Fluorinated diphenylalanine analogue based supergelators: a stencil that accentuates the sustained release of antineoplastic drugs

Article abstract of DOI:10.1080/10610278.2020.1808892

Inspired by the prolonged metabolism displayed by para substituted fluorinated drugs, we intended to design two isomers Fmoc-(4F)-Phe-Phe-OH (hydrogelator I) & Fmoc-(3F)-Phe-Phe-OH (hydrogelator II) to explore the propensity of fluorine substitution in the aromatic ring of phenylalanine, in assisting or disrupting the gelation phenomena. However, our experimental observation reveals that hydrogelator I and II containing fluorines in the aromatic core illustrates excellent hydrogelation ability in comparison to the unsubstituted analogue, in accordance with the computational findings. Indeed, the hydrogelators displayed b-sheet with a fibrillar tape like morphology and were found to be biocompatible. We developed hydrogel nanoparticles (HNPs) that exhibited particle size less than 200 nm, and were found to release the antineoplastic drugs, 5Fluorouracil, curcumin and doxorubicin in a sustained manner depending on the architectural parameters of the drugs. Thus the prospective use of these compounds holds immense promise as a potential tool for future drug delivery applications.

Full text of DOI:10.1080/10610278.2020.1808892

Supramolecular Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gsch20
Fluorinated diphenylalanine analogue based
supergelators: a stencil that accentuates the
sustained release of antineoplastic drugs
Priyanka Tiwari , Arindam Gupta , Radha Rani Mehra , Naureen Khan , Jeena
Harjit , Charles R. Ashby Jr. , Anindya Basu , Amit K. Tiwari , Manju Singh &
Anita Dutt Konar
To cite this article: Priyanka Tiwari , Arindam Gupta , Radha Rani Mehra , Naureen Khan ,
Jeena Harjit , Charles R. Ashby Jr. , Anindya Basu , Amit K. Tiwari , Manju Singh & Anita
Dutt Konar (2020): Fluorinated diphenylalanine analogue based supergelators: a stencil that
accentuates the sustained release of antineoplastic drugs, Supramolecular Chemistry, DOI:
10.1080/10610278.2020.1808892
To link to this article: https://doi.org/10.1080/10610278.2020.1808892
Published online: 30 Aug 2020.
Submit your article to this journal
Article views: 5
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gsch20

SUPRAMOLECULAR CHEMISTRY
https://doi.org/10.1080/10610278.2020.1808892
ARTICLE
Fluorinated diphenylalanine analogue based supergelators: a stencil that
accentuates the sustained release of antineoplastic drugs
Priyanka Tiwaria, Arindam Guptab, Radha Rani Mehraa, Naureen Khana, Jeena Harjitc, Charles R. Ashby Jr.d,
Anindya Basue, Amit K. Tiwarif, Manju Singha and Anita Dutt Konara,e
b
aDepartment of Applied Chemistry, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, India; Department of Chemistry, IISER, Bhopal, India;
d
e
cDepartment of Applied Chemistry, Truba College, Bhopal, India; St. Johns University, USA; School of Pharmaceutical Sciences, Rajiv Gandhi
f
Proudyogiki Vishwavidyalaya, Bhopal, India; Department of Pharmacology and Experimental Therapeutics College of Pharmacy and
Pharmaceutical Sciences, University of Toledo, Toledo, OH, USA
ABSTRACT
ARTICLE HISTORY
Received 20 July 2020
Accepted 6 August 2020
Inspired by the prolonged metabolism displayed by para substituted fluorinated drugs, we
intended to design two isomers Fmoc-(4F)-Phe-Phe-OH (hydrogelator I) & Fmoc-(3F)-Phe-Phe-OH
(hydrogelator II) to explore the propensity of fluorine substitution in the aromatic ring of pheny-
lalanine, in assisting or disrupting the gelation phenomena. However, our experimental observa-
tion reveals that hydrogelator I and II containing fluorines in the aromatic core illustrates excellent
hydrogelation ability in comparison to the unsubstituted analogue, in accordance with the
computational findings. Indeed, the hydrogelators displayed b-sheet with a fibrillar tape like
morphology and were found to be biocompatible. We developed hydrogel nanoparticles (HNPs)
that exhibited particle size less than 200 nm, and were found to release the antineoplastic drugs,
5Fluorouracil, curcumin and doxorubicin in a sustained manner depending on the architectural
parameters of the drugs. Thus the prospective use of these compounds holds immense promise as
a potential tool for future drug delivery applications.
KEYWORDS
Hydrogel nanoparticles; high
mechanical integrity;
biocompatible; efficient drug
delivery vehicle
Introduction
Fluorine (F2), the first member of the halogen family,
is a very rare element. Due to its prevalence in diverse
molecular scaffolds, has been an area of significant inter-
est [17–20]. Fluorine’s high electronegativity, small
atomic radius and strong C–F bond strength produce
significant changes in various molecules [21]. However,
investigations from proteins crystallographic database
provided preliminary data regarding the interaction of
fluorinated drugs with specific proteins [22]. In biomedi-
cal applications, F18 isotope of fluorine have been routi-
nely used in radiolabeling of biomolecules that served as
a diagnostic probe for brain-imaging [23]. Even man-
made fluorinated compounds have served noteworthy
in various environmental concerns [24]. To date, fluorine
biology has been focussed as one of the aspects of
cutting-edge technologies [25–27]. Also, fluorine incor-
poration leads to reduced tooth decay [25–27]. The
metabolism of certain drugs can be decreased by the
presence of fluorine due to the high stability of the
C-F bond [27]. Consequently, the drug is present in the
blood for a longer period of time and thus the half-life is
increased [27]. Furthermore, the presence of fluorine in
drugs enhances the bioavailability due to increased cell
membrane penetration as a result of the enhanced
The intrinsic behaviour of low molecular weight peptide-
based hydrogels has gained significant impetus not only
due to their interests in fundamental science but also
their immense potential in biomedical applications [1–
5]. Peptide-based hydrogels have a decreased capacity
to produce environmental harm, that makes them
appealing synthons for the design and development of
soft materials [6]. Furthermore, they can self-assemble
via the involvement of non-covalent interactions unlike
other cross – linked polymers, that requires the presence
of strong covalent linkages for its formation [7–15]. The
fibrillar constructs that result from the hierarchical pro-
cess of self-assembly, in three dimensions, develops
large internal cavities, which entraps water molecules
inside them, ultimately, leading to the formation of self-
supporting hydrogels [16]. By simply changing the
amino acid residues, one can fine-tune the properties
and subsequently the mechanical strength of the hydro-
gels [16]. Although there are no definitive guidelines to
design hydrogelators, the molecules that fail to form
gels might provide better insights about the structural
aspects of the synthons with high hydrogelation pro-
pensities [16].
CONTACT Anita Dutt Konar
anitaduttkonar@rgtu.net
© 2020 Informa UK Limited, trading as Taylor & Francis Group

