Salt metathesis for developing injectable supramolecular metallohydrogelators as a multi-drug-self-delivery system

Article abstract of DOI:10.1039/c6cc07712a

Salt metathesis has been exploited to generate a series of non-steroidal anti-inflammatory drug (NSAID) based Zn(ii) metallohydrogels displaying both anti-inflammatory and anti-bacterial properties. Relatively low cytotoxicity, rheoreversibility and injec

Full text of DOI:10.1039/c6cc07712a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: P. Dastidar, R.
ROY, M. Bhagyalalitha and P. Choudhury, Chem. Commun., 2016, DOI: 10.1039/C6CC07712A.
This is an Accepted Manuscript, which has been through the
Volume 52 Number
1 4 January 2016 Pages 1–216
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Accepted Manuscripts are published online shortly after
Chemical Communications
www.rsc.org/chemcomm
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
=
3
rsc.li/chemcomm

Please do not adjust margins
ChemComm
Page 1 of 4
View Article Online
DOI: 10.1039/C6CC07712A
Journal Name
COMMUNICATION
Salt Metathesis for Developing Injectable Supramolecular
Metallohydrogelators as Multi-drug-self-delivery System
R. Roy,a M. Bhagyalalitha,a P. Choudhuryb and P. Dastidar*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Salt metathesis has been exploited to generate a series of non-
steroidal anti-inflammatory drug (NSAID) based Zn(II)
metallohydrogels displaying both anti-inflammatory and anti-
bacterial properties. Relatively low cytotoxicity, rheoreversibility
and injectibility of one of these hydrogels make it suitable for
multi-drug-self-delivery application.
Developing low molecular weight gelators (LMWGs)1 capable
of immobilizing (gelling) organic solvents (organogels) or
aqueous solvents (hydrogels) is an important and challenging
research topic because of their various potential applications.2
It is now well-established that the gelator molecules self-
assemble via various supramolecular interactions (H-bonding,
π–π stacking, C-H⦁⦁⦁π interactions, van der Waals interactions,
halogen bonding, etc.) to form self-assembled fibrillar
networks (SAFiNs)3 within which the solvent molecules are
immobilized resulting in gels.
Supramolecular gels having both biomedical4 and rheo-
reversible5 behaviour are of great interest because they are
evolving as promising bio-materials for developing injectable
drug delivery system6 in a self-delivery fashion (popularly
known as self-delivery system). Contrary to conventional drug
delivery, in self-delivery approach, there is no need of delivery
vehicle; instead, the active pharmaceutical ingredients (APIs)
or the drug is converted to supramolecular gelators [either by
chemical (covalent) or supramolecular (non-covalent)
modification] and delivered to the target site either via
invasive (subcutaneous) or non-invasive (topical) route.7 It may
be noted that self-delivery systems involving non-steroidal
Scheme 1 Salt metathesis to generate NSAID based Zn(II) hydrogelators for various
biomedical applications (IND = indomethacin , MEF = mefenamic acid, DIC = diclofenac,
MEC = meclofenamic acid, TOL = tolfenamic acid, FLU = flufenamic acid, IBU =
ibuprofen, FLR = flurbiprofen, KET = ketoprofen, NAP = naproxen, SUL = sulfasalazine,
DIF = diflunisal).
