An auxin-tyrosine derivative based biocompatible supergelator: A template for fabrication of nanoparticles for sustained release of model drugs

Article abstract of DOI:10.1039/c7nj04390b

Bioinspired self-assembling peptides serve as powerful building blocks in the manufacturing of nanomaterials with tailored features. Inspired by the supergelating ability of naphthyl-Phe-OH (hydrogelator I), we synthesized naphthyl-Tyr-OH (hydrogelator II) and naphthyl-Trp-OH (hydrogelator III) with the objective of exploring the propensities of the phenolic OH of Tyr and the NH of the indole for controlling the gelation process. However, our experimental investigation reveals that hydrogelator II, containing Tyr as the aromatic core, shows an excellent gelation ability. But the Trp analogue fails to do so under similar conditions. To validate our results we performed MD simulation in an aqueous environment which significantly justifies that hydrogelator II exhibits a better hydrogelation ability than hydrogelators I and III. The characterisation of hydrogelator II was then performed in detail using various analytical and microscopic techniques and its biocompatibility was tested using an MTT assay. To examine the potentiality of hydrogelator II in drug delivery we developed hydrogel nanoparticles (HNPs) using the concept of self-assembly entirely governed by an ecofriendly approach i.e. weak interactions (like H-bonding, π-π and hydrophobic interactions). Our hydrogel nanoparticles display good release kinetics of the model drugs 5-fluorouracil and curcumin from the hydrogel matrix depending on their chemical structure, molecular weight and hydrophobicity. Thus our research shows that the choice of the core residue has a profound impact on the self-assembly process and thus on the gelation properties. Moreover, nanoparticles generated from our novel biocompatible hydrogelator II hold promise for future drug delivery applications.

Full text of DOI:10.1039/c7nj04390b

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An auxin–tyrosine derivative based biocompatible
supergelator: a template for fabrication of
Cite this:DOI: 10.1039/c7nj04390b
nanoparticles for sustained release of model
drugs†
b
Priyanka Tiwari,a Anindya Basu,
Sonu Sahu,b Sadhna Gound,b
Ryann M. Christman,c Amit K. Tiwari,c Piyush Trivedib and Anita DuttKonar
*
ab
Bioinspired self-assembling peptides serve as powerful building blocks in the manufacturing of nano-
materials with tailored features. Inspired by the supergelating ability of naphthyl-Phe-OH (hydrogelator
I), we synthesized naphthyl-Tyr-OH (hydrogelator II) and naphthyl-Trp-OH (hydrogelator III) with the
objective of exploring the propensities of the phenolic OH of Tyr and the NH of the indole for
controlling the gelation process. However, our experimental investigation reveals that hydrogelator II,
containing Tyr as the aromatic core, shows an excellent gelation ability. But the Trp analogue fails to do
so under similar conditions. To validate our results we performed MD simulation in an aqueous
environment which significantly justifies that hydrogelator II exhibits a better hydrogelation ability than
hydrogelators I and III. The characterisation of hydrogelator II was then performed in detail using
various analytical and microscopic techniques and its biocompatibility was tested using an MTT assay.
To examine the potentiality of hydrogelator II in drug delivery we developed hydrogel nanoparticles
(HNPs) using the concept of self-assembly entirely governed by an ecofriendly approach i.e. weak
interactions (like H-bonding, p–p and hydrophobic interactions). Our hydrogel nanoparticles display
good release kinetics of the model drugs 5-fluorouracil and curcumin from the hydrogel matrix
depending on their chemical structure, molecular weight and hydrophobicity. Thus our research shows
that the choice of the core residue has a profound impact on the self-assembly process and thus on the
gelation properties. Moreover, nanoparticles generated from our novel biocompatible hydrogelator II
hold promise for future drug delivery applications.
