Mar 30, 2017 - Incorporating a photoreactive stilbene moiety, we show that the aggregation state of the material can be tuned by heating and UV exposure in order to control the mechanical as well as the fluorescent properties. Regulating the extent o
Mar 30, 2017 - Among these, supramolecular gels incorporating photosensitive elements have captured the attention of many researchers as a means for creating light responsive materials.(11-14) Capabilities of such systems include ..... (A) Illustrati
Jun 21, 2017 - A supramolecular gelator, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)-substituted cyclohexanediamine derivative, was synthesized, and its excellent charge-transporting capability was explored. The gels with organic solvents and electro
Jun 17, 2014 - Macrocycles often exhibit a distinct hostâguest chemistry and thus ... available by participants in Crossref's Cited-by Linking service. ..... Molecular design of supramolecular polymers with chelated units and their application as .
Jun 17, 2014 - His research interests focus on responsive materials, functional surfaces, and the mass spectrometric characterization of noncovalent complexes. Biography. Christoph ... Furthermore, their hostâguest interactions can be integral to g
Feb 26, 2014 - A new family of discrete hexakis-pillar[5]arene metallacycles with different sizes have been successfully prepared via coordination-driven self-assembly, which presented very few successful examples of preparation of discrete multiple
Feb 26, 2014 - ... Self-Organization of Triply Orthogonal Non-covalent Interactions on ..... Proceedings of the National Academy of Sciences 2016 113 (40), 11100-11105 ..... The marriage of endo-cavity and exo-wall complexation provides a ...
REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, ... i3S − Instituto de Investigação e Inovação em Saúde, Universidade do Porto,.
Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Characterization, Synthesis, and Modifications
Supramolecular Gels Derived from Simple Organic Salts of Flufenamic acid: Design, Synthesis, Structures and Plausible Biomedical Application Rumana Parveen, Bandi Jayamma, and Parthasarathi Dastidar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00153 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Supramolecular Gels Derived from Simple Organic Salts of Flufenamic acid: Design, Synthesis, Structures and Plausible Biomedical Application.
Rumana Parveen, Bandi Jayamma and Parthasarathi Dastidar* AUTHOR ADDRESS: School of Chemical Sciences, Indian Association for the Cultivation of Science, 2A & 2B Raja S .C. Mullick Road, Kolkata700032,West Bengal India, E-mail: [email protected]; Fax: 2473-2805; Tel: +91-33-2473-4971.
ABSTRACT. Following supramolecular synthon rationale in the context of crystal
engineering,
a
non-steroidal-anti-inflammatory-drug
(NSAID)
namely Flufenamic acid (FA) and its -alanine monopeptide derivative (FM) were converted to a series of primary ammonium monocarboxylate (PAM) salts. Majority of the PAM salts (~90 %) showed gelation with various
solvents
including
water
and
methyl
salicylate
(important
solvents in topical gel formulation). Structure-property correlation studies based on single crystal X-ray diffraction (SXRD) and powder Xray diffraction (PXRD) data provided intriguing insights into the structure of the gel network. Furthermore, one of the gelator salts (S7) displayed anti-cancer activity on a highly aggressive human breast cancer
viscoelastic (solid-like soft) materials usually formed by dissolving a little amount of solid (gelator) in a suitable solvent (gelling solvent) followed by external stimuli such as heat, photo-irradiation, sound, change in pH etc. Supramolecular
gelators, especially low
molecular weight gelators (LMWGs, a class of small molecules having molecular weight < 3000) are increasingly becoming important in material science due to their intriguing potential applications in developing electro-optics/photonics,9,10
sensors,11,12
cosmetics,13
structure
directing agents,14 reaction media for catalysis,15 conservation of ACS Paragon Plus Environment
gelator molecules and lack of clear understanding of gelation mechanism in molecular level (despite systematic efforts made by various groups2530)
make it difficult to design a supramolecular gelator a priori. One
of the early attempts to design a gelator molecule was based on structure-property correlation approach proposed by Shinkai et al.;31 based on a few single crystal structures of sugar derivatives and their property (gelation/non-gelation), they proposed that molecules prone to form 1D hydrogen bonding networks (HBNs) were expected to promote gelation as such 1D self-assembly of gelator molecules would promote the formation of network of fibers (known as self-assembled fibrillar networks or SAFiNs)32 that eventually immobilized the solvent molecules due to surface tension or capillary force action resulting in gel. Our group
has
been
supramolecular
developing
synthon33
supramolecular
approach
in
gelators
the
context
following of
crystal
engineering.34 Such link between crystal engineering and gels was first introduced by us and provided a way to occasional prediction of gelation as well as clear understanding of structure landscape of such selfassembled materials.35 Based on more than hundreds of crystal structures of
is the easiest reaction to carry out, b) purification step does not include time consuming and expensive column chromatography, c) yield is often near quantitative, d) 100 % atom economy makes the synthesis of the salts green and sustainable, e) virtually infinite combination of commercially available reactants (acids and amines) allow one to generate a large library of organic salts for gelation sacnning. In the present work, we exploited PAM synthon to develop organic salt based
gelators
for
self-drug-delivery
applications.