anti-inflammatory drugs (NSAIDs) are being explored with
great vigour in recent times.8 The use of conventional therapy
based on a single therapeutic agent capable of only targeting a
single site is insufficient due to complex defence action in
diseases. Thus ‘combination therapy’ using multi-drug-delivery
has become popular due to simultaneous administration of
two or more APIs/drugs with different mechanisms of action.9
Since bacterial infection leads to increase reactive oxygen
species (ROS) responsible for inducing inflammation,10 we
combined various NSAIDs and their derivatives with Zn(II) salts
to develop metallohydrogels as multi-drug-self-delivery system
suitable for combination therapy; while the NSAID is expected
to reduce inflammation,11 Zn(II) is likely to bind well with the
negative charge of the bacterial cell membrane thereby acting
as deterrant to bacterial growth.10
a. Department of Organic Chemistry, Indian Association for the Cultivation of
Science (IACS), 2A and 2B, Raja S. C. Mullick Road, Jadavpur, Kolkata - 700032,
West Bengal (India). E-mail: ocpd@iacs.res.in
b. Department of Biological Chemistry, Indian Association for the Cultivation of
Science (IACS), 2A and 2B, Raja S. C. Mullick Road, Jadavpur, Kolkata - 700032,
West Bengal (India)
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
In this communication, we exploited salt metathesis12 to
develop Zn(II) based supramolecular metallohydrogels derived
from various NSAIDs and their amino acid derivatives capable
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
COMMUNICATION
Journal Name
of injectable multi-drug-self-delivery and cellular imaging as a the cases, the elastic modulus (G') was found to be more than
View Article Online
DOI: 10.1039/C6CC07712A
the viscous modulus (G'') and was almost frequency invariant
model system for combination therapy for the first time.
We first attempted to anchor Zn(II) to NSAID via over the entire range of ω indicating the visco-elastic nature of
carboxylate-Zn coordination bond. However, such attempts to the gels (Fig. S33-S41, ESI†). It was observed that the solid-like
react Zn(NO3)2 with pure NSAID in aqueous Na2CO3 solution behaviour or elastic moduli (G') of all the hydrogels were
resulted in precipitate or turbid solution presumably because ranging from ~3.23x104-2.87x105 Pa. Detailed rheological
of low solubility product of the resulting Zn(OH)2. To avoid studies with IND.SER.TRIS.Zn by variying counter anions and
this, we then considered salt metathesis approach wherein 2- organic salt:metal salt ratio indicated that both these
Amino-2-(hydroxymethyl)-1,3-propanediol(TRIS)-salt of
a
parameter played a crucial role in gelation (Table S2 and Fig.
number of selected NSAIDs were reacted with Zn(NO3)2 in 2:1 S42, S43, ESI†).
(TRIS-salt:Zn-salt) ratio in aqueous medium. While most of the Thus, treatment of Zn(II) salt with twenty eight various NSAID
TRIS-salt, upon such a treatment with Zn(NO3)2, resulted in salts resulted in hydrogels for six cases. At this point, it was
either insoluble mass or gelatinous precipitate, the salt important to understand whether the expected Zn(II)-NSAID
DIF.TRIS produced hydrogels instantaneously at room complex was responsible for gelation or it was the
temperature. Interestingly, Na salt of a few NSAIDs (DIC.Na
,
combination of both NASID salt and Zn(II) salt for such
MEC.Na IBU.Na and NAP.Na) – all commercially available – hydrogelation. For this purpose, we tried to isolate the Zn(II)-
,
resulted in insoluble mass except for MEC.Na which gave NSAID metal complexes for as many cases as possible. Our
instantaneous hydrogels, under identical conditions. The fact efforts resulted in isolation of single crystals of seven Zn(II)
that MEC.TRIS did not produce gel and MEC.Na was capable of coordination complexes namely (DIC)2.Zn, (IND)2.Zn, (TOL)2.Zn
,
gelling aqueous solvent upon identical treatment with IBU)2.Zn, (FLR)2.Zn, (SUL)2.Zn and (DIF)2.Zn obtained by
(
Zn(NO3)2 clearly indicated the role of cationic species in reacting the corresponding NSAID salts with Zn(NO3)2 under
gelation. It is well known that both hydrophobic and various conditions. Single crystal X-ray diffraction (SXRD)