2
P. TIWARI ET AL.
hydrophobicity of C-F bond compared to C-H [28].
A recent study has shown how positional isomerism of
fluorine in the aromatic ring produces supramolecular
heterogeneity in peptidomimetic molecules [29,30].
Currently, there are only a few studies that have deter-
mined the effect of fluorine on supramolecular hydro-
gelation and the use of such molecules as efficient drug
delivery modalities [31–33].
(hydrogelator I) and Fmoc-(3 F)-Phe-Phe-OH (hydrogela-
tor II), by coupling 4/3-fluorinated phenylalanine and
phenylalanine residues. To form a hydrophilic drug deliv-
ery system, we decided to coat the nanoparticles with the
surfactant vitamin E-TPGS, such that they remain in the
body environment for a prolonged period [34]. Our sys-
tematic approach in designing and testing the novel drug
delivery system (DDS) hydrogel nanoparticles from fluori-
nated peptides have been discussed in detail in the sub-
sequent sections (Figure 1).
Ideally, an effective drug carrier should be a user-
friendly delivery system comprising a scaffold with sig-
nificant mechanical integrity and biocompatibility. It
should deliver the drug to the area of therapeutic inter-
est at a concentration that produces efficacy without
exerting adverse toxic effects in the system, and get
rapidly removed from the bloodstream in a secured
pathway, without hampering normal body functioning.
In this regard peptide-based hydrogel nanoparticles
have gained significant impetus in the last few decades
in the area of drug delivery because of the following
reasons: a) these particles possess small size and
enhanced surface area, that increases the solubility and
bioavailability, conferring them the ability to cross the
blood–brain barrier (BBB) and be absorbed through the
tight junctions of endothelial cells of the skin; b) their
simple route of administration by direct injection [34].
One disadvantage associated with these particles is their
hydrophobic nature that easily removes them from the
bloodstream. Therefore, in this study, we sought to: a)
design and synthesise biocompatible fluorine – substi-
tuted diphenylalanine derivatives that have a high hydro-
gelation potential and significant mechanical strength; b)
develop an easy to use hydrophilic delivery system that
would remain in the bloodstream for a prolonged period
and c) determine the release profile of the antineoplastic
drugs, namely 5-FU, doxorubicin and curcumin from the
hydrogel matrix. Thus to meet our goal, we designed and
synthesised two fluorinated phenylalanine derivatives,
fluorenylmethoxy carbonyl (Fmoc)-(4 F)-Phe-Phe-OH
Experiment
Materials and Methods
Fluorenylmethoxy carbonyl (Fmoc) chlorides, 5FU, cur-
cumin, amino acids and other common chemicals/sol-
vents used in the study were purchased from
Spectrochem India. The surfactant vitamin-E-TPGS and
doxorubicin were procured from Sigma Aldrich USA.
1H NMR spectra were recorded using a Bruker Ultra
shield (400 MHz). Mass was measured using (ESI-MS
mode) with a Micro TOF – Q-II instrument, IR with
a Shimadzu, Prestige 21 FT-IR spectrometer and elemen-
tal analysis using CHN Analyser (Elementar, Germany).
Synthesis of hydrogelator I and II
The hydrogelators were prepared using the solution
phase methodology, where dicyclo-hexylcarbodiimide
(DCC)/(1-hydroxybenzotriazole HOBT) was used as
a coupling agent (Scheme 1) [35,36]. Methyl ester hydro-
chlorides of phenylalanine were synthesised utilising the
thionyl chloride–methanol procedure [37,38]. The final
derivatives were purified by column chromatography
and the obtained derivatives were characterised by var-
ious analytical techniques as described.
Figure 1. A) Schematic representation of the fluorinated hydrogelators; B) Other components involved in inverse emulsion technique,
for the synthesis of HNPs.