Self-drug-
delivery41 is an alternative approach to conventional drug-delivery wherein
a
drug
delivery
vehicle
(such
as
vesicles,42 polymer
gel
matrix,43 reverse micelles44 etc.) is not needed to deliver the drug; instead, it can be delivered to the target site by itself (hence the name self-drug-delivery). In this approach, one can avoid the issues related to delivery vehicles (syntheis, cytotoxicity, biodegradability and biostability of the vehicle, drug loading and release kinetics etc.). Self-drug-delivery45 can be achieved by modifying a drug to a supramolecular gelator (without compromising its drug action) so that a suitable gel derived from the gelator drug can be applied either via non-invasive (topical) or invasive (subcutaneous) route.
Scheme 1. An overview of the results described in the present work. For example, a long acting implant in the form of a supramolecular hydrogel
derived
from
a
synthetic
octapeptide
hydrogelator
was
installed via subcutaneous route to treat a growth disorder namely acromagley.46 Pyrene derivative of a highly potent antibiotic namely vencomycin
was
reported
to
form
hydrogel
that
displayed
enhanced
antibiotic activity.47 D-glucosamine derivative hydrogel was effective in treating wound in in vivo studies.48 We have also demonstrated that supramolecular synthon approach was helpful in coverting non-steroidalanti-inflammatory drugs (NSAIDs) to supramolecular gels that could be used to treat inflammation (both in vitro49 and in vivo50). Flufenamic acid (FA) is a well known NSAID containing a reactive monocarboxylic acid functionality. The drug is practically insoluble in water causing poor bioavailability and if administered orally, it causes severe gastrointestinal side effects. Having this bacground in mind, we synthesized a series of PAM salts of FA and its -alanine derivative (FM) by reacting with a number of primary amines with the hope of obtaining gelator salts that could be useful in self-drugACS Paragon Plus Environment
gelation studies, structure-property (gelation) correlation based on single crystal and powder X-ray diffraction data of some of the salts, biocompatibility (MTT assay), anti-inflammatory property (PGE2 assay) and anti-cancer property (cell migration assay) of some of the selected salts are discussed in the context of developing self-drug-delivery system (Scheme 1).
Experimental Section Materials and Methods: All the chemicals including Flufenamic acid and various amines were purchased from Sigma Aldrich and used without further purification. Solvents were AR (Analytical Reagents) grade and were used without any further purification.
Mouse macrophage RAW 264.7 and human breast
cancer cells MDA-MB-231 were purchased from the American Type Culture Collection (ATCC). For PGE2 assay Prostaglandin E2 EIA Kit – Monoclonal (Cayman Chemicals, Ann Arbor, MI) was used. Methods: NMR spectra (both 1H and
13C)
were recorded using both 400 MHz and 500
MHz spectrometer (Bruker Ultrashield Plus-500). FT-IR spectra were recorded
using
Microscopy
(SEM)
instrument was
FTIR-8300,
performed
in
a
Shimadzu. JEOL,
Scanning
JMS-6700F.
Electron
Rheological
experiments were carried out using MCR 102 Anton Paar modular compact rheometer. MTT and PGE2 assay were performed using a multi plate ELISA reader (Varioskan Flash Elisa Reader, Thermo Fisher. For
capturing
images of the cells, Carl Zeiss axio observer z1 fitted with a Hamamatsu ACS Paragon Plus Environment
orca flash 4.0 was used. Fluorescence measurements were done with a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer.
Synthesis of -alanine monopeptide of flufenamic acid (FM) First benzotriazole (357mg, 3mmol) was taken in
dry DCM (20ml) at
room temperature. To this solution thionyl chloride (90µl, 1.25 mmol) was added gradually and the reaction mixture was stirred for 20 minutes. The mother drug flufenamic acid (281 mg, 1mmol) was dissolved in dry THF (15 ml) and added dropwise to the first reaction mixture. The mixture was then stirred for 15 hours at room temperature. The white precipitate, thus obtained, was filtered off. Filtrate was concentrated under reduced pressure to obtain benzotriazole derivative of flufenamic acid as a white solid mass which was then dissolved in THF (20 ml). βalanine (89 mg, 1mmol ) and triethylamine (100 µl) dissolved in H2O and THF (20 ml, 1:4 ratio, v/v) was poured into the solution of bezotriazole derivative
of
flufenamic
acid
and
stirred
for
24
hours
at
room
temperature followed by removal of THF under reduced pressure. The concentrated solution was acidified by HCl (1N) in order to obtain a white precipitate. The white ppt was filtered and washed 3-4 times with distilled water in order to remove the excess acid. The solid product was then dried in a vacuum desiccator and was further purified by column chromatography (using 3% MeOH in DCM) to obtain ~61%) (Scheme 2).
Scheme 2. Synthetic procedure of -alanine monopeptide of flufenamic acid (FM). Synthesis of salts All the salts were prepared by reacting the corresponding acids and amines in 1:1 molar ratio in methanol at room temperature. Solid mass was obtained after the evaporation of the solvent in near-quantitative yield. FM:
FT-IR (KBr pellet): 1623 cm-1 (>C=O stretching for COO-,
Figure S12 in supporting information).
Measurement of Tgel Gel-sol dissociation temperature (Tgel) was measured by dropping ball method; a glass ball (400 mg) was placed on a 0.5 ml gel taken in a test tube (internal diameter = 1.0 cm) which was then immersed in an oil bath and the temperature was gradually increased till the ball touched the bottom of the test tube. The temperature at which the ball touched the bottom of the test tube was recorded as Tgel. Microscopy The SEM images were prepared by smearing a small amount of gel on SEM stub followed by drying overnight at room temperature. Single crystal X-ray diffraction SXRD data were obtained using MoKα (λ = 0.7107 Å) radiation on a BRUKER APEX II diffractometer equipped with CCD area detector. Data ACS Paragon Plus Environment
collection, data reduction, structure refinement were performed using the software package of SMART APEX-II. All structures were solved by direct method and routinely refined. Non-hydrogen atoms were treated anisotropically except for the disordered atoms. Flourine atoms of – CF3 in S2, S5 and S8 were found to be disordered over two positions. Wherever possible, the hydrogen atoms were located on difference Fourier map and refined; in other cases, the hydrogen atoms were geometrically fixed. MTT Assay Cytotoxicity of the gelator salts and the mother drug flufenamic acid was determined in RAW 264.7 as well as MDA-MB-231 cells using MTT (3(4,
5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Cells
were
cultured
in
Dulbecco’s
Modified
bromide)
Eagle’s
assay.
Medium
(DMEM)
supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin in a humidified incubator at 37° C and 5% CO2. In a typical experminent,
the
cells
were
grown
in
96-well
plates
(0.5
×
104
cells/well). After 24 hours, the cells were charged with various concentrations (upto 1 mM) of the gelators or DMEM alone (control) for 72 h in humidified incubator at 37˚C and 5% CO2. Then, the culture medium was replaced with 100 μg of MTT per well and kept the solution for 4 h at 37˚C. Thus formazan was produced by the mitochondrial reductase from live cells which was further dissolved by DMSO (100 μL/ well). The cells were incubated for 40 minutes at room temperature. The colour
intensity
of
the
formazan
solution
was
measured
using
a
multiplate ELISA reader at 570 nm (Varioskan Flash Elisa Reader, Thermo Fisher)., which was directly correlated to the cell viability or amount ACS Paragon Plus Environment
of living cell, The percentage of live cells in gelator-treated sample was thus calculated considering DMEM-treated cells as 100%. PGE2 assay PGE2 assay of S7 was carried out on both Raw 264.7 cells and human breast cancer cells MDA-MB-231. Approximately 1× 106 cells/well was seeded in a six well plate and kept it in a humidified incubator at 37°C saturated with CO2for over night. DMEM (1 ml) was taken in one well which was used as control. For Raw 264.7 to obtain maximum inflammation, the cells (rest wells) were charged with inflammatory agents such as lipopolysaccharid (LPS) and interferon-gamma (IFN-γ) so that final concentrations were 1μg/mL and 100 ng/mL, respectively. Out of
these
inflammated
cells,
two
wells
were
treated
with
0.1
mM
flulfenamic acid and S7 respectively in such a way that the total culture medium (DMEM) volume in each of the cases remained 1 ml. Next, it was kept for 24 hrs at 37°C saturated with CO2. Cell soup was then added to PGE2 kit. After 24 hours, the amount of PGE2 produced was determined using ELMAN’s reagent. For human breast cancer cells MDAMB-231, as the cells were already inflammated, no inflammatory agent such as LPS and IFN-γ was added. Cell imaging For cell imaging studies, MDA-MB-231 cells were cultured in 9 cm2 culture plate and kept it in a humidified incubator at 37°C saturated with CO2 for 24 hours. S7 (0.1 mM) was then added to it and incubated for 15 minutes. The cell medium was sucked out and the MDA-MB-231 cells were treated with 4% paraformaldehyde for 2-3 minutes for cell fixation.
The cancer cells were then washed with 1X PBS. The fluorescence images were captured using 20X objective lens using fluorescence microscope.
Results and Discussion Salts depicted in Scheme 3 were synthesized by reacting FA and FM with the corresponding amines in 1:1 molar ratio in MeOH at room temperature. The resulting white solids after evaporation of the solvent were subjected to FT-IR. Disappearance of –COOH stretching band at 1660 and 1709 cm-1 and appearance of –COO- stretching band at ~1600-1625 and ~1624-1639 cm-1 for FA and FM, respectively confirmed salt formation. The purity of the salts was also supported by 1H and
13C
NMR (Experimental
section). Remarkably most of the salts (9 out of 10 salts) were found to arrest the flow of various selected solvents indicating gel formation (Table 1).
The gels were thermo-reversible over a few cycles. While the minimum gelator
concentration
(MGC)
varied
from
~2.1-4
wt
%,
the
gel
dissociation temperature (Tgel) was found to be moderate (49-73 C). Amongst the gelator salts, S4 and S5 were found to be ambidextrous as it produced both hydro- and organo-gels. While S1, S4 and S5 showed gelation with pure water (hydrogel), S5 and S7 were found to form gel with methyl salicylate (MS) as well. Since MS is an important ingredient of several commercially available anti-inflammatory topical gels, the MS gels derived from S5 and S7 are particularly important in the present context.
As the main purpose of the present study is to develop ACS Paragon Plus Environment
supramolecular topical gel for developing self-drug-delivery system, the hydrogels and MS gels derived from S1, S4, S5 and S7 were studied further.
Scheme 3. Various PAM salts of FA and FM studied herein. The fact that various non-covalent interactions were crucial for gelation was clearly established from the steady increase of Tgel followed by a plateau with the increase in gelator concentration (Figure 1). Such table top rheological response was typical for supramolecular gels.51 Morphological
features
of
the
gel
network
were
revealed
in
SEM
micrograph. In a typical experiment, SEM samples were prepared by smearing a little amount of gel (at MGC concentrations) on a SEM stub followed by drying under room temperature for 12 hours. In all the cases, highly entangled 1D fiber like morphology was observed (Figure 2). ACS Paragon Plus Environment
Figure 1: Tgel vs [gelator] (wt % w/v) plot for hydrogel of S1, S4 and S5
and the MS gels of S5 and S7.
