBS solution of pH 7.4. These glucose
solutions (1 mL) were placed on top of the insulin loaded gel
and incubated for several hours. For control experiment, PBS
solution (without glucose) was layered on the top of the gel
bed. The release of insulin has been monitored by taking the
supernatant solution each time, and subjected to UVꢀVis analꢀ
ysis at λ=276 nm.
TEM sample preparation
The sample for TEM was prepared by smearing the hydrogel
(2.5 wt %) on a carbonꢀcoated Cu (300 mesh) TEM grid. The
grid was dried under vacuum at room temperature for one day
and used for recording TEM images using an accelerating
voltage of 100 kV without staining.
Doxorubicin adsorption within gel matrix:
In a typical experiment, 2 mL solution of 0.1 mg/mL doxoruꢀ
bicin hydrochloride (DOX) has been placed on the top of the
gel bed (2.5 wt%) and kept in dark for one week. After one
week, the supernatant has been decanted and the amount of
DOX adsorbed in the gel bed has been estimated by UVꢀVis
spectroscopy.
SEM sample preparation
SEM sample was prepared by smearing the hydrogel (2.5 wt
%) on a SEM stub. The grid was dried under vacuum at room
temperature for one day and used for recording SEM images.
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DOX release study:
was found to be stable at rt. for several weeks and therꢀ
moreversible over quite a few heatꢀcool cycles. Gradual inꢀ
crease in Tgel with the increase in gelator concentration supꢀ
ported the supramolecular nature of the gel network (Figure
S6b).40
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The release of DOX has been scanned in two different buffer
solution e.g. PBS (pH = 7.4) and acetate buffer (pH = 5.0). 2
ml of each PBS and acetate buffer was placed separately on
the top of DOX loaded gel samples and incubated at 37 °C for
several hours. In each time (3, 6, 9, 12, 24, 48 hours) 200 ꢀL
of supernatant has been taken out from the vials and equal
amount of respective buffer solutions have been added. The
release has been studied from UVꢀVis analysis. These release
profile has been investigated in triplicate.
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Biocompatibility studies
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MTT assay:
RAW 264.7 macrophage cells were purchased from American
Type Culture Collection (ATCC) and maintained following
their guidelines. The cells were cultured in Dulbecco’s modiꢀ
fied eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin–streptomycin and kept
in a humidified incubator at 37° C and 5% CO2.The cytotoxiꢀ
city of HG1 was evaluated in RAW 264.7 cells by using a
standard
MTT
(3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide) assay. In a 96ꢀwell plates, the
cells were seeded keeping density approximately 1×104 cells
per well. After 24 h of seeding, the cells were treated with
various concentrations (0.25, 0.50, 0.75 and 1.0 mM) of the
HG1 or DMEM alone for 72 h in a humidified incubator at
37° C and 5% CO2. The culture medium was then replaced
with 100 mg of MTT per well and kept at 37° C and 5% CO2
for 4 h. The formazan produced by mitochondrial reductase
from live cells was dissolved by adding DMSO (100 ꢀL per
well) and incubated for 30 min at 37° C. The absorbance of
formazan was recorded at 570 nm by using a multiplate
ELISA reader (Varioskan Flash Elisa Reader, Thermo Fisher).
The percentages of survival of cells in HG1 precursor treated
samples were calculated by considering the DMEMꢀtreated
sample to be 100%.
Figure 1: a) Viscoꢀelastic response in frequency sweep, b) thixotꢀ
ropy and c) rheoreversibility of the hydrogel of HG1.
Cell imaging:
For cell imaging, RAW 264.7 cells were cultured by using
DMEM supplemented with 10% FBS and 1% penicillin–
streptomycin on ethanol etched cover slips kept in a 35 mm
tissue culture dishes. The dishes were then kept in a humidiꢀ
fied incubator at 37° C overnight. Then the cells were washed
with PBS and incubated in serumꢀfree media (SFM) for half
an hour. DMSO solution of HG1 at IC50 concentration was
made by mixing it in serumꢀcontaining medium keeping Seꢀ
rumꢀcontaining medium: DMSO = 99:1 (v/v). The mixture
was shaken thoroughly. The solution was poured cautiously
into the cells and incubated for 30 minutes. After incubation,
media was discarded and the cells were fixed by using 4%
paraformaldehyde for 10 minutes at room temperature. Then
the cells were washed with PBS and mounted on glass slides
for microscopy.