hydrophilic interactions play a crucial role in hydrogelation. In revealed that all these crystals belonged to monoclinic crystal
this context, we further reacted Zn(NO3)2 with six selected system wherein the caboxylate moeites were coordinated to
amantadine (AMN) salts of L-amino acid derivatives of IND the metal center displaying all three possible coordination
under identical conditions; L-amino acid with hydrophobic side modes (unidentate, bidentate and bidentate bridging modes)
chain such as ALA, VAL, LEU, PHE and hydrophilic side chain (Table S4, ESI†). In the majority of the cases, the metal center
such as THR and HYP were chosen for this study in order to displayed octahedral or distorted octahedral coordiantion
tune the balance between hydrophobicity and hydrophilicity. geometry. Only in two cases i.e. (IBU)2.Zn, (FLR)2.Zn, the Zn(II)
The salt IND.ALA.AMN turned out to be a hydrogelator. We metal center showed tetrahedral geometry. Interestingly, in
further extended the study with TRIS salts of SER
peptides of IND DIC and MEF under similar conditions; the coordinated to the metal center to produce a secondary
salts DIC.HYP.TRIS
DIC.SER.TRIS and IND.SER.TRIS produced bulding unit of the type Zn3(COO-)6 wherein both tetrahedral
, HYP the case of (TOL)2.Zn, the carboxylate of tolfenamic acid
,
,
hydrogels instantaneously at room temperature (Scheme 1 and octahedral coordination geometry were observed (Fig. 1
and Fig. S29, ESI†). All the hydrogels were found to be thermo- and Fig. S44-S51, ESI†). Each of these crystals was tested for
irreversible. Minimum gelator concentration (MGC) of the hydrogelation simulating the identical conditions as originally
hydrogels derived from the peptide bioconjugate salts (4 wt %) adopted for gelation test (Table S3, ESI†). However, none of
were significantly better than that of the NSAID.TRIS salt (8 wt these Zn-NSAID complexes showed gelation ability under these
%) indicating the role of additional hydrogen bonding condition thereby indicating that both NSAID salt and Zn(II)
interactions coming from the peptide moiety in gelation. salt were responsible for gelation.
Further studies indicated that both counter anion and
stoichiometric ratio did not seem to have much influence on For this purpose, we selected four representative gels derived
gelation for MEC.Na IND.SER.TRIS and IND.ALA.AMN salts from MEC.Na.Zn DIF.TRIS.Zn DIC.SER.TRIS.Zn and
whereas in the rest of the cases, both counter anion and IND.ALA.AMN.Zn and subjected to high-resolution
stoichiometric ratio appeared to be sensitive but no general transmission electron microscopy (HR-TEM). In all the cases,
trend could be obtained (Table S1 and S2, ESI†). the gel networks were made of highly entangled 1D fibers. EDX
Next we investigated the morphology of the gel network.
,
,
,
The gels were characterized by dynamic rheology by data supported the presence of Zn (Fig.
performing frequency sweep experiments. Remarkably, in all
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
ChemComm
Please do not adjust margins
Journal Name
COMMUNICATION
View Article Online
DOI: 10.1039/C6CC07712A
Fig. 2 (A) – (D) Morphologies of various Zn(II) hydrogels under TEM; (E) step-strain
oscillatory switch of the hydrogel derived from DIF.TRIS.Zn; (F) preloaded
metallohydrogel of DIF.TRIS.Zn in a 1 mL hypodermic syringe; (G) hydrogel flowing
freely through the needle of the syringe. (H) hydrosol after passing through the syringe.
(I) formation of hydrogel after a few minutes.
Fig. 1 Single crystal structures of the NSAID-Zn complexes; (A) (IBU)2.Zn, (B) (FLR)2.Zn,
(C) (SUL)2.Zn, (D) (DIF)2.Zn, (E) (TOL)2.Zn, (F) (IND)2.Zn and (G) (DIC)2.Zn.
assessed by MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-
diphenyltetrazolium bromide) assay13 in mouse macrophage
cells (RAW 264.7 cell line); after 72 hr incubation at 37 oC
under 5% CO2 atmosphere, the IC50 value was found to be
50µM (Fig. S56, ESI†). It may be mentioned that the
hydrogelator was quite stable under physiological conditions
and also its solubility in aqueous solvent was found to be
significantly increased (74 times more soluble) as compared to
the parent NSAID i.e. diflunisal (DIF) making it most suited for
biomedical applications (Fig. S52-S53, ESI†).