Flow properties of the gels (4.0 wt % hydrogels of S1, S4 and S5 and MS gels of S5 and S7) were studied by dynamic rheology. Typical viscoelastic response (wherein elastic modulus (Gˊ) is significantly larger than the viscous modulus (Gˊˊ) and remain frequency invariant over a longer time scale in frequency sweep experiment) is observed for all the gels under study confirming true viscoelastic nature of the gels (Figure 3).
Figure 2: Physical appearance of the gels (inset) and morphological feature of the gel network in SEM images; hydrogels of a) S1, b) S4 and c) S5 and MS gels of d) S5 and e) S7.
Figure 3: Rheological responses (frequency sweep) of the 4.0 wt % (w/v) hydrogels of a) S1, b) S4 and c) S5 and MS gels of d) S5 and e) S7 at a given constant strain of 0.1 % at 25°C.
Rheoreversibility indispensable
for
is
an
important
developing
gels
for
material
property
self-healing.52
that and
is
drug-
delivery53 applications. If a gel is rheoreversible, it should display gel-sol-gel transitions over a few high-low strain cycles. In other words, the gel under study should display viscus response (Gˊˊ>Gˊ) under high strain condition and quick elastic response (Gˊˊ>Gˊ) when the strain is removed (relaxation). This property is particularly important if the gel is to be administered topically or subcutaneously. Two selected gels (4 wt % hydrogel of S1 and MS gel of S7) displayed ACS Paragon Plus Environment
constant strain of 20 % (much higher than the corresponding critical strain - 7.5 and 4.4 % for hydrogel of S1 and MS gel of S7, respectively; Figure S13, supporting information) and allowed to relax for 100s (Figure 4).
Figure 4: Oscillatory shear of 4 wt%
a) hydrogel of S1 and b) MS gel
of S7. Structure-property correlation: To get an insight into the role of supramolecular synthons present in these salts and their influence on gelation, we set out to determine the single crystal structures of the salts. Single crystals were obtained for the gelator salts S1, S2, S4, S5 and S8 and subjected SXRD data collection (Table s1). Analysis of the crystal structures revealed that S1, S2, S5 and S8 displayed 1D HBN. On the other hand, S4 showed 2D HBN mainly because of the participation of the hydrogen bond functionalized cationic moiety in the salt. Closer look at the crystal structures showed that S1 possessed a 1D cylindrical HBN instead of a typical PAM synthon (‘X’ or ‘W’); PAM synthon ‘X’ was present in S2, whereas S8 displayed PAM synthon ‘W’ despite having an additional hydrogen bonding functionality (indole NH) that participated in hydrogen bond with the O atom of the carbonyl ACS Paragon Plus Environment
group of adjacent peptide salt; an overall 1D HBN network was observed in S5 despite having lattice occluded water molecules that participated in hydrogen bonding interactions with the carboxylate O and ammonium N atoms; the only gelator salt that displayed 2D HBN was S4; absence of a typical PAM synthon (‘X’ or ‘W’) in this structure could be due to the additional hydrogen bonding moieties (two hydroxyl group of the cationic species) that participated in hydrogen bonding (Figure 5). Powder X-ray diffraction (PXRD) patterns (simulated, bulk solid and xerogel) showed nice correspondence with respect to the peak positions thereby indicating high crystalline phase purity of the bulk as well as revealed the structure of the gel networks in their xerogel state (Figure 6). Thus, SXRD and PXRD data clearly supported Shinkai’s hypothesis
and
validate
our
supramolecular
synthon
approach
for
designing supramolecular gelators derived the NSAID Flufenamic acid.
Figure 5: Crystal structure illustration of the salts; a) cylindrical 1D HBN in S1, b) synthon ‘X’ in S2, c) synthon ‘W’ in S8, d) lattice occluded water mediated 1D HBN in S5 and e) 2D HBN S4 (moieties not participating in HBN are hidden for clarity).
Figure 6: X-ray powder diffraction patterns under various conditions. Biological studies: Owing to the gelation ability of S1, S4, S5 and S7 with solvents like water
and
methyl
salicylate
(both
suitable
for
biomedical
applications), we first evaluated the cytotoxicity profile of these salts in a macrophage cell line namely RAW 264.7 by MTT assay54 for 72 hours at 37 °C in Dulbecco's Modified Eagle's Medium(DMEM). The IC50 values for S1, S4, S5, S7 and FA were 0.1, 0.1, 0.25, 0.25 and 0.1 mM, respectively.
