An easy access to topical gels of an anti-cancer prodrug (5-fluorouracil acetic acid) for self-drug-delivery applications

Article abstract of DOI:10.1039/c9cc03037a

An easy access to topical gels (both hydro- and organogels) derived from an anti-cancer prodrug namely 5-fluorouracil acetic acid (5-FuA) achieved by exploiting a simple salt formation strategy is reported for the first time. Nearly 85% of the salts synthesized were gelators. Single crystal structures of some of the gelator salts revealed an intriguing hydrogen bonding network including double stranded 1D chains stabilized through uracil-uracil complementary interactions and the crystal structures of the gelator salts corroborated well with the hypothesis based on which the gelators were designed. Studies indicated that both the hydrogel and the methyl salicylate gel of the gelator salt FuA-15 were suitable for self-drug-delivery application.

Full text of DOI:10.1039/c9cc03037a

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
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DOI: 10.1039/C9CC03037A
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An Easy Access to Topical Gels of an Anti-cancer Prodrug (5-
Flurouracil Acetic Acid) for Self-drug-delivery Applications
Received 00th January 20xx,
Accepted 00th January 20xx
Poulami Chakraborty and Parthasarathi Dastidar*
DOI: 10.1039/x0xx00000x
An easy access to topical gels (both hydro- and organogel) derived insulin etc. have been found near or within the storage
[
4]
from an anti-cancer prodrug namely 5-fluorouracil acetic acid (5- sites. Significant progress has been made in converting
FuA) achieved by exploiting simple salt formation strategy is drug/prodrug/API to supramolecular gelators for developing
reported for the first time. Nearly 85% of the salts synthesized SDD systems by covalent modification.[
3]
were gelators. Single crystal structures of some of the gelator salts However, designing a supramolecular gelator still remains a
revealed intriguing hydrogen bonding network including double major challenge because of inadequate insights into the
stranded 1D chains stabilized through uracil-uracil complementary gelation mechanism at molecular level.[
5]
Since our
[
6]
interactions and the crystal structures of the gelator salts serendipitous entry to the field nearly two decades ago, we
corroborated well with the hypothesis based on which the have been able to design a large number of gelators derived
gelators were designed. Studies indicated that both the hydrogel from simple organic salts following supramolecular synthon
and methyl salicylate gel of the gelator salt FuA-15 were suitable approach[ in the context of crystal engineering.[9] Such
[
7]
8]
for self-drug-delivery application.
structure-property correlation approach not only provide a
detailed structure landscape of the gelators but also allow
occasional prediction of gelation. Lately, we have been
Self-drug-delivery (SDD) is a relatively new concept that allows
deliveryofthe drug to the target site by itself without the aid of
[10]
actively involved in exploiting such design strategy to develop
[
1]
a drug delivery vehicle. The need for developing such system
has become essential because of the various problems
associated with the delivery vehicle requiredinconventional
[11]
SDD systems.
-fluorouracil (5-Fu) is a well-known anti-cancer drugwidely
5
used for treating various dermatological disorders such as skin
[
2]
drug delivery (CDD) system; synthesis, cytotoxicity,
bioavailability, biodegradability, biodesorbability of the vehicle
and drug loading and its release kinetics are indeed important
parameters that are tobe optimized in order to develop a
successful CDD system.
[12]
cancer, actinic keratosis, keratoacanthomaetc. It undergoes
a series to enzymatic transformation inside the cell to produce
thymidylate synthase inhibitor that eventually causes DNA
[13]
damage resulting in cell death. 5-fluorouracil acetic acid (5-
[14]
FuA)was synthesized many decades ago in order to improve
One of the various ways to develop SDD system is to convert a
drug/prodrug/active pharmaceutical ingredient (API) to a
supramolecular gelator;[ the resulting gel can then be applied
to the target site either via non-invasive (topical) or invasive
on the severe side effects exerted by 5-Fu. Literature reports
suggest that 5-FuA is a prodrug of 5-Fu as various conjugates
of 5-FuA did release 5-Fu in vitro as well as ex vivo (liver
3]
[15]
homogenate) conditions and a polyethylene glycol conjugate
(subcutaneous) route.Highlocal concentration and, slow and
[16]
showed in vivo anti-tumour activity. To develop a SDD
sustained release of the drug from the gel by itself to the
target site make it an attractive alternative.Naturehas
successfully adopted such strategy, e.g. self-assembled
aggregates of a few peptides, proteins, growth hormones,
system for 5-Fu, we therefore decided to work with the
prodrug 5-FuA because it has a monocarboxylic functionality
which is amenable to further modification.
We have demonstrated that one of the easiest and time
economic reactions in chemistry i.e. simple organic salt
formation (providing high yield of the corresponding salts)
turned out to be a remarkable strategy to get an easy access to
a large library of supramolecular gelators and gels displaying
a. School of Chemical Sciences, Indian Association for the Cultivation of Science
(IACS),
2
A and 2B Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India
E-mail: ocpd@iacs.res.in.
Electronic Supplementary Information (ESI) available: Experimental section,
[
7]
intriguing structures and properties. Thus, reactions of 5-FuA
synthesis scheme, IR and NMR data, gelation and crystallography information with a series of primary amines namely n-alkyl amines (Me-
table, gel characterisation by TEM, rheology and tube inversion, MTT assay tables,
DOX release experiment, ORTEP plots of the crystal giving salts and their hydrogen
bonding information table. See DOI: 10.1039/x0xx00000x
(
n 2
CH) -NH ; n = 2-11, 13-15, 17) providedsalts of 5-FuA in near
quantitative yield (Scheme 1).
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Formation of the salts was confirmed in FT-IR by the
disappearance of >C=OCOOHstretching band (1737 cm )of 5-
FuA in the resulting products (Fig. S2, ESI†).
Journal Name
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DOI: 10.1039/C9CC03037A
-1
As envisaged, the salts of 5-FuA studied herein displayed
remarkable ability to produce thermo-reversible gels (both
organo- and hydrogels); nearly 85% of the salts were able to
gel various polar and nonpolar solvents with good to moderate
minimum gelator concentration (MGC) (1-4 wt %) and thermal
stability (Tgel = 55-145  C)(Table S1, ESI†,). Importantly, most
of the saltscould form hydrogel with pure water and methyl
salicylate gel (MS-gel).It is needless to mention that water is
inherently biogenic whereas methyl salicylate is widely used in
many commercial formulation of topical gels for its
Figure 1:MS gel of FuA-14: (left)TEM image of the gel network (inset: photograph
of the gel in an inverted vial) and (right)elastic response (Gˊ> Gˊˊ) in rheology
(frequency sweep).
(acid:amine) salts and crystallized in the centrosymmetric
triclinic space group P-1 with an exception of FuA-14
(crystallized in non-centric space group P1); both FuA-10 and
FuA-12 were 1:2 (acid:amine) salts due to the deprotonation
of the uracil N-H. In the crystal structures, the uracil rings are
either expected to participate in complementary hydrogen
bonding interactions with each other or involve in hydrogen
bonding interactions with both ammonium cation and
vasodilation and anti-inflammatory properties thereby making
applications.[
17]
them suitable for topical
Further
characterization of the gels was, therefore, restricted to hydro-
and MS gels of 5-FuA salts (Fig. 1). Since gels are visco-elastic
materials, they display typical flow characteristics in
rheological studies; for gel materials, elastic modulus (Gˊ) is
larger than loss modulus (Gˊˊ) and nearly frequency invariant
over a longer time scale in a frequency sweep experiment.[18]
Rheological studies of the selected hydro- and MS gels (4 wt %)
of 5-FuA salts carried out at a stress of 0.1 % [as guided by the
linear visco-elastic region (LVE)obtained in amplitude sweep
experiments, Fig. S18, ESI†] clearly established the visco-elastic
nature of the gels (Fig. S19,ESI†).In these gels,bothGˊand tan 
carboxylate anion. Indeed, the 1:1 (acid:amine)
salts
namelyFuA-9, FuA-11,FuA-14 and FuA-15 displayed
complementary hydrogen bonding involving the uracil moiety;
in these structures, the carboxylate and ammonium ions were
…
involved in hydrogen bonding through N-H O interactions
resulting in 1D chain propagating along the a-axis; such 1D
chains formed double stranded 1D HBN through the
(
Gˊˊ/Gˊ)varied widely (~1.7-56.2 kPa and ~0.1-0.6, respectively)
indicating moderate to good mechanical strength of the gels
Table S3, ESI†). TEM images of the dried gels revealed the
…
complementary N-H O hydrogen bonding involving the uracil
moieties of the neighbouring 1D chains. The double stranded
(
1
D chains further self-assembled through hydrogen bonding
presence of discrete short to long tape like morphology in
most of the cases whereasentangled 1D fibrous morphology
was observed in the hydrogels of FuA-11, FuA-15, FuA-16 and
FuA-18 (Fig. S21, ESI†).
To probe the structure landscape of as many gelator salts as
possible, we made efforts to obtain single crystals of the
gelator salts that resulted in X-ray quality crystals of FuA-9 to
FuA-12, FuA-14 and FuA-15 (Table S2, ESI†).SXRD studies
revealed that all the salts (except FuA-10 and FuA-12) were 1:1
involving the >C=O at 4-position of uracil moiety and the
…
ammonium cation through N-H O interactions leading to an
overall 2D HBN. Such 2D sheets were further packed in
interdigital fashion maximizing alkyl-alkyl interactions involving
the long alkyl chains of the ammonium cations (Fig.2).
Scheme 1: List of Salts made from the acid derivative of 5-Fu.
Figure 2: Crystal structure illustrations of the gelator salt FuA-11: a) double
stranded 1D HBN displaying complementary hydrogen bonding involving uracil
moiety; b) side view of the 2D HBN; c) overall packing.
2
| J. Name., 2012, 00, 1-3
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The 1:2 (acid:amine) salts i.e. FuA-10 and FuA-12, on the other
View Article Online
DOI: 10.1039/C9CC03037A
hand displayed cylindrical 1D HBN (propagating along the a-
…
…
axis) through N-H O and N-H N interactions involving the
ammonium cation, carboxylate anion and deprotonated uracil
ring N. Such 1D chains were further packed in an interdigital
fashion presumably driven by alkyl-alkyl interactions of the
long alkyl chains of the ammonium cation (Fig. 3).Thus, the
SXRD data revealed that HBNs in the crystal structures of these
gelator salts were either 1D or 2D that corroborated well with
the hypothesis that 1D or 2D HBN promote gelation under
suitable conditions.[
19]
Having achieved this feat, it was necessary to evaluate the
biocompatibility and anti-cancer behaviour of the gelator salts
and compare the results with that of the parent drug 5-Fu. For
this purpose, we carried out MTT assay in mouse macrophage
RAW 264.7 and melanoma cell line (B16F10) as a model for
human skin cancer. Data revealed that more than 50% RAW
2
64.7 cells survived at 0.03 mM concentration of 5-Fu, FuA-14
and FuA-15 whereas more than ~80% B16F10 cells died at
.02mM of 5-Fu and FuA-15. On the other hand, FuA-14
0
showed relatively poor anti-cancer property (~20% cells died
at 0.02 mM concentration). Thus, the anti-cancer property of
FuA-15 is comparable to that of its parent drug 5-Fu. It may be
noted that the cationic counter part of FuA-15 in its free amine
form i.e. pentadecyl amine was ineffective in killing the cancer
cells; nearly 85% of the cells survived at a concentration of
Figure 4: Cell migration assay in B16F10cell line up to 16hours observation.
Rheo-reversible property of MS gel of FuA-15 was then
evaluated in order to assess its suitability as topical gel if it was
to apply topically for treating dermatological disorders (vide
supra). MS gel of FuA-15 (6 wt %) was thus subjected to such
test that showed rapid recovery on shearing.The gel sample
was sheared at high strain (25 %, higher than the yield strain as
obtained in amplitude sweep experiment, Fig. S18,ESI†) for a
period of 120 s and switched to a low strain of 1% (within LVE
0
.01 mM.
Anti-cancer property of the gelator salts was further evaluated
by cell migration assay carried out on the same cell line. It is
well-known that cancer cells migrate much faster in
uncontrolled fashion resulting in metastasis as compared to
benign cells. From the speed of cell migration under in vitro
conditions, it is possible to assess the anti-cancer activity of an
agent; the slower the speed, the better is the anti-cancer
activity.[ The data revealed that the cell migration speed
under controlled condition (without adding the parent drug 5-
Fu or the gelator salts) was 12.7 M/h which was significantly
faster than that observed in presence of the parent drug 5-Fu
region).At
high
strain,
it
showed
a
viscous
response(G′′>G′)indicating disruption of the gel network.
Reformation of the gel network was quickly attained at low
strain as evident by the elastic response (G′>G′′). Such
transition of gel-sol-gel could be repeated over a few cycles.
The rheo-reversible behaviour was also manifested in its
injectable property(Fig. 5).
20]
(9.1M/h) and the gelator salts FuA-14 (5.0M/h) and FuA-
1
1
5(3.1M/h). Thus, the data revealed that the gelator salt FuA-
5 did possess relatively better anti-cancer property compared
to that of the parent drug 5-Fu(Fig. 4). It may be noted that we
have also evaluated the anti-cancer property of FuA-14 and
FuA-15 on a human lever cancer cell line HepG2. Both the salts
showed anti-cancer behaviour as revealed by MTT and cell
migration assays (Fig. S 23, ESI†).
Figure 5: Rheo-reversible and injectable nature of FuA-15 (methyl salicylate gel).
Figure 3: Crystal structure illustration of FuA-12: a) 1D HBN displaying cylindrical
HBN; b) overall packing.
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When a small amount of the gel was taken in a hypodermic
syringe and pushed into a container, it spontaneously showed
2
DOI: 10.1039/C9CC03037A
3
4
gel-sol-gel
transition
(see
the
video,
supporting
information).Nearly 63 % leaching of the drug salt FuA-15 into
PBS solution layered over a FuA-15 hydrogel bed in 24 h clearly
demonstrated the possibility of application of such hydrogel in
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Conclusions
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28and references cited therein.
Thus, we have demonstrated for the first time that an anti-
cancer prodrug namely 5-FuAcould easily be transformed into
a series of gelators that produced both hydro- and MS gels and
this was achieved by following salt formation strategy
developedby us. Compared to the existing literature reports
wherein various conjugates of 5-FuA were synthesized by non-
trivial, time expensive covalent synthesis for delivering 5-Fu,
the present work is obviously advantageous as it involves salt
formation as the synthetic step which is understandably one of
the easiest reactions to carry producing high yield. Structural
characterization of some of the gelator salts clearly supported
the salt formation strategy based on which the gelators were
designed. Various biological assays clearly established that the
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Whilethe MS gel showed rheo-reversible and injectable
properties, the hydrogel displayed leaching of the prodrug and
release of loaded anti-cancer drug DOX to PBS therebyraising
the hope of developing it further as potential self-drug-
delivery system.
P.C. thanks DST-INSPIRE (IF-150931) for providing research
fellowship. SXRD data were collected at the DBT-funded X-ray
diffraction facility under the CEIB program at the School of
Chemical Sciences, IACS, Kolkata. We thank Mr. Gourav Das for
fruitful discussion regarding for biological experiments.
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Conflicts of interest
There are no conflicts to declare.
Notes and references
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