Received 12th November 2017,
Accepted 20th February 2018
DOI: 10.1039/c7nj04390b
rsc.li/njc
likelihood of oligopeptides to form a hydrogel is difficult, as even
subtle changes in the molecular architecture may exert a profound
Introduction
Bioinspired self-assembling peptides serve as powerful build- impact on its molecular behavior. Although there are no definitive
ing blocks in the manufacturing of nanomaterials with tailored rules for the design of hydrogelators, the existing examples that
features.1–30 Because of their ease of synthesis, biocompatibility failed to form hydrogels or successfully gelate may provide insights
and tunable activity, this emerging class of biomolecules has into the design of oligopeptide-based hydrogelators. Four main
become very popular.1–30 To rationally design peptide based low forces, Coulomb repulsion, hydrogen bonding, hydrophobicity
molecular weight hydrogelators (LMOGs), a profound understand- and p–p interactions, co-operatively interact in guiding peptide
ing of their gelation mechanism is required. In recent years, many self-assembly and thus control the gelation process.31 To date,
efforts have been made towards investigating the mechanism that although significant examples have been documented, a majority
drives peptide self-assembly into 3-D matrices. Predicting the of them consist of longer peptides/peptide amphiphiles as syn-
thons for drug delivery.32–46 However, the exploration of efficient
a Dept. of Chemistry, Rajiv Gandhi Technological University, Bhopal, India.
hydrogels from short peptides/simple amino acid derivatives still
remains in its infancy.47–49
E-mail: anitaduttkonar@rgtu.net
b School of Pharmaceutical Sciences, Rajiv Gandhi Technological University, Bhopal,
Airport Bypass Road, Gandhinagar, Bhopal-462033, India
Henceforth, in this study our aim is to search for simple
derivatives with efficient drug delivery abilities. During the inves-
tigation process, we found that a series of peptide conjugates
possessing polyaromatic protecting groups like fluorenones and
phenanthrenes display an excellent hydrogelating ability.50
c Department of Pharmacology and Experimental Therapeutics, College of Pharmacy
and Pharmaceutical Sciences, University of Toledo, OH, USA
† Electronic supplementary information (ESI) available: Details of spectral data,
1H NMR, IR, and mass. See DOI: 10.1039/c7nj04390b
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slowly by the application of a simple heating–cooling cycle.
But hydrogelator II produced an optically clear gel at room
temperature with a MGC of o0.35 mg mlꢀ1 almost instanta-
neously (Fig. 1). But hydrogelator III fails to produce a gel,
instead forms a translucent viscous aggregate under similar
conditions. The gel thus obtained from hydrogelator II is stable
over a range of pHs, from pH 3.6 to 7 (water), at room
temperature and maintains its state for several months.
Generally, gelators with MGCs below 0.1% (w/v) fall into the
category of supergelators.61 Therefore our hydrogelator II can
also be considered as a supergelator. The gelation characterisa-
tion of hydrogelator II has been dealt with in detail in the
subsequent sections.
Fig. 1 Chemical structures of hydrogels II (left) and III (right).
But their major drawback is their carcinogenic nature, thereby
restricting their applications in drug delivery.51,52 Naphthalene is a
clinically approved fragment and is present in several drug mole-
cules like propranolol, naphazoline, nafronyl, etc.53–55 So we intend
to use this biocompatible and biodegradable fragment naphthyl as
our N-terminal protecting group. To ease the burden, few research
groups have used this moiety for protection and established their
hydrogelation behaviour.56–59
Computational studies for understanding structures of the
hydrogelators
Besides, literature documentation has revealed that peptide
based hydrogel nanoparticles (NPs) have gained momentum in
recent years as a promising drug delivery system.47–49 This is
because of their two different characteristics: (a) hydrophilicity
and extremely high water content of hydrogels and (b) exclusively
small size of the nanoparticles, which allows them to cross the
blood–brain barrier easily and reach the target site. Moreover,
amino acid/peptide hydrogel nanoparticles (HNPs) can be modu-
lated rationally by controlling the hierarchical self-assembly pro-
cess which includes the non-involvement of any potentially
hazardous chemicals such as cross-linkers, which may affect their
biocompatibility. Also their synthesis procedure is very simple and
their in vivo degradation products are non-toxic due to the fact that
they are composed of simple ecofriendly amino acids.