Cell viability of S7 was maximum ( % of survival of S7
at 0.25 mM concentration was ~65% whereas for S5 that was ~56%)
and
therefore, was best suited for further biological studies (Figure 7).
Figure 7: MTT assay of a) S1, b) S4, c) S5, d) S7 and e) FA in RAW 264.7 cell line for 72 hrs. at 37˚C.
It may be mentioned that NSAIDs reduce inflammation by inhibiting PGE2 producing enzymes like cyclooxygenase (COX-1/COX-2). PGE2 assay allows one to measure quantitatively extra-cellular concentration of PGE2; the less the concentration of PGE2, the better is the anti-inflammatory response. We, therefore, carried out prostaglandin E2 (PGE2) assay of the most biocompatible salt S7 in RAW 264.7 cell line following a published protocol55 in order to assess its anti-inflammatory response compared to that of the parent drug FA (see experimental section for details).
External
inflammation-inducing
agents
such
as
lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) were gradually added to obtain maximum inflammation in the cells as measured by the concentration
of
PGE2
in
the
cell
media.
After
24
hours,
PGE2
concentration (2006 pg/mL) was found to be significantly higher in the inflammated cells than that (614 pg/ml) in the control cells (without LPS and IFN-γ) under identical conditions. PGE2 concentration in cell media was significantly reduced to 45 pg/mL and 88pg/mL by S7 (0.1 mM) and FA (0.1 mM), respectively indicating the gelator salt S7 did possess ACS Paragon Plus Environment
anti-inflammatory property which was even better than that of the mother drug FA (Figure 8).
Figure 8: Anti-inflammatory response of FA and S7 in RAW 264.7 cells.
Anti-cancer activities: It has been reported that chronic inflammation at a particular site may cause cancer.56,57 Increased level of concentration of PGE2 has been detected in breast cancer patients58-60 PGE2 is also shown to play important role in progression and metastasis of cancer cells61 Tumor growth and metastasis are also linked to PGE262
Growth and progression
of prostate cancer has been successfully retarded by a combination therapy involving a well-known NSAID namely naproxen and vitamin D3.63 Recent in vitro studies revealed that a naproxen derivative was able to inhibit the cell migration of human breast cancer cells.64 Keeping these backgrounds in mind, we then evaluated the anti-cancer behaviour of the gelator salts S1, S4, S5, S7 and compared with that of the parent drug FA by MTT assay in a highly aggressive human breast cancer cell line MDA-MB-231; the results suggested that S7 had the best ability to ACS Paragon Plus Environment
kill the cancer cells (IC50 0.1 mM) which was even better than that of the mother drug (IC50 0.25 mM) (Figure 9).
Figure 9: Cell viability assay (MTT assay) in human breast cancer cells MDA-MB-231 at 37° C for 72 h in presence a) S1, b) S4, c) S5, d) S7 and e) FA, respectively.
Anti-cancer property of S7 was further supported by its ability to reduce extra-cellular concentration of PGE2 in the same cell line as revealed by PGE2 assay; after 24 hours, the amount of PGE2 production was 466 pg/ml for non-treated cells which was significantly reduced when the cells were charged with FA (24 pg/ml) and S7 (16 pg/ml) in separate experiments (Figure 10).
Cell migration assay: Uncontrolled migration of cells causing metastasis in cancer patients is a common phenomenon. Thus, in vitro inhibition of cancer cell migration (cell migration assay) is a measure of anti-cancer behaviour of any substance. We have thus subjected the gelator salt S7 to cell migration assay64 on the same breast cancer cell line i.e. MDA-MB-231. In a typical experiment, cells were seeded in a 6 well plate in DMEM. After 24 hours, a scratch was introduced on it by a sterile pipette tip (200µl). The cells were then charged with 0.1 mM of FA and S7. Control experiment was also performed where no drug was added. Times resolved still images were captured for 24 hours to measure the migration speed of the cells. After 24 hours, cells covered 40% of the gap for FA, 23% for S7 whereas in the control experiment, 84% gap was covered. The cell migration speed was 8.3, 3.7 and 2.2 µm/h in control, FA, S7 treated cells, respectively, suggesting that S7 was able to inhibit effectively the migration of the cancer cells (Figure 11).
Figure11: Migration of the cell front observed at different time intervals in scratch assay performed on MDA-MB-231 cells. Cellular imaging: Finally, cell imaging experiment was performed in order to probe whether
the
modified
gelator
salt
was
internalized65
or
not.
Interestingly, S7 was internalized in MDA-MD-231 cells as revealed by the green luminescence when it was excited with blue light (ex = 488 nm) under a fluorescence microscope (Figure 12). Green fluorescence (em = ~539 nm) was also observed from the DMSO solution of S7 (ex = 488 nm)(Figure S24).
Figure 12: (a) Bright field image; (b) Fluorescence image; (c) overlay image of S7 (using 0.25 mM concentration)
in MDA-MB-231 cells.
Conclusions Thus, supramolecular synthon rationale in the context of crystal engineering proved to be successful in designing a new series of LMWGs derived from PAM salts of a NSAID namely flufenamic acid. Out of the 10 PAM salts prepared, 9 salts were gelators of various solvents indicating that the hypothesis following which the PAM salts were designed was indeed significant. Single crystal X-ray structures as well as powder X-ray diffraction data of some of the gelators salts under various conditions further supported the hypothesis. The salts S1, S4, S5, and S7 were ambidextrous gelators producing gels with both ACS Paragon Plus Environment
exploiting the gels for self-drug-delivery applications via topical route. Various biological evaluations in the context of self-drugdelivery via topical route revealed that the salt S7 possessed both anti-inflammatory (PGE2 assay) and anti-cancer (PGE2 and cell migration assay) properties comparable to that of the parent drug FA and better than that of the rests. Thus, a topical formulation of S7 in methyl salicylate gel as well as its hydrogel could be useful in the context of self-drug-delivery. ACKNOWLEDGMENT P.D. thanks Department of Biotechnology (DBT), New Delhi for financial support (DBT project number: BT/01/CEIB/11/V/13). R.P. thanks CSIR, New Delhi
for
senior
research
fellowship
(CSIR
grant
number:
09/080(0876/2013-EMR-I). We thank, Parijat Biswas for capturing images in the cell imaging experiments. ASSOCIATED CONTENT Electronic Supplementary Information (ESI) available: spectra hydrogen
of
compounds,
bonding
detailed
parameters,
crystal CCDC
structures
1H
AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] REFERENCES
ACS Paragon Plus Environment
13C
NMR
descriptions,
1590527-1590530.
crystallographic data in CIF See DOI: 10.1039/x0xx00000x
Reinhoudt, D. N. An attempt to predict the gelation ability of hydrogenbond-based gelators utilizing a glycoside library. Tetrahedron 2000, 56, 9595–9599. DOI: 10.1016/S0040-4020(00)00915-7. (32)
George, M.; Weiss, R. G. Molecular Organogels. Soft Matter
Comprised of Low-Molecular-Mass Organic Gelators and Organic Liquids. Acc. Chem. Res 2006, 39, 489–497. DOI: 10.1021/ar0500923. (33)
Organogelators Derived from a Combinatorial Library of Primary Ammonium Monocarboxylate
Salts.
Chem.
Mater.
2006,
18,
3795–3800.
DOI:
10.1021/cm0605015. (38)
Trivedi,
D.
R.;
Ballabh,
A.;
Dastidar,
P.;
Ganguly,
B.
Structure-Property Correlation of a New Family of Organogelators Based on Organic Salts and Their Selective Gelation of Oil from Oil/Water ACS Paragon Plus Environment
Gujrati, V.; Kim, S.; Kim, S. H.; Min, J. J.; Choy, H. E.;
Kim, S. C.; Jon, S. Bioengineered Bacterial Outer Membrane Vesicles as Cell-Specific Drug-Delivery Vehicles for Cancer Therapy. ACS Nano 2014, 8, 1525–1537. DOI: 10.1021/nn405724x. (43)
Sumer
Bolu,
B.;
Manavoglu
Gecici,
E.;
Sanyal,
R.
Combretastatin A-4 Conjugated Antiangiogenic Micellar Drug Delivery Systems Using Dendron-Polymer Conjugates. Mol. Pharmaceutics. 2016, 13, 1482–1490. DOI: (44)
10.1021/acs.molpharmaceut.5b00931.
Lukanov, B.; Firoozabadi, A. Molecular Thermodynamic Modeling
of Reverse Micelles and Water-in-Oil Microemulsions. Langmuir 2016, 32, 3100–3109. DOI: 10.1021/acs.langmuir.6b00100. (45)
Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators
and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165–13307. DOI: 10.1021/acs.chemrev.5b00299.
Hydrophobic Interaction and Hydrogen Bonding Cooperatively Confer a Vancomycin Hydrogel: A Potential Candidate for Biomaterials. J. Am. Chem. Soc. 2002, 124, 14846–14847. DOI: 10.1021/ja028539f. (48)
Yang, Z.; Liang, G.; Ma, M.; Abbah, A. S.; Lu, W. W.; Xu, B.
Parveen, R.; Sravanthi, B.; Dastidar, P. Rationally Developed
Organic Salts of Tolfenamic Acid and Its β-Alanine Derivatives for Dual Purposes as an Anti-Inflammatory Topical Gel and Anticancer Agent. Chem. Asian J. 2017, 12, 792–803. DOI: 10.1002/asia.201700049. (50) Designing
Majumder, J.; Deb, J.; Das, M. R.; Jana, S. S.; Dastidar, P. a
simple
organic
salt-based
supramolecular
topical
gel
capable of displaying in vivo self-delivery application. Chem. Commun. 2014, 50, 1671–1674. DOI: 10.1039/C3CC48513G. (51)
Raghavan, S. R.; Cipriano, B. H. Gel Formation: Phase Diagrams
Using Tabletop Rheology and Calorimetry. In Molecular Gels; Springer, 2006; pp 241–252. ISBN 978-1-4020-3689-7. (52)
Sahoo, P.; Sankolli, R.; Lee, H.; Raghavan, S. R.; Dastidar,
P. Gel Sculpture: Moldable, Load-Bearing and Self-Healing Non-Polymeric
C. Nuclear Factor ΚB Is a Molecular Target for Sulforaphane-Mediated Anti-Inflammatory Mechanisms. J. Biol. Chem. 2001, 276, 32008–32015. DOI: 10.1074/jbc.M104794200. (56)
Balkwill, F.; Mantovani, A. Inflammation and cancer: back to
Virchow? Lancet 2001, 357, 539–545. DOI: 10.1016/S0140-6736(00)040460. (57) Shacter E.; Weitzman S. A. Chronic inflammation and cancer. Oncology 2002, 16, 217 – 226. (58)
Basu, G. D.; Pathangey, L. B.; Tinder, T. L.; Gendler, S. J.;
Mukherjee, P. Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells. Breast Cancer Res. 2005, 7, R422. DOI: 10.1186/bcr1019. (59)
Ristimäki,
A.; Sivula,
A.; Lundin,
J.; Lundin,
M.; Salminen,
T.; Haglund, C.; Joensuu, H.; Isola, J. Prognostic significance of elevated
cyclooxygenase-2
expression
in
Res. 2002, 62, 632-5. ACS Paragon Plus Environment
(60) Mayoral, R.; Fernández-Martínez, A.; Boscá, L.; Martín-Sanz, P. Prostaglandin E carcinoma
2
promotes migration and adhesion in hepatocellular
cells.
Carcinogenesis
2005,
26,
753–761.
DOI:
10.1093/carcin/bgi022. (61)
Chen,
E.
P.;
Smyth,
E.
M.
COX-2
and
PGE
2-dependent
immunomodulation in breast cancer. Prostaglandins Other Lipid Mediat. 2011, 96, 14–20. DOI: 10.1016/j.prostaglandins.2011.08.005. (62)
Timoshenko, A. V.; Xu, G.; Chakrabarti, S.; Lala, P. K.;
Chakraborty, C. Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells. Exp. Cell Res. 2003, 289, 265– 274. DOI: 10.1016/S0014-4827(03)00269-6. (63)
Srinivas, S.; Feldman, D. A Phase II Trial of Calcitriol and