SUPRAMOLECULAR CHEMISTRY
3
O
H
N
O
O
O
H2N
O
ClH .H2
N
CH3O H, SO Cl2
00C, 12 h
NaHCO 3
O H
H2N
OH
O CH3
O CH 3
O
F
DCC, DM F
00C, 12 h
O
H2
N
O H
O
O
H
N
H
N
N
H
TFA
M eO H, 2M NaO H
O
O CH3
O
O
O H
O
N
H
N
H
00C, 12 h
F
O
O
O
F
F
F
O
O
NaHCO 3, 1-4 Dioxane
00C, 12 h, Room Tem p
H
N
O
N
H
O H
O
O
O
Cl
Hydrogelator- I 4F
Hydrogelator- II 3F
Scheme 1. Synthetic Strategy of the hydrogelators, employing conventional solution phase methodology.
sodium carbonate solution (basic, 17 ml) and further
cooled in an ice-water bath. A solution of Fmoc-Cl
(2.81 g), solubilised in dioxane (18 ml) was added to
the cooled mixture from above and was stirred for
24 h. Finally, the pH was adjusted to an acidic level
with concentrated HCl and extracted with ethylacetate.
The organic layer was evaporated to get a white solid.
Yield: 3.43 g (85%, 6.22 mmol); M.p. 113–115°C, LC-MS
: C33H29FN2O5: m/z; 552.21 [M]+ (calculated); m/z; 552.8
[M]+ (obtained); FT-IR: 1442, 1566, 1648, 3232, 3230,
3406, 3482, cm−1; 1H NMR (d6-DMSO, ppm, 25°C,
400 MHz): 3.70–3.75 (CβHs of 4 F-Phe (1), Phe (2) &
Fluoren, 6 H, m), 3.96–3.99 (CH of Fluoren, 1H, m), 5.06–-
5.08 (CαHs of 4 F-Phe (1), & Phe (2), 2 H, m), 7.26–7.38
(Aromatic protons of Fluoren, Phe (2) & NH of 4 F-Phe (1),
& Phe (2), 15 H, m), 7.66 (Aromatic protons of 4 F-Phe (1),
2 H, d, J= 8 Hz), 7.83 (Aromatic protons of 4 F-Phe (1), 2 H,
d, J= 8 Hz). 13C NMR (100 MHz, d6-DMSO, 25°C, 400 MHz):
161.357, 161.190, 154.833, 150.97, 150.874, 145.681,
143.722, 141.264, 141.068, 128.248, 127.679, 127.190,
125.746, 125.361, 124.416, 120.716, 120.278, 69.153,
64.259, 50.564, 46.659; Anal. Calcd for C33H29FN2O5
(552.59): C, 71.722; H, 5.289; N, 5.07. Found: C, 71.700;
H, 5.139; N, 4.88.
Boc-(4 F)-Phe-Phe-OMe (1)
The free base of Phe-OMe obtained from its hydrochlor-
ide (5.34 g, 26.48 mmol) was poured into an ice-cold
solution of Boc-4(F)Phe-OH (3 g, 10.59 mmol) in 10 ml of
DMF, followed by DCC (3.27 gm, 15.88 mmol). The reac-
tion mixture was stirred for 18 hours at room tempera-
ture. Subsequently, the mixture was filtered and taken
up in ethyl acetate. The organic layer was washed with
2 M HCl solution, saturated sodium carbonate and
a brine solution and then dried over anhydrous sodium
sulphate and evaporated in vacuo to obtain a white
solid.
Yield: 4.04 g (86%, 9.09 mmol).
Boc-(4 F)-Phe-Phe-OH (2)
To a methanolic solution of compound 1 (4.04 g,
9.09 mmol), 2 N NaOH (3.5 ml) was added dropwise.
The progress of the reaction was monitored using TLC.
After completion of the reaction, the methanol was
evaporated and the residue was dissolved in water and
washed with diethylether. The aqueous layer was then
acidified with 2 N HCl and extracted with ethyl acetate to
obtain a white solid.
Yield: 3.40 g (87%, 7.89 mmol).
Hydrogelator I
Hydrogelator II
To an ice-cooled solution of (2) (3.40 g, 7.89 mmol) in
DCM, trifluoroacetic acid (5 ml) was added and stirred
until the Boc group was completely removed (based on
TLC results). Next, trifluoroacetic acid was removed
under reduced pressure to afford the crude trifluoroace-
tate salt. This dipeptide was dissolved separately in
Hydrogelator II was prepared using the same synthetic
procedure as that of HydrogelatoraI.Yield: 3.51 g (87%,
6.36 mmol); M.p. 110–113°C, LC-MS: C33H29FN2O5: m/z;
552.21 (calculated); m/z; 580.8 [M+ Na+ 5 H]+ (obtained);
FT-IR: 1418, 1538, 1622, 3250, 3426, 3488, 3564, cm−1;
1H NMR (d6-DMSO, ppm, 25°C): 3.71–3.73 (CβHs of

4
P. TIWARI ET AL.
4 F-Phe (1) & Phe (2), 4 H, m), 3.96–3.99 (CβHs of Fluoren,
2 H, m), 4.22–4.26 (CH of Fluoren, 1H, m), 4.46–4.48 (CαHs
of 4 F-Phe (1), & Phe (2), 2 H, m), 7.26–7.88 (Aromatic
protons of Fluoren, 4 F-Phe (1), Phe (2) & NH of 4 F-Phe
(1), & Phe (2), 19 H, m);). 13C NMR (100 MHz, d6-DMSO,
25°C, 400 MHz): 161.312, 154.833, 145.462, 143.871,
128.189, 127.768, 127.679, 127.555, 127.313, 127.190,
125.733, 125.259, 124.416, 120.667, 120.389, 119.480,
69.200, 64.259, 50.564, 46.635; Anal. Calcd for C33H29
FN2O5 (552.59): C, 71.722; H, 5.289; N, 5.07. Found: C,
71.502; H, 5.126; N, 4.74.
Results and discussion
Design and characterisation of the hydrogelators
Certain aromatic compounds can be biotransformed
into toxic epoxides specific drug metabolising enzymes,
by the body’s native enzymes [27]. Substituting
a fluorine into the para position of the aromatic ring,
however, restricts this process [27]. Inspired by this
event, with the aim of construction of novel hydrogels,
we decided to substitute the para position of first ring of
N-terminally protected diphenylalanine, Fmoc-(4 H)-Phe
-Phe-OH (Reported Molecule) by Fmoc-(4 F)-Phe-Phe-OH
(hydrogelator I), to determine the effect of fluorine sub-
stitution in inducing gelation phenomena (Figure 1).43
To further decipher the outcome of positional isomerism
on self-assembly we synthesised Fmoc-(3 F)-Phe-Phe-OH
(hydrogelator II). The diphenylalanine fragment was
chosen due to its high hydrogel-forming ability [43].
The Fmoc group was introduced to promote self-
assembly by its increased π − π correspondence.
Interestingly, both hydrogelator I and II had excellent
hydrogel-forming capacity at room temperature, with an
mgc of 0.01% and 0.015% w/v, respectively. To confirm
this property, the hydrogelators were initially dissolved
in 1 ml of water under mild heating conditions and
allowed to cool. The solvent system was almost instan-
taneously immobilised into a translucent mass, confirm-
ing the formation of gels.
Computation calculations
The molecules were modelled using Spartan08 software
and energy minimisation was done there itself.
Calculations were done on a single molecule. Further,
optimisation and frequency calculation with tight conver-
gence criteria was done on the obtained minimised struc-
ture in Gaussian 09 software package using B3LYP
functional and 6–31 G basis set. Molecular Orbital calcula-
tions were performed on these optimised structures.
HOMO-LUMO diagrams of the Hydrogelators were gener-
ated using GaussView 5.0 [39].
MTT assay
The cytotoxicity of hydrogelator-I was determined
through MTT (methyl thiazolyl tetrazolium) colouri-
metric assay, which measures cell metabolic activity
through the conversion of tetrazolium dye to insolu-
ble purple formazan crystals as per the method
described [40–42]. The dipeptides were dissolved in
DMSO stock solutions followed by making the dilu-
tions (with DMSO o0.1%) directly using cell culture
media. No precipitation or aggregates were observed
even on storage up to 72 h at 37°C. The cells were
seeded onto flat-bottom 96 well plates at a density of
4000–5000 cells per well. After 24 h, the cells were
drugged with serial dilutions (0, 0.1, 0.3, 1, 3, 10, 30,
100 mM) for each of the compounds in triplicate.
Next, we determined the stability of the gels by
investigating the sol – gel interconversion temperatures
(Tgel) at different hydrogelator concentrations (Figure
2). Importantly, the generated curves indicated an
increase of the Tgel value up to the saturation limit.
Moreover, we postulated that the self-assembly process
After 72
h of incubation, MTT dye (4 mg) was
added to all wells and incubated at 37°C for an addi-
tional 4 h. The medium was carefully discarded after
this incubation period and formazan crystals were
dissolved in 100 ml of DMSO in each well for 15 min-
utes. Absorbance was measured at a wavelength of
570 nm using
a DTX 880 multimode detector
(Beckman Coulter Life, Indianapolis, IN, USA). The
raw data were analysed and plotted using GraphPad
Prismv7.02. Student’s t-test was used to analyse all
the data [40–42].
Figure 2. Tgel curves as
concentration.
a function of hydrogelator

SUPRAMOLECULAR CHEMISTRY
5
is regulated by intermolecular non-covalent interactions,
which might have been optimal at the saturation limit.
Indeed, it has been reported that at the level of satura-
tion, an increase in the hydrogelator concentration did
not significantly alter the Tgel (temperature)
[12,13,40–42].
Generally, gelators with mgcs below 0.1% (w/v) are
categorised as supergelators [11]. Therefore, our mgc
values allowed us to characterise our hydrogelators as
supergelators. Our results are consistent with those of
Mahler et al. who reported that the unsubstituted ana-
logue, Fmoc-Phe-Phe-OH, had an mgc of 0.05% w/v in
water [43]. However, our fluorinated derivatives had
mgcs lower than 0.05% (Hydrogelator I: 0.01% and
Hydrogelator II: 0.015% w/v). Therefore, our initial
experiments suggested that fluorination accentuates
the process of hydrogelation.
hydrophilic (log P = 1.76). Also, the substitution of a fluor-
ine atom at the para position significantly changes the
electrostatic charge distribution compared to the meta
derivative, which could account for the difference in
threshold concentration with respect to the unsubsti-
tuted derivative (Figure 3, Table 1). Additionally, we
observe that the final potential energy and zero point
vibrational energy of the fluorinated derivatives are
lower in comparison to the unsubstituted one. This
makes both of their summation even lower (Table 1).
Overall, based on the results of the experimental and
computation studies, we postulate that the fluorine
introduction not only enhances the hydrophilic–lipophi-
lic balance but also gives rise to the possibilities of new
electrostatic interactions, thereby providing the oppor-
tunity to obtain better hydrogels.
Determination of the mechanical strength,
morphology and conformation of the hydrogelators
Computation investigation about the effect of
fluorine substitution in diphenylalanine in
hydrogelation
Firstly, we determined the mechanical integrity of the
hydrogelators by performing rheological measurements
with freshly prepared hydrogels of concentration 2 mg/
ml. As shown in Figure 4, throughout the viscoelastic
region, the storage modulus (G′), the loss modulus (G″)
(Figure 4(a)), and the complex viscosity (Figure 4(b))
were determined as a function of angular frequency.
The results indicated the formation of a soft gel phase
[11–13]. We then performed an amplitude sweep mea-
surement, where the strain was varied to 100%. We
noticed in the lower viscosity region, the G’ was higher
To search an explanation for the above observation, we
performed computational analysis using Gaussian09
software package with B3LYP functional and 6–31 G
basis set [39]. Figure 3 shows that introduction of
Fluorine seems to have considerable impact on the
HOMO/LUMO energies of the molecules, owing to subtle
changes in the electronic distribution of the system
(Table 1). For example, unlike the unsubstituted analo-
gue (log P = 2.36), Hydrogelators I and II are more
Figure 3. HOMO-LUMO Orbitals of I) reported molecule II) hydrogelator I and III) II.
Table 1. Experimental parameters of the underivatised and derivatised fluorine analogues probable
constructs responsible for driving the gelation mechanism.
Final
Energy
Zero-point vibrational
energy:
Sum of thermal and vibrational
Enthalpy
Hydrogelators HOMO
LUMO
Unsubstituted −5.897 eV −1.5 eV −1685.586
355.871
350.758
350.683
−1684.984
−1784.206
−1784.207
I
−5.842 eV −1.597 eV −1784.800
−5.784 eV −1.697 eV −1784.801
II

6
P. TIWARI ET AL.
Figure 4. Frequency sweep of A) Dynamic Shear Modulus; B) Complex Viscocity; C) Amplitude sweep of the hydrogels made from
hydrogelator I and II at 2 mg/ml concentration at room temperature. D) Bar graph representation of the comparison of the elastic
response (G’) and viscous response (G”) of the hydrogelators.
Figure 5. FT-IR and PXRD spectra of the Hydrogelators showing β-sheet-like conformation. The characteristic peaks in the PXRD curve
are presented in blue.