To verify the self-assembling propensities of hydrogelators I–III
we simulated the geometry optimised structures within an
aqueous environment for 10 ps.62,63 For preliminary under-
standing of the inter-molecular interactions, only four mono-
meric units of the hydrogelators were considered. The lowest
energy conformers thus obtained are shown in Fig. 2. A recent
methodology developed by Ulijn and co-workers is based on
thermodynamic stabilization by a self-assembly process, which
results in amplification of self-assembling properties and thus
accelerates the gelation process.64
The sequences of our hydrogelators accommodate short aromatic
amino acids: phenylalanine (F), tyrosine (Y) and tryptophan (W) in
addition to the naphthyl moiety. So the plausible mechanism of
self-assembly in these simple systems is supramolecular b-sheet
Thus, inspired by the supergelating ability of a 1-naphthyl Phe60
derivative, our objectives are (1) to couple the amino acids Tyr and
Trp with 1-naphthyl acetic acid, with the idea that the phenolic OH
of Tyr and the NH of the indole may enter into additional
H-bonding and accentuate the gelation process; (2) to validate
our experimental results by MD simulation studies; (3) to char-
acterize the formed gels in detail; and (4) to generate hydrogel
nanoparticles from our hydrogelators and explore their implica-
tions in drug delivery (Fig. 1). Interestingly, our experimental
results reveal that hydrogelator II (containing Tyr as the aromatic
core) shows an excellent hydrogelation ability. But the Trp analo-
gue fails to do so under similar conditions. Our computational
study significantly justifies the experimental observation. The
gelation properties of hydrogelator II were thoroughly character-
ized and its biocompatibility was tested using an MTT assay. We
further probed the efficiency of this biomolecule as a tool for drug
delivery by developing hydrogel nanoparticles (HNPs) using the
concept of self-assembly utilizing weak interactions. Our hydrogel
nanoparticles display good release kinetics of the model drugs
5-fluorouracil and curcumin from the hydrogel matrix depending
on their chemical structure, molecular weight and hydrophobicity.
Results and discussion
Upon addition of 1-Naph-Tyr-OH (hydrogelator II) and 1-Naph-
Trp-OH (hydrogelator III) to water, both gelators dissolved very
Fig. 2 Energy optimised structures of hydrogelators I–III and their self-
assembly patterns considering 4 monomeric units.
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organization. We expected that a greater involvement of stabilizing
interactions amongst the monomers would lead to ordered struc-
tures resulting in negative enthalpies of formation. As is evident
from the computational studies, the proposed packing model of
hydrogelator I, containing simple aromatic amino acid side
chains, shows the stabilization of a complex b-sheet with the
involvement of backbone amides and free carboxylate in inter-
molecular hydrogen bonding (Fig. 2a). However, insertion of
phenolic OH at the para position of the aromatic side-chain
(Tyr) resulted in an ordered parallel arrangement. Here the
H-bonds between Tyr NH and naphthalene amide along with
the phenolic OH groups of Tyr confer extra stability to the
supramolecular motif (Fig. 2c). This is reflected by the near-ideal
peptide bond (dihedral-) angles as observed within the reported
conformation (Table S1, ESI†). However, a gradual increase of
aromatic steric bulk in the sidechains (from benzyl to indole)
results in a parallel b-sheet structure with the involvement of the
amide NH of Trp with the carboxylate of 1-naphthoic acid and the
NH of the indole. In fact our results indicate that increased
participation of the indole NH in H-bond formation may lead to
a strain in the planarity of the fused 5-membered ring, where the
corresponding dihedral angle can deviate up to 5 degrees or more
(Table S1, ESI†). These observations demonstrate that hydrogelator
II has the highest self-assembling propensity followed by hydro-
gelators I and III respectively. This is also evident from the
calculated heats of formation of the conformers reported for
Fig. 4 Morphologies of the cell line OV2008 after treatment with hydro-
gelators II.
that an increase in the concentration of the gelator (% w/v)
leads to an increase in Tgel due to enhanced H-bonding and
hydrophobic interactions, until a particular concentration is
reached. This threshold value signifies the arrival of the satura-
tion limit after which no change in the gel melting temperature
occurs (Tgel).65–67
To decipher the efficacy of hydrogelator II as a vehicle for
drug delivery its biocompatibility was tested using in vitro
cellular experiments.68 The MTT assay illustrates that hydro-
gelators II tested up to 100 mM did not produce any significant
cytotoxicity in all four cell lines HEK293/pcDNA, HCT-116,
OV2008 and MDAMB-231 (Fig. 3, right). This investigation
was further supported by morphological analysis showcasing
no significant cytotoxicity of the hydrogelators (Fig. 4). The
higher the IC50 values the lower the cytotoxicity and thus the
higher the biocompatibility. Therefore, hydrogelators II up to
100 mM were determined to be safe in studies for preclinical
development.
hydrogelators I–III: ꢀ945.682, ꢀ1819.885 and ꢀ281.684 kJ molꢀ1
,
respectively. It can therefore be expected that hydrogelator II,
containing Tyr, exhibits a better ability to rigidify water in compar-
ison to hydrogelator I. This premise is in fact in agreement with
our experimental observation where the MGC for the Tyr analogue
was found to be 0.35 mg mlꢀ1 compared to 0.5 mg mlꢀ1 for the
Phe analogue. Furthermore, in accordance with the much
higher heat of formation for hydrogelator III we did not observe
any gelation properties from the Trp derivative. Thus our
simulation studies show that the choice of the core residue
has a profound impact on the self-assembly process and thus
the gelation properties.
Our next attempt was to study the secondary structural
features of hydrogelator II. So we turned to Fourier Transform
InfraRed spectroscopy (FT-IR) using the xerogel.
An IR spectrum first shows peaks around 1657 cmꢀ1 and
3300 cmꢀ1 corresponding to the H-bonded stretching frequencies
of the amide carbonyl group and NH respectively. Besides the
presence of other peaks around 1732 cmꢀ1 (acid carboxylate),
1614 cmꢀ1 (amide I) and 1532 (amide II) affirms the presence of a
b-sheet conformation in hydrogelator II (Fig. S1, ESI†).69,70
We then performed morphological analysis using field
emission scanning electron microscopy (FE-SEM) using the xero-
gels obtained from the corresponding hydrogels. Our images
(Fig. 5) show the formation of flat nano-fibrillar assemblies with
approximately 200 nm width and several micrometres length.
These fibres entangled with each other on large length scales to
Gel characterisation of hydrogelator II
To evaluate the thermal stability of hydrogelator II, gel-to-sol
transition temperatures (Tgel) were determined (Fig. 3, left)
using the ‘‘tube inversion’’ method. From the plot it is evident
Fig. 3 The change in the Tgel profile of hydrogelator II (left) and the cell
survival study (by MTT assay) of four different cell lines after treatment with
hydrogelators II.
Fig. 5 FESEM images of the xerogel from hydrogelator II showing a flat
ribbon like morphology of the fibres: (a) lower magnification; and (b)
higher magnification.
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for drug delivery systems owing to their environmentally benign
nature.47–49 Our hydrogelator II is biocompatible. So to explore its
candidature, we synthesized hydrogel nanoparticles using a mod-
ified inverse emulsion process (water-in-oil) as described in
Fig. 7a.47–49 In this experiment an aqueous solution of hydroge-
lator II was gradually added into a solution of vitamin E-TPGS
dissolved in light paraffin oil to form a heterogeneous mixture.
This mixture was then homogenized at 30 000 rpm for ten
minutes. After this process, the mixture was allowed to self-
assemble at 4 1C for 2 h to permit the attachment of the
surfactant along the surface of the hydrophilic core (Fig. 7b).
Finally, the HNPs were purified using centrifugation. We were
able to control the size of the nanoparticles by standardizing
the parameters of the emulsion process. Our optimized for-
mulation results reflect that hydrogelator II exhibits a bimodal
distribution of average particle sizes of 117 ꢁ 21.6 nm and
35.1 ꢁ 5.2 nm (Fig. 7c).47–49 This bimodal particle size distribu-
tion may have resulted due to the equilibrium between
two opposite processes, droplet fragmentation and droplet
re-coalescence that may have resulted due to the various experi-
mental conditions used in this process, such as surfactant type
and the speed of the homogenization.74 To exemplify our results
we performed morphological analysis using TEM-studies. Our
TEM images display the presence of two types of well discrete
spherical structured nanoparticles, which is in line with the
particle size measurement obtained from a zetasizer (Fig. 8a).
Moreover, the high ꢀve value of the zeta potential (ꢀ30.8 mV)
imparts stability to the formulation.
Fig. 6 Rheological study of hydrogel II showing variation in G0/G00 with
respect to angular frequency (in the left) and complex viscosity (in the right).
form a three dimensional nano-fibrillar network structure, ample
enough to entrap water molecules to form a gel.
To determine the mechanical strength and stability of
hydrogelator II, rheological studies were carried out. In this
experiment the storage modulus G0 (elastic response) and loss
modulus G00(viscous response) were measured against angular
frequency.71–73 As is evident from Fig. 6 (left), throughout the
viscoelastic region, the storage modulusꢀ(G1 0) is higher than the
loss modulus (G00) in the 1 to 100 rad s (angular frequency)
and 10–1000 eta (complex viscosity) ranges, showing no cross-
over point for both cases. This behavior demonstrates a soft gel
phase formation. Moreover, this gel matrix bears good toler-
ance towards external forces. Interestingly, the ratio between G0
and G00 of hydrogelator II is approximately one order of magnitude
higher, denoting significant mechanical stability of the hydrogels.
These data prompted us to explore the efficiency of our hydrogel as
a drug delivery carrier.
This negative charge may be due to the presence of carboxylate
in the derivative and the surfactant coating around the hydro-
phobic core.75,76 Our investigation further shows that the particles
have similar diameters and morphologies even after six months,
thus emphasizing the stability of the formulation (Fig. 8b).
Preparation and characterization of HNPs
It is already known that peptide based hydrogel nanoparticles
(HNPs) have attracted attention recently as promising candidates
Fig. 7 (a) HNP preparation method, (b) cartoon diagram of the details of a nanoparticle, (c) dimensions of a nanoparticle and (d) structures of the
components involved in nanoparticle synthesis.
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Fig. 8 TEM images of the hydrogel nanoparticles (HNPs) from hydro-
gelator II obtained from the optimized formulation (a) instantaneously (b)
after six months.
Fig. 10 Geometry optimized structures of 5-FU/curcumin–hydrogelator
II complexes. All the structures were optimized using the AMBER 99
forcefield. Index of the atom colours – carbon: cyan; hydrogen: white;
oxygen: red; nitrogen: blue. The 5FU is coloured green and curcumin
yellow for easy differentiation between the hydrogelator and drugs.
Drug encapsulation and in vitro release studies
Next our attempt was to study the release kinetics of the model
drugs 5-fluorouracil and curcumin from the hydrogel matrix using
a dialysis membrane as these drugs are known to exhibit chemo-
therapeutic properties.47–49,77,78 Moreover, they widely differ in
their molecular weight and hydrophobicity. Henceforth, to explore
the effects of these characteristics on the release kinetics we chose
these two drugs for our studies. The concentrations of the released
drugs were measured in solution over time, and quantified using
UV spectroscopy in buffer pH 7.45 for 5FU and simulated intestinal
medium (SIM) for curcumin at room temperature under gentle
stirring conditions.47–49,76,77 It has been noticed that the release
kinetics of 5FU loaded HNPs differ considerably from those of
curcumin, which may be because of the difference in their
chemical structures, molecular weights and hydrophobicities.