2A-D and Fig. S30-S32, ESI†). To explore multi-drug-self-
delivery as combination therapy, we focused our attention to
the pure hydrogel obtained from DIF.TRIS.Zn as water is an
essential bio-solvent. In self-delivery applications, injectibility
of the corresponding gel is a very important criterion as the gel
can be delivered directly to the affected site via subcutaneous
routes.6 Thus, the rheoreversibilty of the pure hydrogel of
DIF.TRIS.Zn was probed in a step-strain oscillatory sweep
experiment. For this purpose, we first assessed the critical
strain (γc) for the hydrogel (8 wt %) that turned out to be 0.02.
In a separate experiment, the gel (2 mL, 8.0 wt %) was sheared
at a high strain (above critical strain, 50 %) for 200s and then
went back to low strain of LVE region (1 %). Viscous response
(G''>G') was observed at high strain following immediate
recovery to elastic response (G'>G'') for the sample over
several cycles demonstrating clearly the rheoreversible
behaviour of the metallohydrogel. The high plateau modulus
(G'~100 KPa), high yield stress (10 KPa), instantaneous
recovery under shear for the hydrogel pointed out that this gel
can be used for self-delivery applications as proposed (Fig. 2E).
To evaluate further the suitability of the hydrogel for invasive
(subcutaneous) delivery, the injectable property of the
hydrogel was measured. It was observed that preloaded
hydrogel in a 1 mL hypodermic syringe could be injected out to
a container as a sol which turned into gel within 2-3 minutes
(Fig. 2F-I and video, ESI†).
Prostaglandin E2 assay14 (PGE2 assay) in RAW 264.7 cell line
was then performed in order to evaluate the anti-
inflammatory property of the modified NSAID hydrogelator
DIF.TRIS.Zn
. Well-known inflammatory agents such as
lipopolysaccharide (LPS) and Interferon gamma (IFN-γ) were
used to induce inflammation in RAW 264.7 cells. 1 μg/mL LPS
and 100 ng/mL IFN-γ could produce maximum 299.9 pg/mL
PGE2 in the RAW 264.7 cell culture medium which was reduced
drastically to 21.9, 23.7 and 15.7 pg/mL in the presence of 50
µM (< IC50) of the DIF, DIF.TRIS and the metallohydrogelator
DIF.TRIS.Zn, respectively. The data clearly showed that the
anti-inflammatory response of DIF.TRIS.Zn was marginally
better than the original NSAID i.e. DIF as well as its TRIS salt
(Fig. S56, ESI†).