SUPRAMOLECULAR CHEMISTRY
7
Figure 6. FE-SEM images of hydrogelator I and II, (A, C: low resolution; B, D: high resolution) showing the formation of three-
dimensional dense fibrillar network. The remaining sol was, immobilised within the cavities of the network, thereby forming self-
supporting hydrogels.
Figure 7. Basic Principle of MTT Assay (Top); Biocompatibility Studies of Hydrogelator I in two different cell lines MDA-MB-231 and HEK
293 (Bottom).
than the G”, indicating the stabilisation of the gel phase.
But the sooner, the strain reached to 39.6% and 63% for
hydrogelators I and II respectively, a cross over point
was observed in the experimental frequency region,
where the value of loss modulus G” slightly exceeds
the value of storage modulus G’ and transformed into
solution state (Figure 4(c)). This particular point where
the crossover occurs is called yield stress (σy). These soft
materials displayed the gelation property until the
threshold value of strain is reached which is 39.6% and
63% for hydrogelators I and II respectively (Figure 4(c)).
As the strain was increased beyond this threshold, the
intermolecular forces that held the gel together started
being overcome by the applied strain and the xerogel
fibrils were unable to withstand deformations. The fre-
quency-sweep experiments further showed that both
the G’ and G” values were weakly dependent on the
frequency which was indicative of an entangled net-
work-like system.
Also, the moduli remained parallel to the x-axis, indi-
cating that the obtained hydrogels were stable and rigid
(Figure 4(a-b)). The data further confirmed that
Hydrogelator I (G’: 1700 Pa) had a higher gel strength
compared to Hydrogelator II (500 Pa) for gels of same
concentration, emphasising the importance of position
of fluorine in regulating mechanical integrity (Figure
4(d)).

8
P. TIWARI ET AL.
Figure 8. A) Inverse Emulsion Technique; B) Enlarged cartoon Diagram of Hydrogel Nanoparticle; C) Particle Size Dimension of the
HNPs.
Table 2. Comparative study of particle dimension/release time and percentage of various drugs from the HNPS developed from
Hydrogelators I and II.
Time of
release of
5FU
% of
Particle Size
(nm)
Time of release
of Doxorubicin
% of Doxorubicin
released
Time of release Curcumin
Hydrogelators
% of 5-FU released
50%
of Curcumin
released
Ref
48
Unsubstituted 225.9 3.6
21.5 0.1
5 h
20 h
50%
– – -
– – – – –
I
96.84 90.70
20.84 9.30
50 h
30%
112 h
30%
70 h
– – -
30%
This
This
work
Control (only
gel)
9 h
50% followed by
immediate burst
release
22 h
50% followed by
immediate burst
release
– – – – –
work
work
II
76.98 15.99
10.32 223
50 h
9 h
30%
72 h
22 h
30%
88 h
– – -
30%
This
This
Control (only
gel)
50% followed by
immediate burst
release
50% followed by
immediate burst
release
– – – – –
work
In order to determine the origin of the mechanical
the solid sample as synthesised and the xerogels, respec-
tively. Each molecule of the Hydrogelators contained
two NH protons and three carbonyl moieties (Figure 1).
strength, we performed structural studies using Fourier
Transform–Infra Red Spectroscopy and PXRD Analysis.
Figure 5(a-b) shows the overlapped FTIR spectra from

SUPRAMOLECULAR CHEMISTRY
9
Figure 9. % Cumulative drug release profile of the three antineoplastic drugs from the hydrogel matrix.
Figure 10. Computation investigation of hydrogelator -drug interactions. The model drugs that have been used are 5Fluorouracil
(green), Curcumin (yellow) and Doxorubicin (red). Interactions of 5-FU/Curcumin/Doxorubicin with A/C/D) Hydrogelator I and B/D/F)
Hydrogelator II.

10
P. TIWARI ET AL.
The peaks for the NH protons in both states appeared
provide additional data, supporting the nature of fibril-
lar network (Table 1). Interestingly, the tenacity of
hydrogelator II was more than that of I, as reflected
from Figure 4(c) (cross over point was observed at
39.6% and 63% strain for hydrogelators I and II,
respectively).
at 3390/3330 cm−1 for Hydrogelator I and II. These data
indicated that the NHs were H-bonded in Hydrogelator
I and II. In contrast, the amide I and II carbonyl had
characteristic peaks at 1620/1528/1464 cm−1 for
Hydrogelator
I
and 1612/1556/1448 cm−1 for
Hydrogelator II, respectively (Figure 5).
A closer analysis of the spectra further showed that
although the peak characteristic remained the same in
both states, the peaks were slightly broadened and
shifted for both the Hydrogelators. This could only
occur when the non-covalent interactions, namely
H-bonding and Van der Waals interactions form fibrous
network in three dimensions, that could immobilise the
remaining sol in them and form gels. Overall, the FT-IR
data were indicative of the dominant role of β-sheets in
the respective xerogel state [11–13,44,45].
Biocompatibility studies
Biocompatibility is an important characteristic for
a molecule to be used as a delivery system, which is
generally probed by MTT assay (Figure 7 top) [40–42].
Therefore, using this technique, we determined the
effect of the hydrogelators on the viability of human
embryonic kidney (HEK-293) and epithelial human
breast cancer cells (MDA-MB-231, as shown in Figure 7
(bottom)). Since our hydrogelators are isomers, we con-
ducted the experiment with only hydrogelator I, as iso-
mers are known to have similar behaviour. The IC50
values of hydrogelator I were >100 µM for both cell
lines, thereby allowing us to conclude the molecule to
be biocompatible [11–13,40-42].
To re-confirm our investigations we performed PXRD
experiments with the xerogels of the hydrogelators
[40–42]. The wide-angle PXRD curves showed appar-
ently similar patterns, with periodic diffractions, com-
mon in both the hydrogelators. This data indicated the
presence of ordered structures in the xerogels.
Additionally, the characteristic peaks at 18.49°
(d = 4.9 Å) might have arisen due to the presence of
the β-sheet-like packing in the hydrogelators. We
believe that this β-sheet formation could only be pos-
sible because of an effective overlap between the aro-
matic rings of Phenylalanine and Fmoc moiety,
stabilised by π-π interactions. Indeed, the peak at
24.79° (d = 3.7 Å) further affirmed our analogy. Thus,
taken together the result of FT-IR and PXRD data, our
finding revealed the presence of β-sheet conformation
in the xerogels [40,41]. To obtain additional data about
the morphological arrangement of the xerogels, we
conducted FE-SEM studies with the xerogels obtained
from the corresponding hydrogels (the same concen-
tration and magnification were used for both the
hydrogelators) (Figure 6). The FE-SEM images showed
the formation of dense, fibrillar tape-like structures that
were approximately 500 nm in width and several micro-
metres in length. We postulated that these tapes were
intertwined with one another, producing a three-
dimensional network that served as a template to
immobilise the remaining sol (water) and form self-
supporting hydrogels. Since the hydrogelators bear
isomeric relationship, they had a similar fibrillar net-
work. However, the fibres in hydrogelator I were thick
and more densely packed compared to hydrogelator II,
thus accounting for the differences in mechanical
strength and the minimum gelation concentrations.
Also, the differences in the HOMO/LUMO energies,
final potential energy and zero point vibrational energy
Why peptide-based hydrogel nanoparticles in drug
delivery?
In the last few decades, the development of nanoparti-
cles from various polymers and its use in drug formula-
tion have helped advance the treatment of certain
diseases [46]. However, one of the major problems asso-
ciated with various polymers is that they produce toxi-
city in normal cells.
Peptide – based hydrogel nanoparticles are less toxic
as they consist of biocompatible amino acids. Indeed,
from the literature, we confirmed that for therapeutic
use, the preferred size of the nanoparticles should be
less than 200 nm [47,48]. In the section below, we dis-
cuss the preparation of nanoparticles that contain fluori-
nated hydrogelators and determine their drug delivery
efficiency.
Preparation of hydrogel nanoparticles and its
release kinetics using anti-cancer drugs
The hydrogel nanoparticles (HNPs) were synthesised
using a modified inverse emulsion protocol as shown
in Figure 8(a-b). The hydrogelators had a bimodal dis-
tribution of particle size, with
a
dimension
96.84 21.32 nm and 20.84 3.25 nm for hydrogelator
I and 76.98 15.99 nm and 10.32 2.23 nm for hydro-
gelator II (Figure 8(c)). This bimodal particle size distri-
bution is probably a result of equilibrium between
droplet fragmentation and droplet re-coalescence that

SUPRAMOLECULAR CHEMISTRY
11
occurs due to variation in experimental conditions
[11,12,48]. Indeed, a higher value of negative zeta poten-
tial (−30 mV, the value should be high irrespective of the
charge) indicated the presence of system stability. This
negative charge might be due to the presence of car-
boxylates, as well as the surfactant coating around the
hydrophobic core [11,12]. Since the size of the particles
were less than 200 nm, we hypothesised that our
designed HNPS would release a greater percentage of
the drugs compared to the reported analogue and con-
trol hydrogelators (normal hydrogel, without forming
nanoparticles) for prolonged period of time (Table 2).
To test this hypothesis, we chose three different
drugs: 5-FU, curcumin and doxorubicin, which had vary-
ing properties, structures (structures shown in Figure 7
(b)) and chemotherapeutic efficacy [11–13,40-42,49].
Figure 9 represents the release kinetics of the drugs
over a stipulated period of time, which showed a marked
difference with variation in their respective molecular
weights. As evident from the figure, about 30% of the
5-FU content, from both hydrogelators were released
after a 50-h incubation period. In contrast, it took 70
and 88 hours for the release of 30% of the curcumin
content from Hydrogelator I and II, respectively. The
release of 30% of the doxorubicin took 112 hours from
the matrix of Hydrogeltor I and 72 hours from that of
Hydrogelator II.
To account for the differential behaviour in release
mechanism, we performed computation analysis to inves-
tigate the interactions of the drugs with the hydrogelators.
As revealed from Figure 10, initially two molecules of
the energy optimised hydrogelators could be imagined
to be oriented in the form of a clip, such that Fmoc
moieties lie on one end and Phe-Phe rings on the
other. Now the 5-FU, containing 30% aromatic character,
apart from H-bonding donors and acceptors, inorder to
maximise π-π interactions with the Fmoc units, remains
at the mouth of the clip [50]. So its release from the
matrix of both the hydrogelators becomes easier and
faster (Figure 10(a-b)). Although curcumin/doxorubicin
possessed both H-bond donors and acceptors along
with aromatic units, here the drug–matrix interaction
occurs mainly by hydrophobic and π-π interactions
(Figure 10(c-d)). For curcumin, we observe that in case
of hydrogelator II, the drug becomes sandwiched
between the Fmoc moieties of the clip, in an organised
fashion, stabilised by π-π interactions, unlike hydrogela-
tor I (sandwiched in a disordered manner), which might
be the cause of its slower release from the hydrogel
matrix. Again for Doxorubicin, we observe that for both
the hydrogelators, the drug stabilises itself by entering
deep into the pin pocket, utilising non-covalent
interactions. This stabilisation might be greater in case
of hydrogelator I, than II. Henceforth, the release of the
former gets drastically slowered (Figure 10(e-f)). Taken
together these results, we attribute the entire mechan-
ism of release pattern, to the variation in position of
fluorine in the aromatic ring of diphenylalanine and
physicochemical properties of the drugs. Actually,
these two scaffolds (drug and hydrogelators) interact
with each other in different extents, leading to overall
stabilisation of the motifs, resulting in faster/delayed
release response in different cases.
Conclusions
In summary in this report, we have successfully designed
and developed a set of fluorinated peptides that dis-
played excellent hydrogelation phenomena in aqueous
medium.
Our experimental observations backed by computa-
tional calculations, additionally revealed that fluorine
substitution in the aromatic ring in phenylalanine ring
(hydrogelator I and II) definitely enhanced the gelation
aptitude in comparison to the unsubstituted analogue.
Indeed, the hydrogelators showed significant mechan-
ical strength and biocompatibility, ample enough to be
used for drug delivery applications. So we devised for-
mulation to synthesise hydrogel nanoparticle with the
biocompatible hydrogelators. We believe that mere link-
age of vitamin E-TPGS at the surface could convert the
nature of the nanoparticle from hydrophobic to hydro-
philic, thereby increasing the blood retention time.
Importantly the synthesised hydrogel nanoparticles
exhibit particle size less than 200 nm, thereby account-
ing for their accentuated efficacy to release the antineo-
plastic drugs at physiological conditions. Overall this
study illustrates that through fine-tuning the particle
size and appropriate surface modification, we might be
capable to develop better transport system efficient
enough to deliver drugs for prolonged period with less
frequent dosing (sustained release) along with greater
precision and penetration in tissues difficult to access.
Acknowledgments
PT and RRM acknowledge CSIR/UGC for research fellowships.
ADK is grateful to UGC (F.4-(55)/2014(BSR)/FRP), New Delhi,
and TEQIP 61, for financial support.
Disclosure statement
The author declares no conflict of interest.