The release of 30% of the drug from the HNPs was approxi-
mately within 6 h for 5-FU and 10 h for curcumin, respectively,
after which the kinetics reached a saturation limit (Fig. 9). We
propose that hydrophobic and p–p interactions play a vital role
in determining drug release pathways.
Closer inspection of the structures of the drugs shows that
5FU bears groups for H-bonding participation but lacks aromatic
units (Fig. 7d). In contrast, curcumin being biphenolic in nature
possesses several aromatic moieties apart from H-bond donors
and acceptors (Fig. 7d). Our hydrogelator II prefers a strand like
conformation (Fig. 2c).
In the case of 5-FU (shown in green, Fig. 10), the carboxylates
of two strands of hydrogelator II first form a dimer between
themselves. This entity in turn gets tethered to two 5FU
carbonyls by intermolecular H-bonds, with the amide NH of Tyr.
Therefore effective drug binding interactions per molecule of
naphthyl-Tyr involve one drug molecule per molecule of carrier.
But curcumin being bifunctional in nature holds two hydrogelators
from both sides, utilizing p–p and hydrophobic interactions, giving
extra stability to the entire system. Therefore, here, per drug
molecule, there occur two carriers, which may account for the
slower release of curcumin from the hydrogel matrix (Fig. 10).
Conclusions
In summary, in this report we describe the supergelating ability of
hydrogelator II (containing Tyr as the aromatic core) that rigidifies
water at a very low concentration (0.35 mg per ml). But the Trp
analogue fails to do so under similar conditions. Our MD simula-
tion studies justify that hydrogelator II exhibits a better hydrogela-
tion ability than the others. Furthermore, it also explains why the
Trp analogue fails to display a successful gelation ability. Then we
characterized in detail hydrogelator II using various techniques. We
further explored the efficacy of this biomolecule as a tool for drug
delivery by developing hydrogel nanoparticles (HNPs) using the
concept of self-assembly. To date, literature documentation has
revealed that a large number of longer peptides/peptide amphi-
philes have been used as synthons for drug delivery systems.
However, to the best of our knowledge, our study on hydrogelator
II represents one of the very few reports of HNP formation emanat-
ing from the self-assembly process entirely governed by an eco-
friendly approach i.e. weak interactions. Our hydrogel nanoparticles
display good release kinetics of the model drugs 5-fluorouracil and
curcumin from the hydrogel matrix depending on their chemical
structure, molecular weight and hydrophobicity. The cause of the
slower release of curcumin from the hydrogel matrix has been
evidenced by our computational analysis. Thus our research shows
that the choice of the core residue has a profound impact on the
self-assembly process and thus the gelation properties. Moreover,
our novel biocompatible nanoparticles generated from hydrogelator
II hold promise for drug delivery applications.
Experimental
Materials and methods
Naphthyl 1-acetic acid, 5-fluoro-uracil (5-FU), curcumin and all
other chemicals were purchased from Spectrochem. The surfactant
Fig. 9 Release profiles of the model drug from the hydrogel matrix.
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vitamin E-TPGS was purchased from Sigma Aldrich Chemical by extraction with ethyl acetate (2 ꢂ 50 ml). The extract was
Company Pvt. Ltd. All solvents used in the synthesis were purified, pooled, dried over anhydrous sodium sulfate and evaporated
dried, or distilled, as required. 1H NMR spectra were recorded in vacuo to obtain a white solid.
using a Bruker UltraShield (400 MHz) spectrometer. Mass spectra
Yield: 972 mg, 2.78 mmol (90%); m.p. 132–135 1C; FTIR
were recorded in ESI-MS mode on a MicroTOF-Q-II instrument (KBr pellet, cmꢀ1): 3287, 1732, 1657, 1648, 1532, 1513; LR-MS:
manufactured by Bruker Daltonics; IR spectra were recorded using
a Shimadzu Prestige 21 FT-IR spectrometer.
C
21H19NO4 [M ꢀ H]+ = 348, Mcalcd C21H19NO4 [M]+ = 349;
1H NMR (d6-DMSO, ppm): 9.23 (1H, s, COOH of Tyr), 8.40
Synthesis of an auxin–amino acid conjugate. Amino acid (1H, d, NH of Tyr, J = 8.4 Hz), 7.22–7.88 (7H, m, aromatic Hs of
derivatives were synthesized using a conventional solution phase the naphthyl ring), 6.96 (2H, d, aromatic Hs of Tyr, J = 8.4 Hz),
methodology, with racemization free techniques employing 6.60 (2H, d, aromatic Hs of Tyr, J = 8.4 Hz), 4.32–4.35 (1H, m,
dicyclohexylcarbodiimide (DCC)/(1-hydroxybenzotriazole HOBT) CaH of Tyr), 2.8–2.9 (2H, m, CbHs of Tyr), 2.70–2.76 (2H, m,
as a coupling agent.79 Methyl ester hydrochlorides of tyrosine/ methylene Hs of naphthyl Grp).
tryptophan were prepared via the thionyl chloride–methanol
procedure.15 All the intermediates obtained were checked for
Hydrogelator-III. Hydrogelator-III was synthesized following
purity by thin layer chromatography (TLC) on silica gel. The final a similar procedure to that described for hydrogelator-II.
derivatives were purified by column chromatography using silica
gel (100–200 meshes) as the stationary phase and an ethyl acetate
Yield: 963 mg, 2.58 mmol (86%); m.p. 137–139 1C; LR-MS:
23H20N2O3 [M + H]+ = 373, Mcalcd C23H20N2O3 [M]+ = 372; FTIR
C
and petroleum ether mixture as the eluent. The reported deriva- (KBr pellet, cmꢀ1): 3452, 1733, 1719, 1645, 1636, 1530, 1511;
tives were fully characterised by NMR, IR spectroscopy and mass 1H NMR (d6-DMSO, ppm): 10.83 (1H, s, COOH of Trp), 8.46
spectrometry (Scheme 1).
(1H, d, NH of the indole ring, J = 8 Hz), 7.90–6.92 (13H, m, NH
of Trp and aromatic Hs of naphthyl and Trp), 4.51–4.43 (1H, m,
CaH of Trp), 3.05–2.98 (4H, m, methylene Hs of naphthyl Grp &
CbHs of Trp).
Naph-Tyr-OMe (1). Tyrosine methyl ester obtained from its
hydrochloride (2.12 g, 9.16 mmol) was added to an ice-cooled
solution of 1-naphthyl acetic acid (0.682 g, 3.66 mmol) in 4 ml
of DMF. Then DCC (1.13 g, 5.49 mmol) was added to the cooled
mixture, which was stirred at 1000 rpm approximately for
12 h at room temperature. The progress of the reaction was
monitored by TLC. The residue was then taken into ethyl
acetate and the DCU (N,N-dicyclohexylurea) was filtered off.
The organic layer was washed with 2 M HCl (3 ꢂ 100 ml), 1 M
sodium carbonate (3 ꢂ 100 ml) and brine (2 ꢂ 100 ml), dried
over anhydrous sodium sulfate and evaporated in a vacuum to
obtain a white solid. The crude peptide was used without
further purification.
MTT assay. The cytotoxicity of hydrogelator-II was deter-
mined through MTT (methyl thiazolyl tetrazolium) colorimetric
assay, which measures cell metabolic activity through the
conversion of tetrazolium dye to insoluble purple formazan
crystals as per the method described. The dipeptides were
dissolved in DMSO stock solutions followed by making the
dilutions (with DMSO o0.1%) directly using cell culture media.
No precipitation or aggregates were observed even on storage
up to 72 hours at 37 1C. 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. After 72 h of
incubation, MTT dye (4 mg mlꢀ1) was added to all wells and
incubated at 37 1C for an additional 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 minutes.
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 analyzed and plotted using GraphPad
Prismv7.02. Student’s t-test was used to analyze all the data.
Field emission scanning electron microscopy study (FESEM).
Morphology of xerogels obtained from hydrogelator II were inves-
tigated using FESEM microscope (JEOL JSM - 6700F) and were gold
coated.
Yield: 1.33 g, 3.11 mmol (85%); LR-MS: C22H21NO4 [M]+
=
364, Mcalcd C22H21NO4 [M]+ = 363.
Hydrogelator II. 1 (1.13 g, 3.11 mmol) was dissolved in 30 ml
of MeOH and 1.19 ml of 2 N NaOH was added dropwise to it.
The progress of saponification was monitored by thin-layer
chromatography (TLC). After 10 h, methanol was removed
under vacuum and the residue was taken in 50 ml of water
and washed with diethyl ether (2 ꢂ 50 ml). The pH of the
aqueous layer was then adjusted to 2–3 using 1 N HCl, followed
Rheology. Rheological measurements were carried out on a
Rheoplus MCR302 (Anton Paar) rotational rheometer with a
parallel plate geometry and obtained data were processed with
start rheometer software. For oscillatory shear measurements, a
parallel top plate with a 25 mm diameter and 1.0 mm gap
distance were used. Gels (6 mg mlꢀ1) for rheological experi-
ments were prepared on the bottom plate of the rheometer.
Nanoparticle characterization. The nanoparticle size dia-
meter and surface charge were measured using a Malvern
Scheme 1 Synthetic strategy for the preparation of hydrogelators.
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Zetasizer, with a 4 mW 633 He–Ne laser (DTS version 4.10, Acknowledgements
Malvern, U.K.) with appropriate viscosity and refractive index
settings. The temperature was maintained at 25 1C during the
measurement.
ADK wishes to thank UGC (F.4-(55)/2014(BSR)/FRP), New Delhi,
DST (SR/FT/CS-118/2013), New Delhi, and MPCST (A/RD/RP-2/
2016-17/58), Govt. of Madhya Pradesh, for financial support. AB
is grateful to UGC (F.4-(183)/2014(BSR)/FRP), New Delhi, for
financial assistance. All the authors are very much grateful to
Dr Sanjit Konar, IISER Bhopal, for his valuable advice. We
acknowledge the TEM facility at UGC DAE Consortium for
Scientific Research, Indore.
Transmission electron microscopy (TEM). A total of 500 ml
of HNPs was prepared and suspended in PBS and placed on a
400-mesh copper grid. After 2 min, the excess of fluid was
removed. Negative staining was obtained by covering the grid
with 10 ml of 2% uranyl acetate in water. After 2 min, excess
uranyl acetate solution was removed. Samples were viewed
using a FEI-TECNAI G2 (Netherlands) TEM operating at
200 kV accelerating voltage. Images were acquired digitally
using a Gatan CCD camera.
Notes and references
In vitro release of drugs from HNPs. The in vitro release
profiles of the model drugs 5FU and curcumin from the drug
loaded HNPs were obtained using a dialysis membrane pre-
viously soaked for 24 h in a dissolution medium and stretched
around at one end of the tube. The drug loaded formulations
were prepared using pretreated membranes which were immersed
into 30 ml of phosphate buffer solution at pH 7.4 for 5-FU and SIM
for curcumin at room temperature and magnetically stirred at
50 rpm. At selected time intervals aliquots were withdrawn from
the release medium and replaced with the same amount of
phosphate buffer (1 ml). The samples were analyzed thrice using
a UV-spectrophotometer at 267 nm for 5FU and 432 nm for
curcumin. The percentage of cumulative drug release was plotted
against time to get the release curves.
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Conflicts of interest
There are no conflicts of interest to declare.
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