The next thing was to study the antibacterial properties15 of
the metallohydrogelator DIF.TRIS.Zn in comparison with the
parent drug DIF in order to establish the role of Zn in
antibacterial activity of the metallohydrogelator following a
reported procedure.16 For this purpose, we chose to work with
both Gram-positive [Bacillus subtilis (B. subtilis)] and Gram-
negative bacteria [Escherichia coli (E. coli)]. Colony count in
spread plate method established that the metallohydrogelator
was nearly three times more effective than DIF in killing E. coli
(MIC = 23, 75 mug/mL for DIF.TRIS.Zn and DIF, respectively)
Having evaluated the rheoreversibility as well as injectibility
of DIF.TRIS.Zn derived Zn(II)-hydrogelator, we then assessed
its suitability for self-delivery application in simulated
physiological conditions. It was observed that the
metallogelator was able to gel both 0.9 wt % NaCl solution and
phosphate buffer solution (PBS) at physiological pH 7.4 under
5% CO2 atmosphere at 37 oC. Cytotoxicity of the gelator was
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
COMMUNICATION
Journal Name
whereas killing efficiency was marginally different in the case
of B. subtilis (MIC = 6, 7 mug/mL for DIF.TRIS.Zn and DIF
A new series of Zn(II)-metallohydrogelators derived from
various salts of NSAIDs following salt metathesis is developed
View Article Online
DOI: 10.1039/C6CC07712A
,
respectively). While the role of Zn in killing gram negative as multi-drug-self-delivery system. One such hydrogelators
bacteria was quite clear from the data, such role was not so namely DIF.TRIS.Zn was found to be biocompatible (MTT
evident in killing gram positive bacteria; this could be because assay), anti-inflammatory (PGE2 assay) and anti-bacterial (zone
of the thicker cell membrane of the gram negative bacteria i.e. of inhibition) and displayed rheoreversibility (injectable)
E. coli making it comparatively more difficult to kill
and Fig. S55, ESI†). Zone of inhibition (ZH) studies on agar-
gelatin film subsequently established that the (EMR/2016/00894), New Delhi, and IACS for financial support.
(Table S12 thereby making it suitable for combination therapy.
PD thanks DBT (BT/01/CEIB/II/V/13),
DST
metallohydrogelator was relatively more efficient in killing RR thanks CSIR, New Delhi, for SRF. We thank Prof. Prasanta
both gram positive and gram negative bacteria compared to Kumar Das of Department of Biological Chemistry, IACS for his
the parent drug DIF as evident from the radii of the zone of support in the bacterial experiments.
inhibition (Fig. 3A-D). To demonstrate the suitability of the
metallohydrogelator in self-delivery application, we undertook
leaching experiments wherein 100 μL of PBS (pH 7.4) solution
Notes and references
was placed over a gel bed (100 μL, 8 wt% hydrogel of
DIF.TRIS.Zn) in a 5 mL test tube. Five such test tubes were
incubated at 37 °C for various time intervals (3, 6, 9, 12, and 24
h in each case). The concentration of the gelator in the PBS
layer was quantified in triplicate by UV-spectrophotometric
method. From the data, it was clear that the leaching of
1
(a) P. Terech and R. G. Weiss, Chem. Rev., 1997, 97, 3133; (b)
J. W. Steed, Chem. Soc. Rev., 2010, 39, 3686; (c) M.-O. M.
Piepenbrock, G. O. Lloyd, N. Clarke and J. W. Steed, Chem.
Rev., 2010, 110, 1960; (d) O. Lebel, M. Perron, T. Maris, S. F.
Zalzal, A. Nanci and J. D. Wuest, Chem. Mater., 2006, 18
,
3616; (e) Marleen Haring and David Dıaz Dıaz, Chem.
Commun., 2016, DOI: 10.1039/C6CC06533C.
2
N. M. Sangeetha and U. Maitra, Chem. Soc. Rev., 2005, 34
,
modified drug from the hydrogel was 52% after 24h (Fig. S54,
821.
ESI†).
3
4
5
6
7
M. George and R. G. Weiss, Acc. Chem. Res., 2006, 39, 489.
X. Du, J. Zhou, J. Shi and B. Xu, Chem. Rev., 2015, 115, 13165.
As both Zn(II) and DIF are reported to be emissive,17 we
employed DIF.TRIS.Zn for cellular imaging. For this purpose,
RAW 264.7 cells were incubated in presence of the
E. Carretti, L. Dei and R. G. Weiss, Soft Matter, 2005,
L. Yu and J. Ding, Chem. Soc. Rev., 2008, 37, 1473.
1, 17.
