In search of bioinspired hydrogels from amphiphilic peptides: A template for nanoparticle stabilization for the sustained release of anticancer drugs

Article abstract of DOI:10.1039/c9nj01763a

The development of potent stimuli-responsive hydrogels has rapidly expanded in the last decades due to their diversified applications in the field of biomedicines. In accordance with this drift, herein, we aimed at modulating a series of amphiphilic peptide analogues with the general formula Me-(CH₂)₁₄-CO-NH-CH(X)-COOH, where X = CH₂Ph in hydrogelators I (l-Phe) and II (d-Phe) and X = CH₂Ph(OH) in hydrogelator III (l-Tyr), which displayed an excellent propensity to immobilize water at room temperature with a minimum gelation concentration of 0.04%/0.05%/0.02% w/v for hydrogelators I-III, respectively, regardless of their configuration at the C-terminal centre. To validate this threshold concentration difference, we performed computational analysis that demonstrated the ability of the side-chains of hydrogelators I and III to remain highly planar with the methylene units of the amphiphile and aromatic rings, promoting favourable correspondence through van der Waals forces and pi-pi stacking. Consequently hydrogelators I and III self-assembled in an ordered organisation superior to hydrogelator II. Furthermore, the spectroscopic and microscopic experiments revealed that the hydrogelators manifested a β-sheet conformation and nanofibrous morphology at the supramolecular level. As observed visually and additionally confirmed by differential scanning calorimetry (DSC) and rheological measurements, the hydrogels exhibited thermo-reversibility, injectability and high mechanical strength. Importantly, these biomaterials were also found to be resistant towards proteolytic degradation and non-cytotoxic in the cell line HEK 293 using a dose-dependant cell viability assay. To date, the development of a structured approach for the release of drugs in a predictable manner from an optimised formulation, using peptide-based hydrogel nanoparticles as a delivery system remains in its infancy. Hence, we developed hydrogel nanoparticles (HNPs) with our fabricated amphiphilic peptides that exploited the weak noncovalent interactions for their fabrication, unlike other cross-linked polymers that require strong covalent or ionic bonds for their formation. Interestingly, the as-synthesized nanoparticles showed an unprecedented ability to release the anticancer drugs 5-fluoro uracil/doxorubicin at physiological conditions depending on the physico-chemical parameters of the drugs. We believe that the reported injectable, biocompatible, amphiphilic peptide-based hydrogels hold future promise as a potential tool to transport drugs to a targeted site at a greater concentration, thus relieving the patient from surgical injury and simultaneously aiding in a faster recovery.

Full text of DOI:10.1039/c9nj01763a

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The role of atropisomers on the photo-reactivity and fatigue of
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In Search of Bioinspired Hydrogels from Amphiphilic Peptides: A
Template for Nanoparticle Stabilization for the Sustained Release
of Anticancer Drugs
Received 00th January 20xx,
Accepted 00th January 20xx
Radha Rani Mehra,a Priyanka Tiwari,a Anindya Basu, b and Anita DuttKonar,a,b
*
DOI: 10.1039/x0xx00000x
The discovery of potent stimuli-responsive hydrogels have rapidly expanded in the last decades due to their diversified
applications in the field of biomedicines. In accordance to this drift, herein our effort lies to modulate a series of
amphiphilic peptide analogues of general formula Me-(CH2)14-CO-NH-CH(X)-COOH, where X = CH2Ph in hydrogelator I (L-
Phe) & II (D-Phe) and X = CH2Ph(OH) in hydrogelator III (L-Tyr), respectively, that displays excellent propensity to
immobilize water at room temperature with a minimum gelation concentration of 0.04%/ 0.05% /.02% w/v for
hydrogelators I-III, regardless of their configuration at the C-terminal centre. To validate this threshold concentration
difference, we performed computational analysis that demonstrates the ability of the side-chains of hydrogelator I and III
to remain highly planar with the methylene units of the amphiphile and aromatic rings, promoting favourable
correspondence through Van der Waal's forces and pi- pi stacking. Consequently hydrogelator I and III self-assembles in an
ordered organisation superior to hydrogelator II . Furthermore the spectroscopic and microscopic experiments reveal the
hydrogelators to manifest sheet conformation and nanofibrous morphology at supramolecular level. As observed
visually, additionally confirmed by Differential Scanning Calorimetric (DSC) and rheological measurement the hydrogels
exhibit thermo-reversibility, injectability and high mechanical strength. Importantly these biomaterials were also found to
be resistant towards proteolytic degradation and non-cytotoxic in the cell line HEK 293 using dose dependant cell viability
assay. Till date the development of a structured approach for the release of drugs in a predictable manner from an
optimised formulation, using peptide based hydrogel nanoparticle, as delivery system remains in its infancy. Henceforth
we developed hydrogel nanoparticles (HNPs) with our amphiphilic peptides that exploits weak noncovalent interactions
for its fabrication, unlike other cross linked polymers that requires strong covalent or ionic bonds for its formation.
Interestingly, the synthesized nanoparticles shows an unprecedented ability to release the anticancer drugs 5-Fluoro uracil
/Doxorubicin at physiological conditions depending on the physicochemical parameters of the drugs. We believe that the
reported injectable, biocompatible, amphiphilic peptide based hydrogels hold future promise as a potential tool, to
transport drug to the targeted site at a greator concentration, thus relieving the patient from surgical injury,
simultaneously faster recovery.
.
interactions (H-bonding,  interaction, hydrophobic
interaction etc) in the hierarchical process, unlike other cross
linked polymers that requires strong covalent or ionic bonds
for its formation.17-20
Introduction
Stimuli responsive peptide based hydrogels have rapidly
expanded in the last decades due to their diversified
applications in biomedicines and biotechnology.1-15 The
environmentally benign nature of peptides in addition to their
biocompatibility and biodegradability makes them appealing
synthons for biomaterial design.16 Despite they are
advantaged due to the involvement of non-covalent
Non-covalent interactions are inactuality very weak
interactions, that can be formed or disrupted very easily by
mere application of an external stimuli (pH, salt,
concentration, mechanical stress, heat energy, ultrasound,
solvent polarity etc). Since the process of self-assembly is
governed by non-covalent interaction, any causative factor
that triggers this delicate cruciality leads to imbalance, thereby
failing to form an ordered organisation. Although there are no
definitive guidelines to bring about this balance, the examples
that failed to fulfil the condition, may provide insights and
assist in rational design of successful materials.17,21 In stimuli-
responsive gels, one can induce a gel to sol transition by
stimuli application, and upon its removal leads to
reappearance of the gel phase.
a. Radha Rani Mehra, Priyanka Tiwari and Dr. Anita Dutt Konar
Dept. of Chemistry, Rajiv Gandhi Technological University, Bhopal
b. Anindya Basu and Anita Dutt Konar
School of Pharmaceutical Sciences,Rajiv Gandhi Technological University, Bhopal,
Airport Bypass Road, Gandhinagar, Bhopal-462033; E-mail:
anitaduttkonar@rgtu.net;
Electronic Supplementary Information (ESI) available: [details of spectral data,
Enlarged images of various experimental data, Detailed experimental procedures].
See DOI: 10.1039/x0xx00000x
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Temperature is
a
commonly exploited stimulus, and easily on application of a stimuli. But one disadvantage with
View Article Online
DOI: 10.1039/C9NJ01763A
thermo=reversible polymeric hydrogels that immobilizes water nanoparticle is that they are easily recognized by the lymphatic
at physiological temperature have gained significant impetus system and other components of blood because of their
due to their viability in invivo experiments. The synthons that hydrophobicity. This makes them readily eliminated from the
are precisely used for this purpose are polymers composed of blood stream at a faster rate.36-37
naturally occurring or synthetic materials. However, naturally Indeed literature documentation reveals that numerous
occurring polymers are often disadvantaged by their poor research groups are engaged in developing polymeric hydrogel
mechanical strength and instability owing to proteolytic (natural or synthetic) based delivery system for the transport
degradation inside the cell. 22-24 Although this drawback was to of drugs.38-48 But these reported polymeric motifs are either
some extent resolved by peptoids, yet it faces some new proteolytically stable or biocompatible or injectable or lacks
challenges as the peptoids lack extensive hydrogen bonding sufficient mechanical strength. They do not satisfy all the
ability and backbone chirality as compared to polypeptides.25- prerequisite conditions simultaneously. Therefore our task lied
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Another mandatory criteria that is required for biomedical to search for a single scaffold that possesses all the properties
application is injectability.28-31
together. It is well known that peptide (polymers composed of
Thermo-responsive hydrogels have attracted immense amino acids linked by noncovalent interactions) based drug
significance in this arena.12,32-35 Their mere aptitude to delivery vehicles are cost effective process as the amount of
undergo gel to sol transition and vice versa by heat drug required is low.
application, imparts propensity to withdraw the biomaterial in Moreover peptide based hydrogel nanoparticles are
a syringe and injecting it in the patient. Gradually with advantaged over other cross linked polymers as they are
temperature decrease the injectable hydrogel recovers its formed through self-assembly that utilizes non-covalent
strength and remains localized in the target area after its interactions, which are very weak in nature. Therefore their
injection, thus relieving the patient from surgical injury.
formation and disruption can be easily controlled. Also they
consist of ecofriendly amino acids which makes them
biocompatible and biodegradable. 49-50
Being mindful of all these conditions our idea lies to modulate
the following: Firstly to design and synthesize a set of thermo-
reversible peptide based hydrogels that may display
considerable mechanical strength ; secondly the hydrogelators
should be proteolytically stable, injectable and biocompatible.
thirdly to develop hydrogel nanoparticle transport with the
designed hydrogelators using an optimized formulation such
that the nanoparticle gets converted to hydrophilic nature by
some modification. This in turn would increase the blood
circulation period thereby controlling the clearance time and
fourthly to study the drug release kinetics from the optimized
formulation. Thus, to meet our goal we decided to synthesize a
series of amphiphilic peptide analogues, Me-(CH2)14-CO-NH-
CH(X)-COOH, where X = CH2Ph in hydrogelator I (L-Phe) & II (D-
Phe) and X = CH2Ph(OH) in hydrogelator III (L-Tyr), by coupling
the amphiphile palmitic acid and aromatic amino acid residues.
Our systematic approach in fulfillment of the objective using
the novel delivery system hydrogel nanoparticles from
amphiphilic peptides have been discussed in details in the
subsequent sections (Figure 1).
Figure 1. Schematic representation of the probable self-assembly pattern of the
hydrogelators I – III leading to the formation of self-supporting hydrogels.
Results and discussion
Design and characterization of the Hydrogelators
In this project we judiciously designed a set of peptide
sequences, comprising of the amphiphile palmitic acid and
aromatic amino acid residues.
The basic idea to design an injectable hydrogel for biomedical
application is to select a biocompatible hydrogel scaffold with
considerable mechanical integrity. Then to develop a delivery
vehicle using an optimized formulation with the excipients
such that the delivery vehicle shows drug release ability in a
predictable manner.
Nanoparticles find wide importance in this respect.36 Due to
their small size and enhanced surface area more drug
molecule adheres to the particle surface, that gets released
Why Amphiphiles? Amphiphiles are
a special class of
molecules that bears a hydrophobic head and hydrophilic tail.
Their self-aggregating propensity and ability to induce thermo-
responsiveness and high mechanical strength when used in a
particular system is very well known.51 Palmitic acid falls
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within this category. So we decided to incorporate this unit in
our design considering that the presence of fourteen
methylene units in the backbone, may introduce thermo-
responsiveness and increased hydrophobicity to the material.
In contrary, the aromatic amino acids (L-phenylalanine:
hydrogelator I, L-tyrosine : hydrogelator III) were chosen to
promote the process of self-assembly by its increased π−π and
hydrophobic interactions.52 To study the effect of diversified
configurations on self-assembly we synthesized hydrogelator
II, where we used D-Phe as the aromatic component
(hydrogelator II).
Interestingly, hydrogelators I - III exhibits excellent hydrogel
forming propensity with a minimum gelation concentration of
0.04%/ 0.05% /.02% w/v respectively, without any sonication.
To examine this phenomena, the hydrogelators were initially
dissolved in 1 ml of phosphate buffer (pH:7.5) under mild
heating conditions and allowed to cool. Almost
instantaneously the transparent buffer solution was
immobilized to a transluscent mass for hydrogelators I and III
and after 15 min for hydrogelator II.
of planarity of the aromatic ring in driving the hierarchical
phenomena presumably due to betterDOI-:10.a10n3d9/CH9-NbJo0n1d76in3Ag
interactions.
View Article Online
To validate the difference in minimum gelation concentration
(mgc), we performed computational analysis.56-57 The energy
minimised structures and packing diagrams of the individual
molecules are presented in Figure. 2, S2. As illustrated from
the figure, the hydrogelators I – III show interesting packing
trends. Firstly the backbone of hydrogelator I adopts a -
strand conformation, where the amphiphilic component
remains straight in nature. Interestingly, hydrogelator II – III
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although adopts
a similar -strand architecture, shows
significant kink in the amphiphilic backbone. This may be
attributed to presence of D-configured amino acid in
hydrogelator II and an additional H-bonding donor OH in
hydrogelator III. Moreover, closer inspection in the aromatic
unit demonstrates that the aromatic side chain of both the L-
configured hydrogelators I and III not only remains highly
planar with the long carbon-chain of the amphiphile but also
well aligned to each other (side chains of other molecules).
Consequently interactions through Vander Waal's forces and
pi-pi stacking becomes favourable leading in an ordered -
sheet arrangement. On the other hand in hydrogelator II, this
planarity of the side chains of the aromatic unit and
amphiphilic backbone to some extent gets deviated.
Additionally such fine alignment of the aromatic side chain was
also not observed here. Thus this information sheds some light
for the cause of difference in mgcs in the hydrogelators.
Indeed, the stability of the molecular self-assemblies and
consequently the difference in mgcs seem to be well reflected
by their corresponding Heats of Formation (Hf), where we
observed the following trend: hydrogelator -III (-599.24
kcal/mol)> hydrogelator -I (-506.31 kcal/mol) > hydrogelator -II
(-466.9 kcal/mol).
Demonstration of Thermoreversible Property and
Mechanical Strength of the Hydrogelators
During the procedure for demonstration of thermal stability,
we were interested to examine whether palmitic acid was
eligible enough to induce thermo-responsive behaviour in the
hydrogelators. To check, the same vials in which the Tgel
values were noted, were used. Indeed, the sol in each vial was
transformed into gel almost instantaneously. So we repeated
the process to confirm our analogy. But everytime we found
the same observation. Our next attempt was to withdraw the
sol with a syringe to and fro to investigate the injectability.
Importantly, the hydrogelators revealed both the properties.12
(Figure 3, S3-S4).
To strengthen the thermoreversibility data, we performed
Dynamic Scanning Calorimetric Analysis for all the three
hydrogels in triplicates using the same experimental
parameters (Figure 4). Each cycle was programmed from -30 to
70°C for heating and vice-versa for cooling at a rate of
5oC/min.12,58
Figure 2. Energy optimised structures of hydrogelators I – III and their self-
assembly patters in -sheets considering 3 monomeric units.
The gel formation was confirmed by turning the vial upside
down. The gels did not undergo any deformation for a period
of more than six months. Sol–gel transition temperatures
(Tgel) were plotted against different concentrations of the
hydrogelators (Figure. S1). From the curves it is evident that
the Tgel value increases with increase in concentration of the
amphiphilic hydrogelators, until the arrival of saturation limit.
After this temperature the Tgel values does not significantly
change.53-55 Indeed this data indicates that the self-assembly of
the hydrogelators is governed by strong intermolecular non-
covalent interactions. Moreover the correlation between the
mgc and hydrogelator structure emphasizes the delicate role
Indeed, the DSC thermograms presented a wide endotherm
for the gel - sol transition and an exotherm for the reverse
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phase while cooling (Figure 4A-C, Table 1). The data obtained modulus G’ is higher than the loss modulus (G’’) in the region 1
from the three cycles perfectly superimposed when to 100 rad/s (angular frequency) showing no cross-over point.
overlapped. This finding confirmed the thermo-responsive This behavior demonstrates a soft gel phase formation. 59 – 60
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DOI: 10.1039/C9NJ01763A
nature of the hydrogelators (Figure 3). 12,52
Indeed the storage modulii is of the order ≥ 104 Pa for
hydrogelator III and ≥ 103 Pa for hydrogelator I and II
respectively at the same concentration which suggests that the
designed amphiphilic hydrogelators bears significant
mechanical strength.
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Earlier reports reflect that the D-configured amino acid
derivatives
possessed
higher
mechanical
strength
incomparison to the L- isomer.12 But here we notice opposite
observation. Therefore the importance of overall balance of
intermolecular interactions arising due to the difference in
self-assembly pattern owing to the use of diversified amino
acid configurations and finally the the gelation strength is
highlighted.
Hydrogel III in Inverted Transition of gel Uptake of sol into syringe
glass vial
into sol on heating
Next our interest lied to correlate the thermal properties with
the viscoelastic behavior.58 For this the dynamic viscoelastic
measurements were performed with the same concentration
10mg/ml for all the three hydrogelators (Figure 5B).
During the investigation, the temperature was increased from
room temperature (25°C) to 70°C with heating rates of about 2
°C/min. At this temperature range an angular frequency of 1
rad/s was kept constant along with a strain of .02%.
Self recovered
Hydrogel
Complete transfer Releaseof sol from
of sol into vial
syringeinto glass vial
Figure 3. Demonstration of thermo-responsiveness and injectable behaviour of
hydrogelator III using a model dye Fluorescein.
To further justify the experimental results we conducted
concentration dependant DSC analysis. Figures 4E-F distinctly
shows the effect of hydrogelator concentration on the heating
and cooling thermograms. From the plots we find that
although the endothermic peak increases in height, the peak
temperature remains almost constant in every case.
Additionally, the peak becomes broader.
The endothermic peaks showed similar behavior for
hydrogelators I and III, but slightly different for hydrogelator II,
which may be assigned to the difference in configuration of
the aromatic amino acids used (In hydrogelator I and III: L
configured and II: D). (Figure 4D-F).
Thus we were successful in determining the thermal induced
dissociation and association behavior of all the palmitic acid
derivatives as a function of hydrogelator concentration,
further confirming the ability of palmitic acid to induce
thermo-reversibility in the hydrogels.
Indeed the hydrogels absorb heat energy and gets converted
in sol phase, while cooling results in reappearance of the gel
after release of heat energy. Moreover both the observed
endothermic and exothermic peaks are linear functions of
hydrogelator concentration, demonstrating that the
thermodynamic changes are dependent only and only on the
hydrogelator concentration.58
Table1 : Exothermic Enthalpies for the Hydrogelators as
obtained from the DSC curves:
Hydrogelator I
(Figure 4A)
Cycle 1
Cycle 2
Cycle 3
Heat Flow Max
(mW)
195.1455
-25.4135
Cycle 1
204.9028
-25.4135
Cycle 2
215.108
-25.4135
Cycle 3
Temperature (oC)
Hydrogelator II
(Figure 4B)
Heat Flow Max
(mW)
79.39682
-24.4278
Cycle 1
83.36666
-24.4278
Cycle 2
87.535
Temperature (oC)
-24.4278
Cycle 3
Hydrogelator III
(Figure 4C)
Heat Flow Max
(mW)
100.4896
-19.9401
Cycle 1
110.5385
-19.9401
Cycle 2
211.5924
-19.9401
Cycle 3
Temperature (oC)
Hydrogelator I
(Figure 4D)
Heat Flow Max
(mW)
177.405
-25.4135
Cycle 1
175.9733
-19.8761
Cycle 2
219.9666
-19.8761
Cycle 3
Temperature (oC)
Thus
a completely thermoreversible behavior has been
Hydrogelator II
(Figure 4E)
observed for all the concentrations studied which further
emphasizes the importance of palmitic acid in inducing
thermoresponsive behavior.
Rheological investigations are generally carried out to gain
knowledge about the mechanical strength of the hydrogels,
which allows one to decide its applicability zone. Usually in this
experiment the elastic response G’ and viscous response G’’are
measured in frequency sweep experiments.59 – 60 As observed
from Figure 5A, throughout the viscoelastic region, the storage
Heat Flow Max
(mW)
79.39682
-24.4278
Cycle 1
21.21458
-45.0
24.97099
-29.9359
Cycle 3
Temperature (oC)
Hydrogelator III
(Figure 4F)
Cycle 2
Heat Flow Max
(mW)
Temperature (oC)
100.4896
-17.998
168.8029
-17.998
211.003
-17.998
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Figure 4 : A - C ) Triplicate DSC curves superimposed when overlapped---- as a proof of thermoresponsiveness in hydrogels; D-F) Concentration Dependant DSC
thermograms demonstrating the ability of hydrogelator concentration to largely regulate the amount of heat flow in the system.
Our study reveals three distinct regions. a) The first region is were then subjected to hydrogelation studies. Interestingly,
the gel state region where both the elastic response G’ and both the compounds III and IV failed to exhibit gel forming
viscous response G’’ are parallel to the temperature axis. Also propensity and subsequently, thermoreversible behavior.
here the value of G’ is higher than G’’; b) The second region is Henceforth the role of palmitic acid in inducing thermo-
the cross over region where both the storage modulus G’ and responsive behavior is emphasized.
loss modulus G’’ are equal. This point is the onset of phase The principle guidelines of gelation involves a balance between
transition i.e from gel to sol and is known as yield stress (); c) enthalpy and entropy, the nature of the weak interactions
The third region is predominantly the sol region where the involved, such as hydrogen bonds and  correspondence
value of loss modulus G’’ is higher than the storage modulus resulting in thermodynamic minimum conditions. Both
G’. The temperatures at which G crosses over G are hydrogelator I and III are L-configured amino acids. We
55/46/57 °C for hydrogelators I- III respectively. Figure 5C and speculate that in L-configured amino acids the aromatic units
D represents the change in complex viscocity with respect to (phenylalanine) are in such an orientation, that it forms
temperature and time. Traditionally the cross over is indicated favourable  interactions with each other resulting in gels of
as a sol-gel transition point. But the method by which it is higher strength incomparison to D-configurd amino acids
determined in rheological analysis depends on the frequency (hydrogelator II). Considering the case of hydrogelator III , the
of the measurement.58 Again for DSC, the rates of heating and presence of an additional H-bond donor (OH of Tyrosine) may
cooling determines the transition temperature, i.e it is have further assisted in the formation of gels of higher
dependant on the experimental parameters.58 So in both the mechanical strength in comparison to hydrogelator I.
experiments we observe difference in transition temperatures.
But the expected pattern obtained in either cases are identical Structural and Morphological Properties
which allows us to arrive at a logical conclusion.
To explore the structural aspect of the hydrogelators,
Taken together the result of DSC and rheological measurement especially the H-bonding interactions, we turned into FTIR
, our investigation reveals that all the three hydrogelators spectroscopy.61-63 Figure 6A-C shows the amide I and amide II
exhibit excellent thermoresponsive behavior.
bands in the FTIR spectra from the solid sample as synthesized
To affirm whether the thermo-responsive property have and the xerogels (Figure S5-S7). It is well-known that the
emanated from the palmitic acid component, we have location of amide I absorption is primarily determined by the
synthesized and characterized two different control molecules, backbone conformation irrespective of the amino acid
Compounds IV (Dodecanoic acid as the amphiphile): Me- sequence.
(CH2)10-CO-NH-CH(X)-COOH, where X = CH2Ph (L-Phe) & Each molecules contain one NH proton and two carbonyl
Compound V (Stearic acid as the amphiphile): Me-(CH2)16-CO- moieties (Figure 1). Peaks for NH protons in the hydrogelators
NH-CH(X)-COOH, where X = CH2Ph (L-Phe). The molecules
in the powdered and xerogel state appear at 3390/3376 cm−1
−
1
(hydrogelator I), 3302/3300 cm
(hydrogelator II) and
3370/3350 cm− 1 (hydrogelator III) respectively (Figure S5-S7).
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These data signifies that in both the states the NHs are H- Thus the dominant role of -sheets in the respeVcietwivAertixcleerOonglineel
bonded. Similarly the amide I and II carbonyl, gives distinct state is focussed.62-63 The rotational degree of freedom of the
DOI: 10.1039/C9NJ01763A
peaks in the region at 1648/1562cm−1 (xerogel) & 1648 / 1542 aromatic groups in phenyl alanine may have attributed to the
cm− 1 (solid) for hydrogelator I ; 1650/1548 cm− 1 (xerogel) & existence of -sheet like extended conformation, which is
1646 / 1558 cm−1 (solid) for hydrogelator II ; 1650 / 1548 cm−1 further exemplified by the broadness of the amide frequencies
(xerogel) & 1650/1558 cm− 1 for hydrogelator III respectively of the xerogels.
(Figure 6). However the IR spectra reflects that the peak To re-confirm our analysis we performed PXRD experiments
heights for both the NHs and amides get noticeably diminished with the xerogels of the hydrogelators.12,32,66 The wide angle
in the xerogel state. This is only possible when the hydrogen PXRD patterns of all the xerogels show periodic diffractions
bonding and van der waals interactions are engaged in which are common in all the three hydrogelators,
interconnecting the molecules to form an entangled fibrous demonstrating the presence of ordered structures. The
network leading to hydrogel formation through -sheet like characteristic peaks at 18.06 - 18.09° (d = 4.9 Å) is attributed to
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extended conformation.64-65
the presence of the -sheet like extended conformation in the
hydrogelators.66
Moreover the peak at 23.5-24.0° (d = 3.7 Å) confirms the
presence of  stacking which may have risen due to the
presence of ordered arrangement of phenyl rings of aromatic
amino acid residues (Figure6D-F, S8-S10). 66
In order to further delineate the solution conformation, we
performed CD measurement in methanol. The hydrogelators I
- III show similar CD patterns with a distinct minima at 230 –
245 nm (for hydrogelator I: 232 nm ; hydrogelator II: 231 nm;
hydrogelator III: 232 nm ). This signature was reported as a
characterestic of -sheet like extended structures (Figure 7). 67
Thus taken together the result of computational studies
combined with the FT-IR, PXRD and CD data, our investigation
affirms the presence of -sheet like extended conformation in
the hydrogelators.
To crosscheck the morphological arrangement we performed
FE-SEM studies from the xerogels obtained from the
corresponding hydrogels (same concentration and same
magnification has been used for all the three hydrogelators)
Figure 8. The FE-SEM images reveal that in all the three cases
formation of filamentous-tape formation occurred with
dimension of width approximately 500 nm and length in
Figure 5. Rheological measurement of the hydrogelators A)The variation of elastic
response G’ and viscous response G’’ in angular frequency sweep experiments,
confirming the formation of hydrogels; B-C) Demonstration of Thermoreversible
property by the variation of Modulus/Complex viscocity with respect to temperature
D) Variation of Complex viscocity with respect to time.
;
Figure 6 : A - C ) Triplicate DSC curves superimposed when overlapped---- as a proof of thermo-responsiveness in hydrogels; D-F) Concentration Dependant DSC
thermograms demonstrating the ability of the hydrogelator concentration to largely regulate the amount of heat flow in the system.
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several micrometers. Closer view reveals that in hydrogelator This is a colorimetric protocol that quantifies theVrieewdAurtcictleioOnnlionef
II, we noted filamentous-tape which self-assembled to a three yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl
DOI: 10.1039t/eCt9rNaJz0o1l7i6u3mA
dimensional network, that served as a template to encapsulate bromide (MTT) by mitochondrial succinated dehydrogenase.
water molecules, assisting in the formation of self-supporting Initially the MTT enters the cells and passes into the
hydrogels. But in hydrogelator I and III, these fibrous tape like mitochondria where it is reduced to an insoluble, coloured
structures are interlinked by a porous mesh, accounting for the (dark purple) formazan product. The cells were then
difference in mechanical strength and minimum gelation solubilised using DMSO and the released, solubilised formazan
concentration.32 Also we correlate this difference in the 3D reagent is measured spectrophotometrically. Since reduction
network due to the attainment of planarity between of MTT occurs in metabolically active cells, the level of activity
amphiphilic backbone and aromatic side chains in is a measure of the viability of the cells. Henceforth, we have
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hydrogelators I and III, confirmed by computational studies.
assessed the cytotoxicity of the compounds by MTT assay in
HEK 293 (Human Embryonic Kidney cell) cell line by incubating
the latter for 48 h as shown in Figure 9B.
The IC50 values of the hydrogelators I /II /III are 352/ 294/ 242
M respectively. Since the IC50 values of the hydrogelators are
greator than 100 M, it can be concluded that the
hydrogelators are biocompatible in nature.12,49,68
Hydrogelator I
Hydrogelator II
500
Hydrogelator III
400
Thus the above investigations allow us to infer that our
hydrogelators are proteolytically stable and biocompatible.
300
200 225 250 275 300
Wavelength (nm)
Preparation and characterization of HNPs
We synthesized hydrogel nanoparticles using the modified
inverse emulsion process as described in Figure 10A.49-50 In this
experiment the hydrogelators were first dissolved in water to
form a homogeneous solution. This phase was then dropwise
added into the organic phase containing the surfactant vitamin
Figure 7. CD spectra of the hydrogelators I – III showing -sheet like extended
conformation in methanol.
Proteolytic Stability and Biocompatibility Studies
In order to gain insight about the proteolytic stability of the
hydrogelators I - III containing the nonproteinogenic palmitic
acid residue, at the N-terminus and a proteinogenic alpha
amino acid (Phe/Tyr) of diversified configuration at the C-
terminus we incubated the hydrogelators with proteolytic
enzyme proteinase K as previously described for 48 hrs.32,68
Enzymatic degradation was monitored by measuring the
corresponding mass spectra till 48 hrs at regular intervals of
6hr as shown in Figure 9A.
The absence of the peak corresponding to palmitic acid (256)
(i.e. the degradation product), and the observance of 100%
peptide peak corresponding to (M)+ (Hydrogelator I and II:
403; III: 418) after 48 hrs, affirms the resistance of our
hydrogelators towards proteolysis (Figure 9A, S11-S19).32,68
Next our attempt lied to decipher the biocompatibility of the
hydrogelators, as this is one of the prerequisite condition for
any molecule to be used as drug delivery vehicle. Therefore we
conducted cell viability experiments.69-70
E-TPGS, solubilized in light paraffin oil resulting in
heterogeneous mixture as shown in Figure 10A.
a
The latter was then homogenized for fifteen minutes using a
high speed homogeneizer where the stirring speed was
adjusted to 30,000 rpm.
After this process, the mixture was allowed to self-assemble at
0oC for 2 h to permit the attachment of the surfactant along
the surface of the hydrophilic core (Figure. 10A). Figure 10B
and 10C shows the structure of the components involved in
nanoparticle formation and cartoon diagram of the latter
respectively.
Traditionally, hydrogel nanoparticles are hydrophobic in
nature. The more hydrophobic a nanoparticle is, its circulation
time in blood is less, so more likely it gets cleared from the
blood stream.36 To convert this hydrophobic nature to
hydrophilic, we decided to coat the nanoparticle with the
biocompatible surfactant Vitamin E-TPGS.49,68,71 Ultimately the
HNPs were purified using centrifugation. Our optimized
Porous Mesh
Porous Mesh
Filaments
Filaments
Filaments
Figure 8 : FE-SEM images of the xerogels of hydrogelators I – III showing filamentous-tape like entangled three dimensional network (hydrogelator II) and
filamentous tape further interconnected by a porous mesh (hydrogelators I and III).
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formulation results reflect a bimodal distribution of average Release profile from the Hydrogel matrix
particle sizes of 304.6± 47.12 nm & 21.32± 4.15 nm for
View Article Online
DOI: 10.1039/C9NJ01763A
hydrogelator I, 171.6± 30.01nm & 15.71± 2.046 nm for Next we attempted to test the versatility of our hydrogelators
hydrogelator II and 136.1± 21.16
23.27± 4.93 for as drug delivery vehicles.72-73 So we chose 5-FU and
&
hydrogelaor III (Figure. 10D). This bimodal size distribution may doxorubicin as the model drugs owing to their widely varied
have arisen due to equilibrium between two opposite physicochemical properties namely chemical structure,
phenomena arising because of variation in experimental molecular weight and hydrophobicity.32
parameters. Moreover, a high negative zeta potential (-18.7 Besides these drugs exhibit excellent chemotherapeutic
mV) conferred formulation stability. This negative charge is activities.32 The concentrations of the released drugs were
considered to be a contribution from carboxylate functionality measured in solution over time using a dialysis membrane, and
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as well as the hydrophilic surfactant coating (Figure. 10A-B).
quantified using UV spectroscopy in buffer pH 7.45 at room
We hypothesize that the drug molecules would remain temperature under gentle stirring conditions.32,49-50,71 It has
entrapped within the core and would be released after been observed that the release kinetics of 5FU loaded HNPs
exposure to a stimulus (Figure 10C)
differ considerably from that of doxorubicin, which may be
accounted by the difference in their physicochemical
properties.
Figure 11A illustrates the release of 45% of the low molecular
weight drug, 5FU from the HNPs occurred within 36 h for
hydrogelator II and 48 h for hydrogelator I and III respectively,
after which the kinetics attained a saturation limit. In contrary
the release of 30% of doxorubicin took place at 90h for
hydrogelator III and 105 h for hydrogelator I and II respectively
(Figure 11B). We propose that the mechanical strength of the
hydrogels might have played a vital role in determining the
release kinetics. Since hydrogelators I and III possessed higher
mechanical strength (Figure 5), we find slower release of both
the drugs from the hydrogel matrix.55,69-70
Figure 9. A) Proteolytic Stability Study of the Hydrogelators I – III, after treatment with
the proteolytic enzyme proteinase K ; B) Dose dependant cell viability measurement
employing MTT assay using the cell line HEK293.
Figure 10. A) Inverse Emulsion Technique showing the formation of HNP, B) Structures of the components involved in nanoparticle synthesis, C) Cartoon diagram of the details of drug
embedded hydrogel nanoparticles, D) Physical Dimensions of the nanoparticle as obtained from zeta-sizer .
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To gain deeper insight about the drug - hydrogelator with our biocompatible hydrogelators, that lacks the use of
View Article Online
interaction we performed computation studies using covalent or ionic bonds during its formaDtiOoIn: 1.0W.10e39f/eCe9lNtJh0a17t6b3Ay
Hyperchem software. For this, initial inspection of the drug simple attachment of Vitamin E-TPGS at the surface, will
structures reveal that 5FU bears only H-bonding promoting convert the nanoparticle to hydrophilic from hydrophobic
groups but lacks stacking moieties (Figure 10B). In contrast, nature, which might enhance the blood circulation time
doxorubicin possesses additional aromatic units for  thereby allowing higher concentration of the drug to be
interactions apart from H-bond donors/acceptors (Figure 10C). delivered at the site of action. Interestingly our hydrogel
Secondly when we scrutinize the hydrogelator conformations nanoparticles displayed an unprecedented ability to release
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we observe a -strand in every case. (Figure 2).
the anticancer drugs at physiological conditions depending
Incase of drug 5FU, at first two -strands arrange in an ordered upon the physicochemical parameters of the drug molecules.
environment to maximize  and hydrophobic interactions We feel our systematic attempt in rational design of this
between them. Under such circumstances 5-FU gets palmitic acid based amphiphilic thermoreversible, injectable
sandwitched between two hydrogelator strands. Hydrogelator and proteolytically resistant drug delivery system represents
I stabilizes by both intermolecular H-bonding and vander waals an attractive perspective and can be used to transport myriads
interactions but hydrogelator II/III by only Vanderwaals forces of therapeutic agent belonging to different families for the
(Figure 12A) .
treatment of diversified diseases, which is being currently
Incontrast, doxorubicin comprises of aromatic scaffolds that investigated by our group.
promote  correspondence in addition to H-bond donors
and acceptors. The case of doxorubicin can be imagined as a
“Nut Bolt System”. In the presence of this drug, two strands of
Experimental
Materials and methods:
Palmitic Acid, 5-Fluoro-uracil (5-FU), Doxorubicin and other
hydrogelators arranges in the form of a nut or better a clip,
and the drug molecule to maximize noncovalent interactions
bolts or hooks itself inside the cavity. In other words, the weak
interactions, nut bolts the drug molecule within the cavity of
the strands. Henceforth release of doxorubicin occurs at a
much slower rate in comparison to the smaller drug, which
lacks this extra stabilization (Figure 12D-E).
components were purchased from Spectrochem. The
surfactant Vitamin E-TPGS was purchased from Sigma Aldrich.
1H NMR / Mass and IR spectra were recorded on a Bruker Ultra
shield (400MHz) spectrometer/ MicroTOF-Q-II instrument and
Shimadzu, Prestige 21 FT-IR spectrometer respectively. All the
compounds were synthesized using conventional solution
phase methodology using racemization free techniques as
described in Scheme 1. All the original spectra of the
molecules are enlisted in Supplementary Section (Figure S5-
S22)
Synthesis of the Compounds I - V
L-Phe-OMe.HCl/D-Phe.HCl/ L-Tyr-OMe.HCl (1): The Methyl
ester hydrochloride was prepared by dissolving the
corresponding amino acids (10/5 gm, 59.17/29.586 mmol of
L/D Phenylalanine and 5gm, 24.48 mmol of L-Tyrosine) in 50ml
of methanol. Then after some time thionyl chloride was
dropwise added to the cooled mixture, and was stirred for 18
hours . After the stipulated period of time, the methanol was
removed under vaccum, and the obtained solid residue was
washed with diethyl ether and used for further reactions.
Yield: 12.39 gm (57.51 mmol, 95%: L-Phe-OMe.HCl); 5.54 gm
(25.73 mmol, 85%: D-Phe-OMe.HCl); 5.75 gm (24.83 mmol,
90%: L-Tyr-OMe.HCl).
Figure 11. Release kinetics of the two anticancer drugs 5-Fluoro uracil and doxorubicin
from the hydrogel matrix of hydrogelators I – III.
Conclusions
In summary in this report we have successfully designed and
synthesized a set of amphiphilic peptides that exhibits
excellent hydrogelating propensity with slight variance in
minimum gelation concentrations. The cause of this threshold
difference in mgcs have been nicely demonstrated by our
computational studies. Indeed our experiments reveal that the
Palmitic Acid L-Phe-OMe (2):74 The Phe-OMe obtained from its
hydrochloride (3.16 gm, 14.66 mmol) was added to an ice-cold
solution of Palmitic Acid (1.5 gm, 5.86 mmol) in 10 ml of DMF.
Then the very strong coupling reagent DCC (1.8 gm, 8.79
mmol: 1, 3-dicyclohexylcarbodiimide) was mixed to the cooled
solution, and stirred for 18 hours. The reaction progress was
monitored by TLC. After the completion of the reaction the
residue was taken up in ethyl acetate and the byproduct DCU
was filtered off. Next, the work up was done by washing the
hydrogelators
displayed
high
mechanical
integrity,
proteolytical stability, injectablity and biocompatibility,
optimal for drug delivery applications. Till date the
development of innovative formulation technologies using
peptide based hydrogel nanoparticles that releases drugs at
the site of action at higher concentration, preserving their
original properties remains in its infancy. So we devised
optimised formulation to synthesize hydrogel nanoparticle
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Figure 12: Computation analysis of hydrogel-drug interactions. A-B-C: shows correspondence of hydrogelators I-III with 5-fluorouracil and D-E-F with
doxorubicin. All the geometry optimized structures both for the hydrogelators and drugs were performed using AMBER 99 forcefield. Colour of the atoms
are as follows: Carbon: Cyan; Hydrogen: white; Oxygen: Red; Nitrogen: Blue. The 5-fluorouracil is coloured green: and doxorubicin red for easy
differentiation of the drugs from the hydrogelators.
organic layer obtained after removing DCU, with 2 M HCl, 1 M Yield: 1.06 gm, (2.64 mmol, 65%); m.p. 57 -60 0C LC-MS:
sodium carbonate and brine successively, and dried over C25H41NO3: 404.5 [M-H]+; MS (calculated) m/z: 403.59 [M]+; FT-
anhydrous sodium sulphate. The organic layer obtained was IR: 940, 1296, 1434, 1465, 1542, 1644, 1703, 2851, 2918,
evaporated in vacuum to obtain a white solid. This crude 3405, 3455, 3566, 3595, 3623 cm-1; 1H NMR (d6-DMSO, ppm):
peptide was used for the next step without further 12.17 (1H, br, COOH of Phe(2)), 8.04 (1H, d, J = 8Hz, NH of Phe
purification.
Yield: 1.7 gm (4.07 mmol , 70%)
(2)), 7.25- 7.13 (5H, m, Aromatic Hs of Phe(2) ), 4.37-4.43 ( 1H,
m, CαH of Phe (2)), 3.00 - 3.05 (1H, m, CβHs of Phe(2) ) , 2.83 -
Hydrogelator I : 74 To obtain Hydrogelator I, Compound 2 (1.7 2.78 ( 1H, m, Cβ Hs of Phe(2)), 2.17-2.13 (2H, m, CαHs of
g, 4.07 mmol) was dissolved in methanol (28 ml) and requisite Palmitic acid(1)), 1.44-1.47 (2H, m, CβHs of Palmitic Acid(1)),
amount of NaOH (2M NaOH) was added to the mixture. The 1.21 (10H, br, 12- methylene Hs of Palmitic Acid (1)), 0.85 -
progress of the reaction was monitered by TLC. After 0.84 (3H, m, CH3-Hs of Palmitic Acid (1)).
13C NMR (d6-DMSO, ppm): 175.14, 173.61, 172.83, 138.32,
129.73, 128.80, 126.73, 53.81, 37.222, 35.72, 34.38, 31.78,
29.51, 29.40, 29.29, 29.20, 29.04, 28.93, 25.66 24.97, 22.58,
14.40.
completion of the reaction, as determined by TLC, the
methanol was evaporated. The residue containing the sodium
salt was dissolved in water and extracted with diethylether to
remove the unreacted stuff. The aqueous layer obtained was
cooled, acidified with 2N HCl and extracted with ethyl acetate.
The solvent was evaporated in vacuo to obtain a white solid
Palmitic Acid D-Phe-OMe (3):74 This compound was prepared
following the same synthetic procedure as that of 2.
Yield: 1.95 gm (4.67 mmol, 80%)
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Hydrogelator II:74 To obtain Hydrogelator II, Compound 3 (1.95
ARTICLE
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C
O
C
DOI: 10.1039/C9NJ01763A
g, 4.67 mmol) was dissolved in methanol (28 ml) and requisite
amount of NaOH (2M NaOH) was added to the mixture. The
progress of the reaction was monitered by TLC. After
completion of the reaction, as determined by TLC, the
methanol was evaporated. The residue containing the sodium
salt was dissolved in water and extracted with diethylether to
remove the unreacted stuff. The aqueous layer obtained was
cooled, acidified with 2N HCl and extracted with ethyl acetate.
The solvent was evaporated in vacuo to obtain a white solid
O
O
R
R
R
C
OH
O
OCH3
A
B
D
OCH3
+
OH
n
NH2
NH2 .HCl
NH2
C
R
O
R
9
OH
C
OCH3
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N
N
H
C
O
n
n
H
O
D = CH3OH, 2M
NaOH, RT, 12 Hr
A = CH3OH,
SOCl2, 00C, 12 Hr
C = DMF,DCC,
00C, 12 Hr
B= NaHCO3
Yield: 1.57 gm, (3.90 mmol, 78%); m.p. 56 - 580C LC-MS:
C25H41NO3: 404.50 [M-H]+; MS (calculated) m/z: 403.59 [M]+;
FT-IR: 940, 1297, 1434, 1465, 1543, 1644, 1704, 2659, 2851,
1
2919, 3296, 3665 cm-1; H NMR (d6-DMSO, ppm): 12.03 (1H,
R =
R =
R =
n = 14
n = 14
n = 14
OH
Hydrogelator III
br, COOH Of Phe(2)), 8.04 (1H, d, J = 8Hz, NH of Phe(2)), 7.25-
7.16 (5H, m, Aromatic Hs of Phe (2)), 4.37-4.42 ( 1H, m, CαH of
Phe (2)), 3-.05 (1H, m, CβH Of Phe (2)), 2.77 - 2.83 ( 2H, m, CβH
of Phe(2)), 2.14 - 2.73 (2H, m, CαHs of Palmitic acid (1)), 1.44 -
1.47 (2H, m, CβHs of Palmitic Acid (1)), 1.26 - 1-21 (10Hs, m,
12- methylene Hs of Palmitic Acid (1)), 0.81 - 0.85 (3Hs, m,
CH3-Hs of Palmitic Acid(1)) ; 13C NMR (d6-DMSO, ppm): 134.93,
173.82, 172.45, 138.38, 129.52, 128.89, 34.13, 31.78, 29.52,
29.39, 29.50, 29.22, 29.19, 29.03, 24.97, 22.57, 14.15.
Hydrogelator I
Hydrogelator II
R =
n = 16
R =
n = 10
Compound V
Compound IV
Scheme 1. Synthetic strategy for the preparation of the
Compounds I – V.
Palmitic Acid L-Tyr-OMe (4): 74 This compound was prepared
following the same synthetic procedure as that of 1 and 3.
Yield: 2.69 gm, (6.20 mmol, 90.0%)
Dodecanoic Acid-L-Phe-OMe (5):71 This compound was
prepared following the same synthetic procedure as that of 1-
4.
74
Yield: 1.68 gm (4.67 mmol, 80%).
Hydrogelator III: To obtain Hydrogelator III, Compound 4
(2.69 g, 6.20 mmol) was dissolved in methanol (28 ml) and
requisite amount of NaOH (2M NaOH) was added to the
mixture. The progress of the reaction was monitered by TLC.
After completion of the reaction, as determined by TLC, the
methanol was evaporated. The residue containing the sodium
salt was dissolved in water and extracted with diethylether to
remove the unreacted stuff. The aqueous layer obtained was
cooled, acidified with 2N HCl and extracted with ethyl acetate.
The solvent was evaporated in vacuo to obtain a white solid
Compound IV : 74 This compound was prepared following the
same synthesis procedure as that of Hydrogelator I – III from
(5)
Yield: 1.37 gm, (2.88 mmol, 85%,); LC-MS C21H33NO3: 346.25
[M-H]+; MS (calculated) m/z: 347.25 [M]+; H NMR (d6-DMSO,
ppm): 12.04 -12.30 (1H, br, COOH of Phe(2)); 8.08 (1H, d, NH
of Phe(2), J = 8 Hz); 7.16 – 7.28 (5H, m, Aromatic Hs of Phe(2));
4.41-4.46 (1H, m, CαH of Phe (2)); 2.81 – 3.09 (2H, m, CβHs of
Phe(2)); 2.16-2.21 (4H, m, CH2-Hs of Do-decanoic acid (1));
1.98 – 2.06 (2H, m, CH2-Hs of Do-decanoic acid (1)); 1.46 – 1.52
(2H, m, CH2-Hs of Do-decanoic acid (1)); 1.35 – 1.41 (2H, m,
CH2-Hs of Do-decanoic acid (1)); 1.18 – 1.27 (10H, m, CH2-Hs of
Do-decanoic acid (1)); 0.85 – 0.90 (3H, m, CH3 H’s of Do-
decanoic acid ).
1
Yield: 1.87 gm, (4.46 mmol, 72 %); LC-MS: C25H41NO4: 420.49
[M-H]+; MS (calculated) m/z: 419.59 [M]+; m.p. 55 -570C ;FT-IR:
941, 1228, 1297, 1465, 1530, 1648, 1701, 1745, 2851, 2919,
1
3473 cm-1; H NMR (d6-DMSO, ppm): 11.93 (1H, br, COOH of
Tyr(2)), 9.16 (1H, br, NH of Tyr(2) ), 6.95 (2H, d, J = 8Hz), 4.30 -
4.37 ( 1H, m, CαH of Tyr(2)), 3.55 (1H, br, OH of Tyr(2)), 2.83 -
2.91 ( 1H, m, CβHs Of Tyr(2)), 2.69-2.83 (1H, m, CβHs of Tyr(2)),
2.14-2.17 (2H, m, CβHs of Palmitic Acid(1)), 1.44 - 1.47 (2H, m,
CβHs of Palmitic Acid(1), 1.21 - 1.26 (10H, 12-methylene Hs of
Palmitic Acid(1)), 0.85 – 0.81 (3H, m, CH3-Hs of Palmitic
Acid(1)); 13C NMR (d6-DMSO, ppm): 174.90, 130.40, 127.70,
115.50, 34.20, 31.70, 29.50, 29.40, 29.30, 29.20, 29.10, 25.01,
22.58, 14.40.
74
Stearic Acid-L-Phe-OMe (6): This compound was prepared
following the same synthetic procedure as that of compound
1-3.
Yield: 2.26 gm, (5.23 mmol, 90%).
Compound V : This compound was prepared following the
same synthesis procedure as that of Hydrogelator I – III from
(6)
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Yield: 1.87 gm, (4.33 mmol, 80%); LC-MS: C27H45NO3: 430.2 [M-
H]+; MS (calculated) m/z: 431.34 [M]+; 1H NMR (d6-DMSO,
ppm): 12.62 (1H, br, COOH of Phe(2)); 8.06-8.04 (1H, d, NH of
Phe(2), J = 8 Hz); 7.24-7.14 (5H, m, Aromatic Hs of Phe(2));
3.06-3.04 (1H, m, CαH of Phe (2)); 3.01-3.00 (2H, m, CβHs of
Phe(2)); 2.77 - 2.83 (8H,m, CH2-Hs of Stearic Acid(1)) ; 1.9 -
2.01 (2H, m, CH2-Hs of Stearic Acid(1)); 1.07 -1.38 (20H, m, 11
CH2-Hs of Stearic Acid(1)); 0.81-0.85 (3H, m, CH3s of Stearic
Acid (1)).
Notes and references
View Article Online
DOI: 10.1039/C9NJ01763A
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View Article Online
DOI: 10.1039/C9NJ01763A
Textual Abstract
This report represents the efficiency of palmitic acid based
proteolytically stable, biocompatible hydrogelators in
sustained release of anticancer drugs.
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