To study the morphology of the gel network, we then perꢀ
formed highꢀresolution transmission electron microscopy
(HRꢀTEM) and scanning electron microscopy (SEM) on the
hydrogel. Entangled tape like morphology was observed in
both the experiments (width 220ꢀ525 nm in HRꢀTEM and
320ꢀ650 nm in SEM) (Figure S5).
Dynamic rheology (frequency sweep) of the hydrogel (2.5
wt %) revealed that the storage modulus G′ was largely freꢀ
quency invariant and higher than the loss modulus G″ – a typiꢀ
cal characteristics of viscoꢀelastic material like gel. The hyꢀ
drogel was found to be rheoreversible – an important criterion
for biomedical applications such as in drug delivery via topical
or subcutaneous routes. In a typical rheoreversibility test, we
applied a constant strain (50 %) higher than that of the critical
strain (12.8 %) and allowed the sample (2.5 wt %) to relax for
200 s followed by lowering the strain to 0.5 %. At higher
strain the sample showed a viscous response (G″ > G′) indicatꢀ
ing disruption of the gel network and at lower strain it disꢀ
played an elastic response (G′ > G″) confirming gel like beꢀ
havior; these data clearly established the rheoreversible propꢀ
erty of the hydrogel. Such thixotropic behavior of the hydrogel
was further confirmed by converting mechanically (vigorously
shaken by hand) the hydrogel to a sol which readily reverted
back to hydrogel within 15 mins upon resting and such gelꢀ
solꢀgel conversion was possible over a few cycles (Figure 1).
RESULTS AND DISCUSSION
The hydrogelator HG1 was synthesized by acid catalyzed
trimerization of 4ꢀcyanophenyl boronic acid at ambient condiꢀ
tion in more than 90 % yield (Supporting Information). HG1
was then subjected to gelation studies. Fourteen different solꢀ
vents (polar, nonpolar and aqueous) were scanned for gelation
(Table S1). Interestingly, it could gel only aqueous solvent
(1:9 v/v DMSO/water) resulting in hydrogel. The minimum
gelator concentration (MGC) was found to be 2.5 wt % with a
gel to sol dissociation temperature (Tgel) 57 °C. The hydrogel
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To probe structureꢀproperty correlation based on which
inclusion compound from a hot solution of NG1 in 1,4ꢀ
dioxane wherein one of the –NH2 group was found to be inꢀ
volved in NꢀH…O hydrogen bonding interaction with the O
atom of 1,4ꢀdioxane (Figure S10).
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HG1 was designed (vide supra), attempts were made to grow
single crystal of HG1. After several trials, we could crystallize
HG1 by slow cooling of a hot solution (DMSOꢀwater) of the
hydrogelator. Single crystal Xꢀray diffraction (SXRD) reꢀ
vealed that the crystal was nonꢀmerohedrally twinned (59:41)
and belonged to the noncentrosymmetric orthorhombic space
group Pna21. The asymmetric unit comprised of fourteen molꢀ
ecules of HG1 and quite a few scattered electron densities
which could not be modeled. Analyses revealed that HG1 selfꢀ
assembled to form a hitherto not reported 2D honeycomb netꢀ
work sustained by boronic acid dimer hydrogen bonding interꢀ
actions resulting in a large pore (~27 x 30 Å); each pore was
further interpenetrated by seven 2D honeycomb network disꢀ
playing a 8ꢀfold interpenetrated hydrogen bonded network
(Figure 2). The scattered electron densities were attributed to
disordered solvent molecules (1 DMSO and 4 H2O per asymꢀ
metric unit) located within the interstitial space of the network
as suggested by the SQUEEZE calculations.41 TGA data on
freshly grown crystals of HG1 indicated ~25.8 % weight loss
in the temperature range of ~31ꢀ132 °C which corroborated
well with the SQUEEZE calculation (calc. weight loss for 1
DMSO and 4 H2O ~25.4 %).
To probe further the role of boronic acid hydrogen bonded
dimer in hydrogelation, we carried out gelation experiments as
a function of pH. HG1 was able to produce hydrogel at pH
7.4, 5 and 3 whereas it produced clear solution at pH 10. Boꢀ
ron being a strong Lewis acid, it is expected to be coordinated
by hydroxyl ion at alkaline pH (10) thereby changing the coꢀ
ordination geometry of B (in boronic acid) from planar to tetꢀ
rahedral consequently disrupting the boronic acid hydrogen
bonded dimer resulting in a transition from gel to sol. Underꢀ
standably, the hydroxyl group of boronic acid is not readily
available for protonation at lower pH (e.g. 3 and 5) thereby
keeping the gel undisturbed. The corresponding morphology
in HRꢀTEM revealed intriguing results; highly entangled 1D
tape type of morphology at pH 7.4 was observed indicating
stability of the gel network. On the other hand, the tape type of
fibers appeared to have been broken into small pieces at lower
pH (5 and 3). The effect of lowering the pH on the morpholoꢀ
gy was more prominent at pH 3 compared to that at pH 5. This
could be because of the partial disruption of gel network arisꢀ
ing due to protonation of the ring N of triazine moiety of
HG1. Such observation was corroborated well with the correꢀ
sponding viscosity data; a plot of average viscosity (η) vs. pH
showed that η gradually increased with the increase in pH up
to pH 7.4 and then abruptly dropped to a minimum at pH 10
(Figure 3).
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Figure 2: a) Honeycomb network, b) eight fold interpenetration
of the honeycomb network in the crystal structure of HG1.
To provide further support to the existence of occluded solꢀ
vent molecules, freshly grown crystals of HG1 were soaked in
1
MeOD and H NMR of the filtrate was recorded; the appearꢀ
ance of a quintet peak at δ 2.05 supported the existence of
DMSO (Figure S11ꢀS12). Elemental analysis also supported
the SQUEEZE calculations (see experimental section).
The forgoing data clearly indicated that the hydrogelator
HG1 indeed formed lattice occluded crystalline solid which
was in agreement with the hypothesis based on which HG1
was designed (vide supra). However, it was not possible to
decipher the exact supramolecular structure of the network in
the xerogel; while the PXRD of the bulk solid and the xerogel
displayed excellent match, the simulated pattern calculated
from SXRD data showed several discrepancies with the other
two patterns.6 This may be attributed to the loss of the occludꢀ
ed solvents from the bulk solid as well as the xerogel (Figure
S13).
Figure 3: Variation of average viscosity and morphology with
pH.
To probe the role of boronic acid hydrogen bonding in crysꢀ
talline network formation as well as hydrogelation, we exꢀ
ploited cisꢀdiolꢀboronic acid chemistry.42 It is well known that
cis diol reacts with boronic acid by coordinating the lone pair
of electrons of the hydroxyl groups to B atom thereby elimiꢀ
nating one hydroxyl group from B resulting in expected disꢀ
ruption of the hydrogen bonding interactions. Thus, an aqueꢀ
ous solution containing glucose was carefully layered over a
hydrogel bed of HG1 at rt. After 48 hrs., the gel bed started
breaking and after one week, it turned out to be a colloidal
mass indicating complete disruption of boronic acid hydrogen
bonding resulting in the breakage of the gel. It may be noted
that HG1 was photoluminescent displaying emission band at
(λem ≈ 400 nm, λex ≈ 330 nm in DMSO) and the negative
The data presented thus far clearly indicated that boronic acꢀ
id dimeric hydrogen bonding in HG1 played a crucial role in
gelation. To support this further, we synthesized a trisꢀamine
analogue of HG1 (devoid of boronic acid functionality, desigꢀ
nated as NG1). Expectedly NG1 was unable to gel any solvent
studied herein. Since –NH2 functionality cannot form dimeric
hydrogen bonding akin to boronic acid moiety, NG1 was unaꢀ
ble to form the similar 2D honeycomb network required for
gelation. In fact, NG1 was found to crystallize as 1,4ꢀdioxane
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charge generated on B atom after cisꢀdiol reaction in presence
Page 6 of 9
The ratio of both these bands (Φ209/Φ223) provides a qualitative
idea of the overall conformation of insulin.37, 44 The ratio for
excreted insulin from the hydrogel under various glucose conꢀ
centration and PBS alone was found to be within the range
1.25ꢀ1.21 which is nearly equal to that of native insulin (Table
S3). Therefore, no significant conformational change took
place for the released insulin from the hydrogel network comꢀ
pared to that of the standard one.
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of glucose was expected to increase the PL intensity of HG1.
Interestingly, HG1 in solution could easily sense glucose as
revealed by PL studies (Figure S15ꢀS16).
These results prompted us to study glucose responsive reꢀ
lease of insulin from the hydrogel matrix.37 HG1 was found to
produce gel in DMSOꢀPhosphate buffered saline (1:9, pH =
7.4) with slightly higher MGC (3.3 wt %) as compared to that
of in DMSOꢀwater (2.5 wt %). The increase in MGC could be
because of the increase in polarity of the medium due to the
presence of salts in PBS. We first made hydrogel (3.3 wt %
containing insulin) in DMSOꢀPBS in such a way that the final
concentration of insulin remained 0.05 mg/ml. Remarkably
insulin containing hydrogel thus prepared was found to be
stable for more than a week and HRꢀTEM data revealed that
the gel network retained the tape like morphology observed
for the hydrogel alone (Figure S17b). We then assessed the
glucose responsive insulin release from the hydrogel using
varying concentration of glucose in PBS. It was observed that
the release of insulin gradually increased with the increase in
concentration of glucose (32% in PBS only, 39 % in 5 mM
glucose, 58 % in 10 mM glucose and 75 % in 20 mM gluꢀ
cose). Thus, under simulated diabetic conditions43 (10 and 20
mM glucose in human blood serum), the insulin entrapped
hydrogel could release a significant amount of insulin. From
The data presented thus far clearly indicate the crucial role
of boronic acid hydrogen bonding in gelation. Therefore, the
hydrogel matrix is may be exploited as a pH responsive drug
delivery vehicle. In a model study, we loaded an antiꢀcancer
drug doxorubicin in its hydrophilic form i.e. doxorubicin hyꢀ
drochloride (DOX) within the hydrogel matrix and monitored
its pH responsive release by UVꢀVis spectroscopy. It may be
noted here that DOX which is devoid of any cisꢀdiol is not
expected to react with boronic acid moiety. In a typical experꢀ
iment, 0.1 mM aqueous solution of DOX was layered on the
top of 400 ꢀL gel bed (2.5 wt%) and kept at rt. for one week.
The data revealed that the maximum loading of the drug was
76% (Figure S20). The DOX entrapped hydrogel bed was then
subjected to be in contact with phosphate buffered saline
(PBS, pH = 7.4) and acetate buffer (pH = 5.0) by layering the
corresponding buffer solution on the top of the hydrogel bed in
separate experiments.
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Figure 5: a) Image of DOX adsorption on the HG1 gel matrix, b)
release of DOX from HG1 gel matrix in a pH responsive manner.
Time dependent UVꢀVis data of the supernatant solution reꢀ
vealed that the pH responsive release of DOX was much highꢀ
er (~56 %) in the case of acetate buffer compared to that of in
PBS (~25 %) (Figure 5). The result is quite intriguing as it is
well known that in cancer cells, the pH is quite acidic45 and
such DOX loaded hydrogel may be used in treating skin canꢀ
cer (for example Kaposi sarcoma) via topical route.46
To demonstrate further the slow and sustained release of
DOX from the hydrogel matrix, we carried out in vitro studies
using RAW 264.7 cell line. In these experiments, we first
loaded a known amount of DOX in the hydrogel matrix, preꢀ
pared a xerogel (DOX@HG1) and carried out MTT assay on
RAW 264.7 cells. The results were then compared with the
MTT assay data obtained using only DOX. It was observed
that the percentage survival of the cells was much better in the
case of DOX@HG1 (∼62% at 1 ꢀM) compared to that of free
DOX (∼46% at 1 ꢀM) indicating slow and sustained release of
the drug from the gel matrix (Figure S21). Photoluminescence
of HG1 (vide supra) prompted us to undertake cell imaging
studies. MTT assay on RAW 264.7 cells revealed that the IC50
of HG1 was 1 mM after 72 hrs (Figure S22). Thus for cell
imaging studies, we kept the concentration of HG1 to 1 mM.
The cells treated with 1 mM HG1 could be nicely observed
under fluorescence microscope with green emission (λem = 560
nm) when excited with blue light (λex = 480 nm) upon 30 mins
Figure 4: a) Release of insulin from the gel matrix of HG1 in
presence of varying concentration of glucose, b) CD spectra of
standard and released insulin.
circular dichroism (CD) spectra it was evident that no signifiꢀ
cant denaturation of insulin was taken place under the experiꢀ
mental conditions (Figure 4). Native insulin displays two negꢀ
ative bands at λ= 209 and 223 nm in the CD spectra due to the
existence of α and βꢀhelical structure of insulin, respectively.
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Langmuir
and partly solved by Dr. Dyanne Cruickshank, UK. Single crystal
Xꢀray diffraction facility at the Department of Organic Chemistry,
IACS supported by CEIB program of DBT
(BT/01/CEIB/11/V/13) was used for data collection of NG1. We
thank Mr. Saswat Mohapatra, IICB Kolkata for helping in cell
imaging.
incubation at 37 °C (Figure 6). It may be mentioned here that
such experiments with 4 hrs incubation did not produce any
emission which may be because of the phagocytic behavior47
of RAW 264.7 cells (Figure S23). Manual Z stacking also
ruled out the possibility of adherence of HG1 on the cell and
confirmed the uptake of the compound within the cells (for Z
stacking animation video, see supporting information).
The possibility of auto fluorescence was also ruled out by a
control experiment in which imaging was done in the absence
of HG1 (Figure S24). To probe that the green emission was
indeed due to internalization, we excited the solution of HG1
at 480 nm (blue) and the corresponding emission peak was
observed at ~540 nm (green) (Figure S25). This observation
clearly established that HG1 was indeed internalized. It may
be noted that DMSO in low concentration (~1%) is routinely
being used in various biological assays.48, 49
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Figure 6: Fluorescence microscopic images of RAW 264.7 cells
incubated with HG1; a) bright field, b) fluorescence and c) overꢀ
lay.
CONCLUSION
Thus, we presented a unique design of a boronic acid funcꢀ
tionalized C3ꢀsymmetric molecule (HG1) that was capable of
forming hydrogel. The hitherto unreported 8ꢀfold interpeneꢀ
trated honeycomb network sustained by hydrogen bonded
boronic acid dimer left enough void space to occlude solvent
molecules (DMSO/water) in the crystal lattice of HG1 – an
observation that corroborated well with the strategy of designꢀ
ing the hydrogelator. The hydrogel displayed glucose responꢀ
sive release of insulin and pH responsive release of doxorubiꢀ
cin in in vitro studies. HG1 was also found to be internalized
in RAW 264.7 cell line as revealed by the cell imaging studꢀ
ies. Thus, the result presented herein is an elegant example of
the design of a functional hydrogelator based on a rational
approach.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental section, gelation studies, ORTEP plots, crystal data,
TGA, PXRD patterns and biological studies.
AUTHOR INFORMATION
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Corresponding Author
* Eꢀmail: ocpd@iacs.res.in
ACKNOWLEDGMENT
K.S thanks CSIR (Grant No. 09/080(0882)/2013ꢀEMRꢀ1) for
research fellowship. P.D thanks DST (Grant No.
EMR/2016/000894) for financial support. Single crystal Xꢀray
data of HG1 has been collected in a Rigaku oxford diffractometer
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Boronic acid hydrogen bonding interactions have been exploited to design a supramolecular hydrogel
displaying glucose responsive and pH responsive release of insulin and doxorubicin, respectively.
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