12
P. TIWARI ET AL.
Funding
[16] Du X, Zhou J, Shi J, et al. Supramolecular hydrogelators
and hydrogels: from soft matter to molecular
biomaterials. Chem Rev. 2015;115:13165–13307.
[17] Siegemund G, Schwertfeger W, Feiring A, et al. Organicin
Ullmann’s encyclopedia of industrial chemistry Wiley-
VCH. Weinheim: Wiley Intersciences Germany; 2005.
[18] Aigueperse J, Mollard P, Devilliers D, et al. Inorganic in
Ullmann (ed.), encyclopedia of industrial chemistry Wiley-
VCH. Weinheim: Wiley Intersciences Germany; 2005.
[19] Bondi A. Van der Waals volumes and radii. J Phys Chem.
1964;68:441–451.
[20] Stabel A, Dasaradhi L, Hagan DO, et al. Scanning tunnel-
ing microscopy imaging of single fluorine atom substi-
tution in stearic acid. Langmuir. 1995;11:1427–1430.
[21] Booth H, Grindlev TB, Khedhair KA. The anomeric and
exo-anomeric effects in 2-methoxytetrahydropyran.
Chem Comm. 1982;8:1047–1048.
[22] Zhou P, Zou J, Tian F, et al. Fluorine bonding — how
does it work in protein−ligand interactions? J Chem Inf
Model. 2009;49:2344–2355.
[23] Bustamante E, Pedersen PL. High aerobic glycolysis of rat
hepatoma cells in culture: role of mitochondrial
hexokinase. Proc Natl Acad Sci U S A. 1977;74:3735–3739.
[24] Greenwood NN, Earnshaw A. Chemistry of the elements.
Butterworth Heinemann; 1988. USA: Elsevier. ISBN 978-
0-7506-3365-9.
[25] Centers for Disease Control and Prevention.
Recommendations for using fluoride to prevent and con-
trol dental caries in the United States. MMWR
Recommendations Rep. 2001;50:1–42.
[26] Pizzo G, Piscopo MR, Pizzo I, et al. Designing with natural
materials. Clin Oral Invest. 2007;11:189–193. .
[27] Emsley J. Nature’s building blocks: an A-Z guide to the
elements. USA: Elsevier, Oxford University Press. ISBN:
9780199605637. 178. 2011.
[28] Swinson J. Fluorine - A vital element in the medicine
chest. Pharma Chem. 2005;4:26–27.
[29] Sharma A, Goswami S, Rajagopalan R, et al.
Supramolecular Heterogeneity in β-turn forming syn-
thetic peptides nucleated by isomers of fluorinated phe-
nylalanine and Aib as corner residues. Supramol Chem.
2015;27:669–678.
[30] Sharma A, Tiwari P, DuttKonar A. The dominant role of
sidechains in nucleation of double helical organization
in synthetic peptides. J Mol Struct. 2018;1161:44–54.
[31] Wu FY, Hsu SM, Cheng H, et al. The effect of fluorine on
supramolecular hydrogelation of 4-fluorobenzyl-capped
diphenylalanine. New J Chem. 2015;39:4240–4243.
[32] Wu C, Zheng Z, Guo Y, et al. Fluorine substitution
enhances the self-assembling ability of hydrogelators.
Nanoscale. 2017;9:11429–11433.
[33] Ryan DM, Anderson SB, Nilsson BL. The influence of
side-chain halogenation on the self-assembly and
hydrogelation of Fmoc-phenylalanine derivatives. Soft
Matter. 2010;6:3220–3231.
[34] Rizvi SAA, Saleh AM. Applications of nanoparticle sys-
tems in drug delivery technology. Saudi Pharm J.
2018;26:64–70. .
This work was supported by the TEQIP, RGPV Bhopal , India
[61]; University Grants Communication, New Delhi, India
[F.4-(55)/2014(BSR)/FRP].
References
[1] Sun L, Zheng C, Webster TJ. Self-assembled peptide
nanomaterials for biomedical applications: promises
and pitfalls. Int J Nanomedicine. 2017;12:73–86.
[2] Schnaider L, Brahmachari S, Schmidt NW, et al. Self-
assembling dipeptide antibacterial nanostructures with
membrane disrupting activity. Nat Commun. 2017;8:1365.
[3] Collier JH, Rudra JS, Gasiorowski JZ, et al. Multi-
component extracellular matrices based on peptide
self-assembly. Chem Soc Rev. 2010;39:3413–3424.
[4] Segarra-Maset MD, Nebot VJ, Miravet JF, et al. Control of
molecular gelation by chemical stimuli. Chem Soc Rev.
2013;42:7086–7098.
[5] Rutgeerts LAJ, Soultan AH, Subramani R, et al. Robust
scalable synthesis of a bis-urea derivative forming thix-
otropic and cytocompatible supramolecular hydrogels.
Chem Commun. 2019;55:7323–7326.
[6] Ulijn RV, Smith AM. Designing peptide based
nanomaterials. Chem Soc Rev. 2008;37:664–675.
[7] Nandi N, Gayen K, Ghosh S, et al. Amphiphilic
peptide-based
stimuli-responsive hydrogels with antibacterial activity.
Biomacromolecules. 2017;18:3621–3629.
supramolecular,
noncytotoxic,
[8] Basu K, Baral A, Basak S, et al. Peptide based hydrogels for
cancer drug release: modulation of stiffness, drug release
and proteolytic stability of hydrogels by incorporating
D-amino acid residue(s). Chem Comm. 2016;52:5045–5048.
[9] Gavel PK, Dev D, Parmar HS, et al. Investigations of
peptide-based biocompatible injectable shape-memory
hydrogels: differential biological effects on bacterial and
human blood cells. ACS Appl Mater Interfaces.
2018;10:10729–10740.
[10] Gavel PK, Permar HS, Tripathi V, et al. Investigations of
anti-inflammatory activity of a peptide-based hydrogel
using rat air pouch model. ACS Appl Mater Interfaces.
2019;10:2849–2859.
[11] Tiwari P, Basu A, Sahu S, et al. An auxin–tyrosine deriva-
tive based biocompatible supergelator: a template for
fabrication of nanoparticles for sustained release of
model drugs. New J Chem. 2018;42:4915–4922.
[12] Tiwari P, Rajagopalan R, Moin M, et al. Can
self-assembling smart amino acid conjugate hydrogel
nanoparticles (HNPs) function as vehicles for drug
delivery? New J Chem. 2017;41:308–315.
[13] Tiwari P, Verma R, Basu A, et al. Proteolysis-resistant
self-assembled-amino acid dipeptide-based biocompa-
tible hydrogels as drug delivery vehicle. Chem Sel.
2017;2:6623–6631.
[14] Fichman G, Gazit E. Self-assembly of short peptides to
form hydrogels: design of building blocks, physical
properties and technological applications. Acta
Biomaterialia. 2015;10:1671–1682. .
[15] Das AK, Collins R, Ulijin RV. Exploiting enzymatic
(reversed) hydrolysis in directed self-assembly of pep-
tide nanostructures. Small. 2008;4:279–287.
[35] Sharma Gangele A, Goswami S, Bar A, et al. Can side
chain interactions nucleate supramolecular heterogene-
ity in synthetic tripeptides? Cryst Growth Des.
2016;16:2130–2139.

SUPRAMOLECULAR CHEMISTRY
13
[36] DuttKonar A. The unique crystallographic signature of a β-
turn mimic nucleated by N- Methylated Phenylalanine
and Aib as corner residue: conformational and self-
assembly studies. CrystEngComm. 2013;15:10569–10578.
[37] DuttKonar A. mABA inserted supramolecular triple helix
formation in the solid state in synthetic tripeptides con-
taining β-cyano alanine and Aib as corner residues. Cryst
Eng Comm. 2012;14:6689–6694.
[43] Mahler A, Reches M, Cohen S, et al. Rigid, self-assembled
hydrogel composed of a modified aromatic dipeptide.
Adv Mater. 2006;18:1365–1370.
[44] Mutter M, Maser F, Altmann KH, et al. Sequence-depen-
dence of secondary structure formation: conformational
studies of host–guest peptides in α-helix and β-structure
supporting media. Biopolymers. 1985;24:1057–1074.
[45] Moretto V, Crisma M, Bonora GM, et al. Comparison of
the effect of five guest residues on the beta-sheet con-
[38] DuttKonar A. Can a single pyridine dicarboxylic acid
ample enough to nucleate supramolecular double
formation
of
host
(L-val)n
oligopeptides.
helices
in
enantiomeric
pseudo
peptides?
Macromolecules. 1989;22:2939–2944.
CrystEngComm. 2013;15:2466–2473.
[46] Han J, Zhao D, Li D, et al. Polymer-based nanomaterials
and applications for vaccines and drugs. Polymers.
2018;10:31–45.
[47] Singh R, Lillard JW. Nanoparticle-based targeted drug
delivery. Exp Mol Pathol. 2009;86:215–223.
[48] Ischakov R, Abramovich LA, Buzhansky L, et al. Peptide-
based hydrogel nanoparticles as effective drug delivery
agents. Bioorg Med Chem Lett. 2013;21:3517–3522.
[49] Feng SS, Zeng WT, Lim YT, et al. Vitamin E TPGS-emulsified
poly(lactic-co-glycolic acid) nanoparticles for cardiovascu-
lar restenosis treatment. Nanomedicine. 2007;2:333–344.
[50] Galvao TLP, Rocha IM, Ribeiro de Silva MDC, et al. Is uracil
aromatic? The enthalpies of hydrogenation in the gaseous
and crystalline phases and in aqueous solution, as tools to
obtain an answer. J Phy Chem A. 2013;117:5826–5836.
[39] Gaussian 09, Revision A. 1. Frisch MJ, Trucks GW,
Schlegel HB. et al. Gaussian, Inc.: Wallingford CT; 2009
[40] Mehra R, Tiwari P, Basu A, et al. In search of bioinspired
hydrogels from amphiphilic peptides for sustained
release of anticancer drugs. New J Chem. 2019;43:
11666.
[41] Mehra R, Basu A, Christmann R, et al. Mechanoresponsive,
proteolytically stable and biocompatible supergelators
from ultra short enantiomeric peptides with sustained
drug release propensity. New J Chem. 2020;44:6346–6354.
[42] Tiwari P, Basu A, Vij A, et al. Rationally designed bioin-
spired - Amino valeric acid based hydrogel: one shot
solution for drug delivery and effluent management.
ChemistrySelect. 2019;4:6896–6905.