(a) F. Zhao, M. L. Ma and B. Xu, Chem. Soc. Rev., 2009, 38
,
hydrogelator at 37 C under 5% CO2 for 15 mins and subjected
883; (b) S. Bhuniya, Y. J. Seo and B. H. Kim, Tetrahedron Lett.,
2006, 47, 7153; (c) M. J. Webber, J. B. Matson, V. K. Tamboli
and S. I. Stupp, Biomaterials, 2012, 33, 6823; (d) K. J. C. van
Bommel, M. C. A. Stuart, B. L. Feringa and J. van Esch, Org.
to observation under fluorescence microscope excited with
blue light. Significantly intense green fluorescence could be
seen from the cells. Manual z-stacking of each layer of the
treated cells ruled out the possibility of mere adherence of the
hydrogelator on the cell membrane and supported the uptake
of the drug-derived-hydrogelator within the cells (Fig. 3E-G
and z-stacking animation video, ESI†).
Biomol. Chem., 2005, 3, 2917; (e) P. K. Vemula, J. Li and G.
John, J. Am. Chem. Soc., 2006, 128, 8932; (f) R. V. Ulijn and A.
M. Smith, Chem. Soc. Rev., 2008, 37, 664; (g) R. Langer,
Nature, 1998, 392, 5; (h) J. H. Jung, J. H. Lee, J. R. Silverman
and G. John, Chem. Soc. Rev., 2013, 42, 924; (i) S. Fleming
and R. V. Ulijn, Chem. Soc. Rev., 2014, 43, 8150.
(a) J. B. Matson and S. I. Stupp, Chem. Commun., 2011, 47,
7962; (b) R. Roy, J. Deb, S. S. Jana and P. Dastidar, Chem. Eur.
J., 2014, 20, 15320 and references therein.
8
9
M. S. Aw, M. Kurian and D. Losic, Chem. Eur. J., 2013, 19
12586; and references therein.
,
10 T. Kern, M. Giffard, S. Hediger, A. Amoroso, C. Giustini, N. K.
Bui, B. Joris, C. Bougault, W. Vollmer and J.-P. Simorre, J. Am.
Chem. Soc., 2010, 132, 10911.
11 B. J. Lee and J. R. Lee, Arch. Pharmacal Res., 1995, 18, 22.
12 T. Kundu, S. C. Sahoo, S. Saha and R. Banerjee, Chem.
Commun., 2013, 49, 5262.
13 Q.-S. Wang, Y. Xiang, Y.-L. Cui, K- M. Lin and X.-F. Zhang, PLoS
ONE, 2012, 7, e34122.
14 E. Heiss, C. Herhaus, K. Klimo, B. Bartsch and C. Gerhauser, J.
Biol. Chem., 2001, 276, 32008.
15 (a) A. Shome, S. Dutta, S. Maiti and P. K. Das, Soft Matter,
2011,
7, 3011; (b) S. K. Mandal, S. Brahmachari and P. K. Das,
ChemPlusChem, 2014, 79
,
1733; (c) A. Makovitzki, D.
Avrahami, and Y. Shai, Proc. Natl. Acad. Sci., 2006, 103
15997.
,
16 S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao
and D. Dash, Nanotechnology, 2007, 18, 225103.
17 (a) H. G. Brittain, B. J. Elder, P. K. Isbester and A. H. Salerno,
Pharmaceutical Research, 2005, 22, 999; (b) M. Tsuchiya, R.
Sakamoto, M. Shimada, Y. Yamanoi, Y. Hattori, K. Sugimoto,
E. Nishibori and H. Nishihara, Inorg. Chem., 2016, 55, 5732.
Fig. 3 Antibacterial activity of DIF (A, C, ZH = 10, 9 mm, respectively) and DIF.TRIS.Zn (B,
D, ZH = 15, 11 mm, respectively) in fused agar–gelatin films against B.subtilis and E.coli
respectively; fluorescent microscopy images of the RAW 264.7 cells incubated with the
metallohydrogelator DIF.TRIS.Zn. (E) bright field, (F) fluorescence and (G) overlay
images.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins