Can self-assembled hydrogels composed of aromatic amino acid derivatives function as drug delivery carriers?

Article abstract of DOI:10.1039/c6nj02125e

Low molecular weight hydrogelators (LMOHGs) have attracted recent attention due to their diversified applications. In an attempt to artificially imitate their importance in the design of drug delivery carriers, we have synthesized two simple N-terminally protected aromatic amino-acid derivatives that form efficient stable hydrogels at room temperature. The gelation properties of the hydrogels have been thoroughly investigated using various techniques and their strength has been determined by rheological studies. In order to explore the efficacy of the hydrogels as tools for drug delivery, we have developed hydrogel nanoparticles (HNPs) using a surfactant and high-speed homogenization approach. Interestingly, our hydrogel nanoparticles display good entrapment efficiency and release kinetics of the model drug 5-fluoro uracil from the hydrogel matrix. Our experimental results reveal that hydrogel II displays slightly higher efficiency as a drug delivery carrier, which may be due to the presence of an aromatic ring in the backbone in comparison to hydrogel I. This increased strength may be attributed to the increase in π-π interactions when the aromatic residue is present in the backbone. Therefore the nanoparticles generated from hydrogel II may have better hydrogen bonding abilities with drugs in comparison to hydrogel I; thus resulting in a slightly slower release of drug from the hydrogel matrix. This fact may shed some light on the candidature of our hydrogels as future carriers for drug delivery. However, further studies to evaluate the candidature of these novel types of aromatic amino acid hydrogel nanoparticles for nano-medical applications are under investigation.

Full text of DOI:10.1039/c6nj02125e

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: P. Tiwari, R.
Rajagopalan, M. Moin, R. Soni, P. Trivedi and A. Dutt Konar, New J. Chem., 2016, DOI:
10.1039/C6NJ02125E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Volume 40 Number
1 January 2016 Pages 1–846
NJC
Accepted Manuscripts are published online shortly after
New Journal of Chemistry
www.rsc.org/njc
A journal for new directions in chemistry
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

Please do not adjust margins
New Journal of Chemistry
Page 1 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
Can Self-Assembled Hydrogels Composed of Aromatic Amino Acid
Derivative function as drug delivery carrier?
Received 00th January 20xx,
Accepted 00th January 20xx
Priyanka Tiwari,a Ramanathan Rajagopalan,bMohammad Moin,bRohit Soni,bPiyush Trivedib*and
Anita DuttKonar,a,b
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Low molecular weight hydrogelators (LMOHGs) have attracted recent attention due to their diversified applications. In an
attempt to artificially imitate their importance in the design of drug delivery carrier, we have synthesized two simple N-
terminaly protected aromatic amino-acid derivatives that form efficient stable hydrogels at room temperature. The
gelation property of the hydrogels have been thoroughly investigated by various techniques and its strength has been
determined by rheological studies. Inorder to explore the efficacy of the hydrogels as tools for drug delivery, we have
developed hydrogel nanoparticles (HNPs)using a surfactantand high speed homogenization approach. Interestingly,
ourhydrogel nanoparticles display good entrapment efficiency and release kinetics of the model drug 5-Fluoro Uracil from
the hydrogel matrix . Our experimental results reveal that hydrogel II displays slightly higher efficiency as a drug delivery
carrier may be due to the presence of aromatic ring in the backbone incomparison to hydrogel I. Thisincreased strength
may be attributed to the increase of π−π interaction when the aromatic residue is present in the backbone. Therefore the
nanoparticles generated from hydrogel II, may have better hydrogen bonding ability with drug in comparison to the
hydrogelI. Henceforth resulting in slightly slower release of drug from the hydrogel matrix.This fact may shed some light
about the candidature of our hydrogels as future carriers for drug delivery.However, further studies to evaluate the
candidature of these novel type of aromatic amino acid hydrogel nanoparticles for nano-medical applications is under
investigation..
behavior. They can form specific secondary, tertiary and
quaternary structures which provides unique opportunities for
the design of nanomaterials,that doesnot exist with traditional
Introduction
The principle of self-assembly involves the construction of
complex architectures from biological building blocks which
exhibits diversified applications in drug delivery, tissue
engineering, electronic devices etc.1-4The building blocks that
have been extensively known for generation of novel structures
are nucleic acids, phospholipids and large peptidyl building
blocks.5-8Like large polypeptides, short amino acid derivatives
and peptides too, can self-assemble into various nanostructures
as well as nanoscale ordered hydrogels.9-18
Low molecular weight hydrogelators(LMOHGs) composed of
amino acids and short peptides represent a promising approach
to drug delivery pathways.19This is because of the fact that
these molecules are of biological origin and possesses non-toxic
organic molecules.20
Peptide based hydrogel nanoparticles (HNPs) have gained
momentum in recent years as promising nanoparticulate drug
delivery system.21-24 This occurs due to a combination of two
different characterestics a) Hydrophilicity and extremely high
water content of hydrogels and b) nanoparticle which is
exclusively small in size. The amino acid/peptide hydrogel
nanoparticles (HNPs) can be modulated rationally by
controlling the hierarchical self-assembly process which
includes the non-involvement of any potentially hazardous
chemicals such as cross-linkers, that may affect its
biocompatibility.24-28Moreover, their synthesis procedure is
very simple and in vivo degradability is non-toxic due to the
fact that they are composed of simple ecofriendly amino acids.
Although, till date significant examples are documented in the
literature, majority of them either involves synthetic and natural
polymers or longer peptides/peptide amphiphiles as building
blocks for drug delivery systems.29-41. The exploration of
hydrogel nanoparticles from short peptides/simple aromatic
amino acid derivatives, where the concept of self-assembly has
been explicitly exploited remains in a rudimentary stage.42
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
As a part of the investigation, in this report our effort lies in for purity by thin layer chromatography (TLC) on silica gel.The
modulating the excellent hydrogelating ability of two final derivatives were purified by column chromatography
terminally protected aromatic amino acid derivatives, using silica gel (100-200 mesh) as the stationary phase and
N
-
Benzoyloxy
(Hydrogelator
benzoic acid (Fmoc-
carbonyl
) and Fluorenylmethoxy carbonyl meta amino reported derivatives
-ABA-OH) (HydrogelatorII) (Figure 1) IR spectroscopy and mass spectrometry.
that forms efficient, stable hydrogel in water, at room
phenylalanine
(Z-Phe-OH) ethyl acetate and petroleum ether mixture as the eluent. The
I
I
and II were fully characterised by NMR,
m
temperature with a minimum gelator concentration of 0.02 % Z-Phe-OH (Hydrogelator I): Phenylalanine (2.0gm, 12.12
and 0.05 % w/v, respectively. The gelation properties of the mmol) was dissolved in 20 ml of 20% sodium bicarbonate
hydrogel have been investigated by scanning electron solution and 4.0 gm of benzoyl chloride was added to it and
microscopic studies, FT-IR, and rheological studies. Inorder to shaken vigorously in a stoppered flask. The stopper was
explore the efficacy of the hydrogels as tools for drug delivery, removed from time to time since carbon dioxide is evolved.
we have developed hydrogel nanoparticles (HNPs) using a When the odour of benzoyl chloride completely disappeared, it
surfactant and high speed homogenization approach. Our was acidified with dilute hydrochloric acid and extracted with
hydrogel nanoparticles display good entrapment efficiency and ethylacetate as the organic phase. The organic layer was
release kinetics of the model drug 5-Fluoro Uracil from the evaporated to get a solid brownish derivative.
hydrogel matrix.This fact may shed some light about the Yield: 3g (83%); m.p. 343 - 344°C. FTIR (KBr pellet, cm-1):
candidature of our hydrogels as future carriers for drug 1538, 1643, 1696, 3329.1H-NMR (CDCl3, ppm): 2.8(1H,d,CβH
delivery.
of Phe), 3.08(1H,d, CβH of Phe), 4.3(2H,m,CβHs of
benzyloxycarbonyl),
7.4(10H,m,Aromatic
5.0
(1H,m,CαH
H’s of
of
Phe),
7-
and
Phe
O
Benzoyloxycarbonyl),7.6(1H,d,NH of Phe), 12.8(1H,br, COOH
of Phe).
O
OH
OH
O
N
H
O
N
H
O
O
Fmoc-mABA-OH (Hydrogelator II):mABA-OH (2 g, 14.58
Hydrogel IHydrogel II
Figure 1. Chemical structures of HydrogelI and II
mmol) was dissolved separately in basic sodium carbonate
solution (36.4 ml) and cooled in an ice-water bath. Then to it,
cooled solution of Fmoc-Cl (5.6 g) in dioxane (36.4 ml) was
added. The reaction mixture was allowed to come to room
temperature and stirred for 24 hrs. The pH was adjusted to
acidic condition with 1:3 HCl and extracted with ethylacetate as
the organic phase. The organic layer was evaporated to get a
solid brownish product of Fmoc-mABA-OH.
Experimental
Materials and methods:
Benzoyloxy carbonyl (Z) and Fluorenylmethoxy carbonyl
(Fmoc) chlorides, 5-Fluoro-uracil (5-FU) and all other
chemicals were purchased from Spectrochem chemicals. The
surfactant Vitamin-E-TPGS was purchased from Sigma Aldrich
Chemicals Company Pvt. Ltd. All solvents used in the synthesis
Yield: 4.19 g (80%); m.p. 220-225°C. FTIR (KBr pellet, cm-
1):1536, 1694, 3306. 1H NMR (CDCl3, ppm): 4.28
–
1
were purified, dried, or distilled, as required. H NMR spectra
4.30(1H,m,CH of Fluoren), 4.48(1H, d,
Fluoren), 7.30-7.41 (8H,m,Aromatic protons of Fluoren), 7.41
(2H,br, ABAHb and Hd),7.56-7.66(3H,m, ABAHb, Hd&Hc)
, 7.72-7.88 (8H,m,Aromatic protons of Fluoren), 8.13(1H,s,
ABA Ha), 9.89(NH of ABA), 12.91(COOH of ABA).
J = 6.4 Hz, CH2 of
were recorded by using Bruker Ultra shield (400MHz)
spectrometer. Mass spectra were recorded in ESI-MS mode on
MicroTOF-Q-II instrument manufactured by Bruker Daltonics;
IR spectra were recorded by using Shimadzu, Prestige 21 FT-IR
spectrometer.
m
m
m
m
m
Hydrogelator
I
Gel-melting temperature (Tm)
The melting temperature of resultant gel in water was
determined by the ‘inverse flow method’. Samples were
gelled in 10 mm diameter glass vials and attached inverted to a
thermometer near the bulb end. This assembly was immersed in
a stirred water bath, the temperature of which was raised at 5˚C
Hydrogelator II
-1
min using a hot plate. The temperature at which the solid
mass falls from top was recorded as Tm. This procedure was
repeated on three independent samples, and the average values
are reported.
Synthesis of Amino Acid Derivatives I and II
Field Emission scanning electron microscopic study (FESEM)
Morphology of xerogels obtained from amino acid derivatives
and IIwere investigated using FESEM microscope (JEOL JSM
- 6700F) and were gold coated.
I
The amino acid derivativesI and II were synthesized using
conventional solution phase methodology, with racemization
free techniques. All the intermediates obtained were checked
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 2
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 3 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
Synthesis of HNPs
of pH 7.4 at room temperature and magnetically stirred at 50
rpm. At selected time intervals aliquots were withdrawn from
release medium and replaced with same amount of phosphate
buffer (1 ml). The samples were analyzed thrice using UV-
spectrophotometer at 267 nm. The percentage of cumulative
drug release was plotted against time to get the release curves..
The amino acid derivative based hydrogel nanoparticles
(HNPs) were prepared using self-assembly and modified
inverse emulsion technique in which vitamin E-TPGS was
used as an emulsion stabilizer. In short, HNPs were formulated
by the dilution of stock solution (100 mg/mL) in pure water to
a final peptide concentration of 10 mg/mL. The resulting
solution was added drop-wise into 50 mL slightly warmed
mineral oil containing vitamin E-TPGS at concentration of
0.4% wt/v and homogenized using high speed homogenizer.
Next, nanoparticles were allowed to self-assemble for 2 h with
Rheology
Rheological measurements were carried out on a Rheoplus
MCR302 (Anton paar) rotational rheometer with parallel plate
geometry and obtained data were processed with start
rheometer software. For the oscillatory shear measurements,
parallel top plate with a 25 mm diameter and 1.0 mm gap
distance were used. Gels (6 mg/ml) for rheological experiments
were prepared on the bottom plate of the rheometer. The shear
modulus (storage modulus G` and loss modulus G``) were
measured against % strain from 0.001% to 1 %. Frequency
sweep experiment was performed from 0.11 to 100 rad/s at
constant strain of 1 %.
continuous stirring at 4ºC to allow the surfactant monolayer
accumulation on the surface of the nanoparticle. Upon the
completion of the self-assembly process, the resulting
suspension was mixed with
a non-polar solvent, and
centrifuged to obtain phase separation. Supernatant was
removed and HNPs were washed again to remove the
remaining residues of mineral oil and finally vacuum dried. The
obtained HNPs were used immediately, or stored at 4ºC for
later use.
Nanoparticle characterization
Results and discussion
Nanoparticle size diameter, polydispersity, and surface charge
were measured using Malvern Zetasizer, with 4mW 633 He-Ne
Laser, (DTS version 4.10, Malvern, U. K.) with appropriate
viscosity and refractive index settings. The temperature was
maintained at 25oC during the measurement.
Preparation and Characterization of the Hydrogels
From the literature documentation, it is evident that
π−π interactions play
presence of aromatic groups in the molecule is an obvious
choice. So our first design consists of Z-F-OH (Hydrogel
(Figure 1). This molecule consists of a single aromatic unit at
the -terminus and another aromatic unit in the sidechain. In
a vital role in gelation. Therefore
I)
N
Transmission electron microscopy (TEM)
addition the insertion of two methylene units in between the
two aromatic groups provides two rotational degrees of
freedom to the aromatic units which was expected to accelerate
the gelation process.43Inorder to decipher whether the position
and number of aromatic groups present in the conjugate plays
A total of 500 ꢀL of HNPs was prepared and suspended in PBS
and placed on a 400-mesh copper grid. After 2 min, the excess
of fluid were removed. Negative staining was obtained by
covering the grid with 10
ꢀL 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 Gatan CCD camera.
any role in gelation process, we have designed Fmoc-mABA-
OH (Hydrogel II), (Figure 1). In this conjugate the aromatic
group in the sidechain has been shifted to the backbone (Phe to
m
ABA-OH) and the Z-group (1 aromatic unit) in the
terminus has been replaced by Fmoc group (2 aromatic units).
In this study, the aromatic amino acid derivatives and II
undergoes hydrogelation, with minimum gelation
N-
Entrapment efficiency of the hydrogel HNPs
I
The entrapment efficiency of hydrogel nanoparticles were
determined by separation of nanoparticles from the aqueous
medium containing non-associated drug by centrifugation at
15000 rpm at 4oC for 40 min. The amount of free drug was
measured by UV spectrophotometer at 267 nm. The entrapment
efficiency of the nanoparticles were calculated as per the
equation given below:
a
concentration of 0.02% w/v and 0.05% w/v respectively
without any sonication at room temperature. At first the
hydrogelators were dissolved in a drop of DMSO under mild
heating condition and allowed to cool. Then to it required
amount of water was added to maintain the volume upto 1 mL.
It was allowed to stand at room temperature for 8-10 min to
obtain an opaque gel.The formation of the gel was confirmed
by inverted test tube method (Figure. S1).
The gels are stable over a range of pH, from pH 5.5 to 7 (water)
and also over a period of six months. Sol–gel transition
In vitro drug release of 5-fluorouracil from HNPs
temperatures (Tgel) for both the hydrogelators
were plotted against different concentrations
hydrogelators(Figure 2). These plots show that the Tgel values
of the aromatic amino acid derivatives increases with an
increase in concentration (% w/v) of the hydrogelator until the
plateau region is reached. This plateau region indicates that the
I
and II (% w/v)
The in vitro release profile of the model drug 5FU from the
drug loaded HNPs were performed using dialysis membrane
previously soaked for 24 hrs in the dissolution membrane and
stretched around at one end of the tube. The drug loaded
formulations were carried out using pretreated membrane
which were immersed into 30 mL of phosphate buffer solution
of
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
Rheology Characterisation
formation of the hydrogel network has reached the saturation
limit. No further addition of any molecule is capable of
Inorder to address the mechanical strength and stability of the
hydrogelators and II, rheological studies were performed
increasing the gel melting temperature (Tgel value).44
I
66
60
54
48
where the storage modulus G’(elastic response) and loss
modulus G’’(viscous response) were measured in frequency
sweep experiments. 47
60
54
Hydrogelator I
Hydrogelator II
48
G' of hydrogelator I
42
G' of hydrogelator I
42
G" of hydrogelator I
100
10
1
0.02 0.03 0.04 0.05 0.06
100
10
1
0.05
0.06
0.07
0.08
Hydrogelator concentration / % w/v
Hydrogelator concentration(%w/v)
G' of hydrogelator II
G" of hydrogelator I
G" of hydrogelator II
G' of hydrogelator II
G" of hydrogelator II
Figure 2. The change in Tgel profile of theHydrogelator
respect to the concentration
I (left) and II (right) with
To understand the mechanism governing the self-assembly
propensities of the aromatic amino acid derivatives and II
FTIR measurement was carried out (Figure S2). The FTIR
spectra of the xerogels obtained from the amino acid
derivatives show
major band at 3300–3340 cm_1,
characteristic feature of hydrogen bonded NH stretching. No
band was observed around 3400 cm-1 for both the derivatives,
which indicates the absence of any free NH group. Both the
I
,
1
10
100
0.01
0.1
1
-1)
s
Angular Frequency in
(
Strain (%)
a
a
Figure 4.Rheological study of the hydrogels I and II showing the frequency sweep study
(in the left)and Amplitude sweep study (in the right )
As is evident from Figure 4 (left), in the lower viscoelastic
region (LVR), the storage modulus G’ is higher than the loss
modulus (G’’) for both the amino acid derivatives. The
xerogels of the derivativesI and II further exhibited peaks in the
region 1696 , 1694 cm_1 (amide I) and 1538, 1536 cm-1 (amide
II) respectively, characteristic feature of C=O stretching and
NH bending frequencies respectively. These data therefore
observation suggests
a
soft ‘solid-like’ gel phase
formation.48Moreover a cross over point is noticed in the
experimental frequency regions, where the value of loss
modulus G’’ slightly exceeds the value of storage modulus G’
and transformed into solution state (Figure 4 right). This
particular point where the crossover occurs is called yield stress
suggests the presence of
β-sheet conformation for both the
conjugates in their corresponding dried gel state.45-46
In order to gain insight about the morphology of the hydrogels,
scanning electron microscopic (FESEM) investigation was carried
out. The FE-SEM images (Figure 3) of the xerogels of hydrogelator
I and II exhibits flat ribbon like morphology with width ranging
from 600–800 nm and 100–120 nm respectively. The fibers were
( σy). These gelators showed a G’(Storage modulus) value that
was sufficiently larger than G’’(Loss modulus); this difference
remained almost constant up to about 0.5% and 0.05 % strain
respectively for both the amino acid derivative
I and II
found
to
be
several
micrometers
long.
respectively (Figure 4 right). As the strain was increased
beyond this point, both the storage modulus (G’) and the loss
modulus (G’’) started to fall because intermolecular forces that
held the gel together started being overcome by the applied
strain and the fibrils were unable to withstand large
deformations. The frequency-sweep experiments further
showed that both the G’ and G’’ values were weakly dependent
on the frequency (Figure 4 left), which was indicative of an
entangled network-like system.
Interestingly,hydrogelatorIIexhibits dense mesh-like aggregates
(Figure 3d at higher magnification)formed due to overlapping of flat
ribbon like structures incomparison to hydrogelator I.This difference
of morphology may be attributed to the less number of π-surface as
well as fewer hydrogen bonding elements in hydrogelatorIwith
respect to hydrogelator II.
.
The principle guidelines of gelation involves a balance between
enthalpy and entropy, the nature of weak interactions, such as
hydrogen bonds and π−π forces involved, resulting in
thermodynamic minimum. In Hydrogelator I, two methylene units
are being inserted in between the aromatic moieties, incomparison
to hydrogelatorII (that contains one methylene unit). Insertion of
methylene units between aromatic groups are already known to
accelerate the gelation process.43 Although more π−π interactions
are observed in hydrogelator I, due to the presence of aromatic
residue in the backbone, we observe lower melting point of
hydrogelator II than hydrogelator I, at same concentration. This may
be attributed to the more entropic contribution from the two
methylene unitsof hydrogelator I, which might overrule the extra
stability gained due to π−π interaction,due to presence of aromatic
moiety in the backbone, resulting in thermodynamic minimum
Figure 3.FESEM images of the xerogelIand IIshowing flat ribbonlike morphology of
thefibris(a,c: lower magnification; b,d: higher magnification).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 4
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 5 of 10
Journal Name
View Article Online
DOI: 10.1039/C6NJ02125E
ARTICLE
conditions. Interestingly, the ratio between storage modulus and
loss modulus of both the aromatic amino acid derivative and
washings with a non-polar solvent.The particle dimensions
were then recorded using a zetasizer. For pharmaceutical
application, the physical dimension of the nanoparticles (like
I
II is approximately one order of magnitude higher which
indicates significant mechanical stability of the hydrogels. This
data prompted us to explore the candidature of our hydrogels in
drug delivery application. Similar observation where gels of
similar strength have been utilized for various applications has
been reported by other groups.49-53
particle
size,
polydispersity
index,
zetapotential)
influencesbiodistribution and in vivo efficacy.We were able to
control the size of the HNPs by standardizing the parametersof
the emulsion process, such as the stirring speed, stirring
time,concentration of gelators and concentration of the
surfactant.Our optimized formulation results reflect that the
Preparation and characterization of HNPs
hydrogelatorIand II exhibit an average particle size of 254 nm
and 482 nm respectivelys. Furthermore, we analyzed the HNPs
using transmission electron microscopic (TEM) analysis. The
TEM images obtained demonstrates the presence of well-
discrete spherical structure nanoparticles with various sizes
(Figure. 7 and 8). The Zeta potential indicates the degree of
repulsion between adjacent, similarly charged particles in
dispersion. For molecules and particles that are small enough, a
highZeta potential will confer stability, i.e. the solution or
dispersion will resist aggregation. When the potential is low,
attraction exceeds repulsion and the dispersion will break and
flocculate.The Zeta potential value of hydrogel nanoparticles
for both the hydrogelators were found to be -25.5 mV for
optimized formulation, which indicates significant stability of
the nanoparticles.The pKa of 5-FU is 8.1. Under the conditions
of the experiment, the pH of the medium remains neutral (since
water has been used for gelation) or slightly acidic (which may
occur due to ionization of the organic acids that has been used
as hydrogelators). Henceforth the drug is expected to remain
un-ionized under this condition as the pH of the medium is
much below the pKa value.The negative charge of the zeta
potential could be attributed to the presence of carboxyl groups
in the amino acid derivatives as well as the emulsion stabilizer,
vitamin E TPGS coating around the hydrophobic core.57
Literature documentation reveals that hydrogel nanoparticle
(HNP) provides a promising alternative approach towards drug
delivery applications.16-18,34 But most of the report demonstrates
the use of extremely complex techniques.
Our aromatic amino acid derivative I and IIbased hydrogels are
prepared using the concept of self-assembly involving weak
interactions. Henceforth are ecofriendly in nature. Therefore
we became interested to explore whether our hydrogelscould be
utilized for the synthesis of hydrogel nanoparticles. As
illustrated in Figure 5 modified inverse emulsion technique
(water-in-oil) was used for HNP formulation. In order to
synthesize the hydrogel nanoparticles, the peptide was first
dissolved in its monomeric state in organic solvent (DMSO),
followed by dilution in water. This solution is considered to be
the aqueous phase. This aqueous phase was drop-wise added
into the organic phase containing the surfactant vitamin E-
TPGS dissolved in light paraffin oil. Vitamin ETPGS was
chosen as a surfactant due to its enormous advantage.54-
55Moreover,the hydroxylic groups in vitamin E-TPGS may
provide sites for the attachment of targeting units.
The heterogeneous mixture was then subjected to
homogenization using a high speed homogeneizer operating
at25000 rpm for ten minutes. During this process the
hydrogelator
I and II mixture were dispersed well and allowed
to self-assemble into particle aggregates.56
Figure 5: Schematic representation of Hydrogel Nanoparticle Formulation (HNP) preparation by modified Inverse Emulsion Method.
The mixture was then allowed to stir at 4oC for 2 hrs to allow
the attachment of the surfactant along the surface of the
hydrophilic core.
The self-assembled aromatic conjugate occupies the
hydrophilic core of HNPs, and vitamin E-TPGS monolayer in
outer hydrophobic periphery as in micelle (Figure 6).Finally,
the HNPs were purified using centrifugationand a series of
Generally, stresses caused by lyophilization may cause fusion
of the HNPs.58 Henceforth before subjecting to
lyophilization,cryoprotectants are added to protect the fragile
structure of the nanoparticles.In our case we have used vitamin
E-TPGS, which not only serves as a surfactant but also as a
cryoprotectant.59After lyophilisation we determined the particle
size distribution after three months (Figure 9a and b). The
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 6 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
results show that the particles have similar diameters and release studies of a model drug 5-fluorouracil (5-Fu)from the
morphology even after three months, thus emphasizing the hydrogel matrix. Therefore, the drug loading efficiency and
stability of the formulation. These results further indicate release kinetics were determined by dialysis membrane in HBS
thatafter formulation, the particles could be readily turned into at 37oC under gentle stirring, to mimic physiological
dry form, and re-suspended prior to use.
conditions. The encapsulation of the model drug 5-flourouracil
(5-Fu) from the HNPs matrix showed an entrapment
efficiency of 77.57% and 84.52% for hydrogelatorsI and II
respectively. The concentration of the released drug was
measured in solution over time, and quantified using UV
spectroscopy. The resulting release profiles of 5-Fu from both
the derivatives are presented in Figure 10. It has been noticed
that the release profile of 5-Fu loaded HNPs slightly differs for
both the derivatives. The release of 50% of 5-Fu from the HNPs
Figure 6.Cartoon diagram of drug encapsulated hydrogel nanoparticle formed after
self-assembly.
was within 3 h/4 hr for hydrogelatorIand II respectively, and
after 7 h/9h the kinetics of release reached a plateau. This
difference in release kinetics between 5-Fu for both the
derivatives might be due to their dissimilar chemical structures.
We propose that aromatic interactions and hydrogen bond
formation between the amino acid derivativeand drug may play
a crucial role in its efficient encapsulation. The slightly higher
1000nm
500nm efficiency as a carrier of hydrogelatorII may be attributed to the
presence of aromatic ring in the backbone which may increase
the extent of hydrogen bonding and π−π interaction to a greator
Figure 7: TEM images of the hydrogel nanoparticles (HNPs) from
extent with the drug molecule (incomparison to hydrogel I),
thereby resulting in slightly slower release of drug from the
hydrogel matrix.
optimized formulation a) Hydrogelator
I
and b) Hydrogelator II
Hydrogelator I
Hydrogelator II
500±10nm
200±10nm
100
Hydrogelator I
60
400±10nm
50
0
450±10nm
30
0
Hydrogelator II
0
10
20
Figure 8:Particle size distributionof the hydrogel nanoparticles (HNPs)
Time in Hrs
obtained from optimized formulation
Figure 10: Release profiles of the model drug from the hydrogel matrix
Conclusions
In summary, we have demonstrated the formation of two simple
amino acid derivatives that display excellent hydrogelating
ability under mild conditions. The gelation property of the
hydrogel was investigated by various microscopic techniques
and its strength was determined by rheological measurements.
Interestingly,these amino acid conjugate based hydrogels have
been nicely tuned to prepare and stabilize hydrogel
nanoparticles (HNPs) utilizing inverse emulsion procedure.The
method proposed here is environmentally benign, as simple
amino acid conjugates have been employed. The approach
adopted is the concept of self-assembly utilizing weak
interactions, unlike other polymers that involves harsh
conditions and complex techniques for its fabrication.Till date
significant examples are documented in the literature where
500nm
200nm
Figure 9.TEM images of the stored hydrogel nanoparticles (HNPs) after
three months a) HydrogelatorIand b) Hydrogelator II
Drug Encapsulation and in vitrorelease studies
As a part of the investigation, thereafter our idea was to
explorethe efficacy of our nanoparticles for encapsulation and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 6
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 7 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
15. A. Mahler, M. Reches, M. Rechter, S. Cohen and E. Gazit,
Adv. Mat, 2006 , 18, 1365.
16. R. Orbach, LA-Abramovich, S. Zigerson, IM. Harpaz,
D.Seliktarand E. Gazit, Biomacromol.,2009 ,10, 2646.
17. A. M. Jonker, D. W. P. M. Lo¨wik and J. C. M. Van Hest,
Chem. Mater., 2012, 24, 759–773.
synthetic and natural polymers, longer peptides/peptide
amphiphiles have been used as building blocks for the drug
delivery systems. However, to the best of our knowledge,
thesederivatives represent one of the very few reports of HNP
formation resulting from the self-assembly of simple amino
acid derivatives, solely driven by an environmentally benign
approach i.e. weak interactions, unlike other polymers that
requires drastic conditions and complex techniques for its
fabrication. The obtained HNPs were characterized and
evaluated for their size, zeta-potential and post-formulation
stability.Our gelators display good entrapment efficiency and in
vitro release kinetics of a model drug 5-FU.The obtained results
clearly indicate that these amino acid derivative based HNPs
may be suitable as a potential drug delivery system. However,
further studies to evaluate the candidature of these novel type of
amino acid conjugate based HNPs for nano-medical
applications is under investigation .
18. M. Zelzer and R. V. Ulijn, Chem. Soc. Rev., 2010, 39, 3351–
3357.
19. M. Hamidi, A. Azadi and P. Raife, Adv. Drug Del Rev
60, 1638-1649.
., 2008,
20. Z. Yu, Q. Xu, C. Dong, S. S. Lee, L. Gao, Y. Li, M. D.
Ortenzio and J. Wu, Curr. Pharmaceutical. Des., 2015, 21
,
4342-4354.
21. T. R. Hoare and D. S. Kohane, Polymer, 2008,
22. A. Dasgupta, J. Hassan Mondal and D. Das, RSC. Adv., 2013,
, 9117-9149.
23. X. Gong, Nano Life., 2015,
5, 1993-2007.
3
5, 1542002-
24. D. N. Woolfson and M. G. Raydnov, Curr
2006, 10, 559-567.
. Opin. Chem. Biol.,
25. L. Zhang, J. Zhong, L. Huang, L. Wang, Y. Hong, and Y. Sha,
J. Phys. Chem., 2008, 112, 805-816.
26. M. Ma, L. Zhong and W. Li, Soft Matter, 2013, 9, 11325-
Acknowledgements
11333.
27. M. Suzuki and K. Hanabusa, Chem. Soc. Rev. 2010, 39, 455 –
466.
28. M. Suzuki, C. Setoguchi, H. Shirai and K. Hanabusa, Chem.
ADK wishes to thank UGC (F.4-(55)/2014(BSR)/FRP) and DST,
New Delhi, India (Project No. SR/FT/CS-118/2013), for financial
Eur. J. 2007, 13, 8193 – 8200
support. All the authors sincerely
acknowledges
29. Y. Osada and J. P. Gong, Adv. Mater. 1998, 10, 827 - 837.
30. S. Nayak and L. A. Lyon, Angew. Chem. Int. Ed., 2005, 44
7686- 7708.
31. J. M. Chan, L. Zhang, K. P. Yuet, G. Lio, J. W. Rhee, R.
Langer and O. C. Farokhzad, Biomaterials., 2009, 30, 1627-
1634.
Dr.SanjitKonar, IISER Bhopal, and Dr. C. Karthikeyan,RGPV for
valuable suggestions. ADK is very grateful to Prof. N. K. Jain
and Dr. AnindyaBasu, RGPV, Bhopal for very valuable inputs
during manuscript preparation. The authors would like to
thank Mr. DigambarWaiker and Mrs.Vandana Gupta (SOPS,
RGPV) and Mr Amit Kumar Mondal (IISER Bhopal) for various
help during manuscript preparation. We thank Mr. Manoj
Sharma and Mr. MaharsiGajjar, Antonn Parr New Delhi for
rheological measurements and the TEM facilities at AIIMS New
Delhi for recordingTEM images.
,
32. Y. Nagai, L. D. Unsworth, S. Koutsopoulos and S. Zhang, J.
Controlled Release, 2006, 115, 18-25.
33. R. V. Rughani and J. P. Schneider, MRS Bull., 2011, 33, 530–
535.
34. C. Yan, M. E. Mackay, K. Czymmek, R. P. Nagarkar, J.P.
Schneider and D. J. Pochan, Langmuir, 2012, 28, 6076–6087.
35. C. Yan, A. Altunbas, T. Yucel, R. P. Nagarkar, J. P. Schneider
and D. J. Pochan, Soft Matter, 2010, 6, 5143–5156.
36. S. Zarhitsky and H. Rapaport, J. Colloid Interface Sci., 2011,
360, 525–531.
Notes and references
1. S. Zhang, Nat. Biotechnol. ,2003, 21, 1171.
2. M. Reches andE. Gazit, Curr. Nanosci.,2006, 2, 105.
3. C. Mao, D. J. Solis, B. D. Reiss, S. T. Kottmann, R. Y. Sweeney,
A. Hayhurst, G. Georgiou, B. Iverson and A. M. Belcher, Science,
2004,303, 213.
37. L. Mao, H. Wang, M. Tan, L. Ou, D. Kong and Z. Yang,
Chem. Commun., 2012, 48, 395–397.
38. Y. Zhao, T. Tan, H. Yokoi, M. Tanaka and T. Kinoshita, J.
Polym. Sci., Part A: Polym. Chem., 2008, 46, 4927–4933.
39. C. Y. Yang, B. Song, Y. Ao, A. P. Nowak, R. B. Abelowitz,
R.A. Korsak, L. A. Havton, T. J. Deming and M. V.
Sofroniew, Biomaterials, 2009, 30, 2881–2898.
40. R. Ischakov, L. A. Abramovich, L. Buzhansky, T. Shekhter
and E. Gazit, Bioorganic & Med. Chem. Lett., 2013, 21, 3517-
3522.
41. A. Reddy, A. Sharma and A. Srivastava, Chem. Eur. J., 2012,
18, 7575-7581.
42. C. S. Love, A. R. Hirst, V. Chechik, D. K. Smith, I. Ashworth
and Brennan, Langmuir, 2004, 20, 6580 – 6585.
4. A. B. Sanghvi, K. P. Miller, A. M. Belcher and C. E. Schmidt,
Nat. Mater.,2005, 4, 496.
5. M. R. Ghadiri, J. R. Granja, R. A. Milligan, D. E. Mcree and N.
Khazanovich, Nature, 1993, 366, 324.
6. Y. V. Zastavker, N. Asherie, A. Lomakin, J. Pande, J. M.
Donovan,J. M. Schnur andG. B. Benedek, Proc. Natl. Acad.
Sci.USA ., 1999, 96,7883.
7. S. Vauthey, S. Santoso, H. Y. Gong, N. Watson andS. Zhang,
Proc. Natl.Acad. Sci. USA.,2002, 99, 5355.
8. M. Hamidi, A. Azadi and P. Raife, Adv. Drug Del Rev., 2008, 60,
1638-1649.
9. A. Dutt, M. G. B. Drew and A. Pramanik, Org. Biomol.Chem,
2005,2250 .
10.aP. Koley and A. Pramanik, Soft Matter, 2012, 8, 5364.
11.aP. Koley and A. Pramanik, Adv. Funct. Mater., 2011, 21, 4126.
10. A. Sharma Gangele, S. Goswami, R. Rajagopalanand A.
DuttKonar,Supramol. Chem., 2015, 27,10, 669.
11. A. Sharma Gangele, S. Goswami, A.Bar, P. Tiwari, S. Konar
and A. DuttKonar,Cryst Growth Des., 2016, 16, 2130.
12. A. DuttKonar, Cryst Engg and Commun, 2013 ,15,10569.
13. A. DuttKonar,Cryst Engg and Commun, 2012 ,14,6689.
14. A. DuttKonar,Cryst Engg and Commun, 2013 ,15, 2466.
43. M. Mutter, F. Maser, K. H. Altmann, C. Toniolo and G. M.
Bonora, Biopolymer, 1985, 24
44. V. Moretto, M. Crisma, G. M. Bonora, C. Toniolo, H. Balaram
and P. Balaram, Macromol., 1989, 2939-2944.
, 1057-1074.
22,
45. A. Reddy, A. Sharma, Q. Maqbool and A. Srivastava, RSC.
Adv., 2013, 3, 18900-18907.
46. A. K. Das, R. Collins and R. V. Ulijn, Small, 2008, 4, 279 –
287.
47. R. Avalos-Diaz, C. Long, E. Fontano, M. Balbirnie, R. Grothe,
D. Eisenberg and D. L. D. Caspar, Fiber Diffrac. Rev., 2003,
11, 79– 86.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 8 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
Journal Name
ARTICLE
48. J. J. Balbach, Y. Ishii, O. N. Antzutkin, R. D. Leapman, N. W.
Rizzo, F. Dyda, J. Reed and R. Tycko, Biochemistry, 2000, 39
13748 – 13759.
,
49. B. Adhikari, J. Nanda and A. Banerjee, Soft Matter, 2011, 7,
8913–8922.
50. G. Palui, A. Garai, J. Nanda, A. K Nandi and A. Banerjee, J.
Phys. Chem. B, 2010, 114, 1249–1256.
51. B. Adhikari, J. Nanda and A. Banerjee, Chem. Eur. J., 2011,
17, 11488 – 11496.
52. L. Mu and S. S. Feng, J. Control Release, 2002, 80, 129 -144.
53. S. S. Feng, W. T. Zeng, Y. T. Lim, L. Y. Zhao, K. Y.Win, R.
Oakley, S. H. Teoh, R. C. H. Lee and S. R. Pan,
Nanomedicine, 2007,
54. B. Abismaïl, J. P. Canselier, A. M. Wilhelm, H. Delmas and C.
Gourdon, Ultrason. Sonochem.,1999, , 75-83.
2, 333-344.
6
55. W. Abdelwahed, G. Degobert, S. Stainmesse and H. Fessi,
Adv. Drug Delivery Rev.,2006, 58, 1688-1713.
56. Y. Fu and W. Kao, J. Expert Opin Drug Deliv., 2010, 7, 429 –
444.
57. M. Rehman, A. Madni, A. Ihsan, W. S. Khan, Imran Khan, M.
Ahmad, M. Mahmood, S. Z. Ashfaq and I. Shakir, Int J.of
Nanomedicine, 2015, 10, 2805 – 2814.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 8
Please do not adjust margins

Page 9 of 10
New Journal of Chemistry
View Article Online
DOI: 10.1039/C6NJ02125E
Textual abstract
This result reflects the efficient candidature of our hydrogelators and nanoparticles generated therefrom as
excellent carriers for drug delivery.

New Journal of Chemistry
Page 10 of 10
View Article Online
DOI: 10.1039/C6NJ02125E
TOC for NJC