Unsaturation and Polar Head Effect on Gelation, Bioactive Release, and Cr/Cu Removal Ability of Glycolipids

Article abstract of DOI:10.1021/acs.langmuir.0c00349

Designing of multifunctional soft and smart materials from natural sources is a useful strategy for producing safer chemicals having potential applications in biomedical research and pharmaceutical industries. Herein, eight glycolipids with variation in unsaturation of hydrophobic tail and polar headgroup size were designed. The effect of unsaturation in the tail group and headgroup size on gelation ability, and mechanical and thermal stability of glycolipid hydro/organogels was studied to understand structure and property relationship. Glycolipids are functional amphiphilic molecules having potential applications in the field of drug delivery and metal removal. The encapsulation capacity and kinetic release behavior of hydrophobic/hydrophilic bioactives like curcumin/riboflavin from the hydrophobic/hydrophilic pockets of glycolipids hydro/organogels was examined. A significant observation was that the glucamine moiety of the glycolipid headgroup plays a vital role in removal of Cr and Cu from oil/water biphasic systems. Typical functions of the glycolipid hydrogels are metal chelation and enzyme-triggered release behavior, enabled them as promising material for Cr, Cu removal from edible oils and controlled release of water soluble/insoluble bioactives.

Full text of DOI:10.1021/acs.langmuir.0c00349

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Article
Un-saturation and Polar Head Effect on Gelation, Bioactive
Release, and Cr/Cu Removal Ability of Glycolipids
Kanaparedu P C Sekhar, Deepak Kumar Swain, SNEHAL
Ashokrao Holey, Sreedhar Bojja, and Rati Ranjan Nayak
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.0c00349 • Publication Date (Web): 05 Mar 2020
Downloaded from pubs.acs.org on March 9, 2020
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Un-saturation and Polar Head Effect on Gelation,
Bioactive Release, and Cr/Cu Removal Ability of
Glycolipids
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Kanaparedu P. C. Sekhar, Deepak Kumar Swain, Snehal Ashokrao Holey, Bojja Sreedhar‡
†
,§
†
†,§
and Rati Ranjan Nayak*,†,§
†
‡
Centre for Lipid Science and Technology, Department of Analytical & Structural Chemistry,
CSIR-Indian Institute of Chemical Technology, Hyderabad-500 007, India
§Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India
ABSTRACT: Designing of multifunctional soft and smart materials from the natural sources is
a useful strategy for producing safer chemicals having potential applications in biomedical
research and pharmaceutical industries. Herein, eight glycolipids with variation in un-saturation
of hydrophobic tail and polar headgroup size were designed. The effect of un-saturation in tail
group and headgroup size on gelation ability, mechanical, and thermal stability of glycolipid
hydro/organogels were studied to understand structure and property relationship. Glycolipids are
functional amphiphilic molecules having potential applications in the field of drug delivery and
metal removal. The encapsulation capacity and kinetic release behavior of
hydrophobic/hydrophilic bioactives like curcumin/riboflavin from the hydrophobic/hydrophilic
pockets of glycolipids hydro/organogels was examined. A significant observation was that the
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glucamine moiety of the glycolipid headgroup plays a vital role in removal of Cr and Cu from
oil-water biphasic systems. A typical function of the hydrogels of glycolipid is metal chelation
and enzyme-triggered release behavior, enabled them as promising material for Cr, Cu removal
from edible oils and controlled release of water soluble/insoluble bioactives.
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KEYWORDS
Glycolipids, self-assembly, hydrogel, organogel, metal-ion scavengers, controlled release
INTRODUCTION
Nowadays, chemists, pharmacists, and biotechnologists are facing challenges in designing of
efficient technologies in view of global sustainability. Semi-synthetic materials derived from
natural sources have offered suitable methodologies to design safer chemicals and offering
advancement in process chemistry, biomedical research, and pharmaceutical industries.1
Amphiphiles derived from the plant sources are green alternative to ecologically-hazardous
petrochemicals. Self-assemblies of amphiphiles can fabricate soft nano-architectures by
combination of non-covalent interactions like H-bonding, hydrophobic, and π-π interactions,
etc.48 The soft materials obtained by amphiphiles with diverse functional groups exhibits
3
mechanical tunable and multi-stimuli-responsive behavior for biomedical applications.9 In the
area of soft materials, the studies of molecular gels have gained immense importance due to their
unique potential industrial applications.
12
Nature provides a lot of opportunities to prepare amphiphilic molecules from vegetable oils,
amino acids, and carbohydrates with multifunctional applications.13,14 Amphiphiles with a polar
sugar headgroup and a hydrophobic fatty acid chain can act as gelators.1517 Molecular structure
of the glycolipid gelators strongly influences the mechanical stability and gelation behavior of
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glycolipids in various solvents.16,18 Multiple hydroxyl groups bearing sugar polar head and π-
systems in the gelator enhances interactions between gelator−gelator and gelator−solvent via H-
bonding and π-π stacking.1
924
In case of glycolipids, the extent of unsaturation present in the
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hydrophobic moiety strongly affects the stability of the gel and self-assembly behavior in various
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solvents. Therefore, the systematic study on gelation behavior of sugar-based amphiphiles is
useful for strategic design of glycolipids with various applications in soft-optical devices, oil-
spill recovery, lectin binding, CO absorption, and sequestration of alkali metal ions.26
2
The organo/hydrogels of the amphiphilic molecules are capable to encapsulate
hydrophilic/hydrophobic drugs in their self-assembled 3-D networks towards controlled release
of bioactives.5,6,27 Further, hydrogels and gel emulsions showing numerous applications in
preparation of food gels based on their characteristic properties such as spooning, cutting,
mastication, and slicing.7
,2830
The release behavior of bioactive from the gel depends on the
response of amphiphilic supramolecular self-assemblies towards the external stimuli (light,
temperature, electric field, pH, and enzymes).4,8,31-33 Moreover, the supramolecular self-assembly
of amphiphiles acts as metal and dye-binding sites, they are capable to bind dyes and metal
ions.34,35 Thus, the porous and guest binding nature of amphiphilic self-assemblies can find
potential applications in water purifications by removing heavy metal ions/dyes and enabled
them as smart materials.31,36-41
Organo/hydrogels are having well-ordered structure which can be used to execute many
important functions. Previously, we prepared glycolipids with various hydrophobic tails and
headgroup geometry from the natural sources and their surface behavior, self-assembly pattern,
and surfactant applications were studied.14,42,43 Encouragingly, glycolipids have shown formation
of stable (W/O and O/W) emulsions and gel emulsions with sunflower oil and water. The formed
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gel emulsions exhibited controlled enzymatic release of bioactives from the bioactive-loaded
formulation. The design of glycolipids with variation in un-saturation and polar headgroup for
constructing gels with fascinating and novel structure is important to get its further applications.
In this work, we describe the preparation and gelation behavior of asymmetrical
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(18:XM;X=0,1,2,3) and symmetrical (18:XD;X=0,1,2,3) branched glycolipids with the saturated
and unsaturated (mono-, di-, and trienes) hydrophobic tails (Figure 1). Based on hydro/organo
gelation ability of glycolipids, encapsulation and release studies of oil/water-soluble bioactives
from the prepared gels having amide bond containing gelator molecule were studied. Later,
Cr/Cu removal capacity of the hydrogels of these N-methyl-D-glucamine containing glycolipids
from Cr/Cu containing sunflower oil solution was explored here.
EXPERIMENTAL SECTION
Materials:
Asymmetrical (18:XM;X=0,1,2,3) and symmetrical (18:XD;X=0,1,2,3) branched glycolipids
with increase in number of double bonds (0-3) in their hydrophobic tail were employed in this
study (Figure 1) and synthesized as per the method described in our previous work (Scheme 1).14
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The structural confirmation of the glycolipids under investigation was done by using their H,
13C NMR, and MS data. Pyridinium chlorochromate, copper (II) acetate monohydrate, riboflavin
(
vitamin B ), curcumin, and trypsin from porcine pancreas were purchased from Sigma Aldrich,
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USA. Solvents like n-pentane, n-hexane, cyclohexane, n-heptane, benzene, toluene, xylene,
chloroform, 1,4-dioxane, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and other
analytical grade reagents were obtained from SRL, Mumbai, India. Sunflower oil was procured
locally and used directly. Water with resistivity ~ 18.2 MΩ cm was obtained from Siemens,
Labostar 7, Milli-Q system.
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Synthesis of Glycolipid Amphiphiles.
O
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a
d
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OH
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NH2
O
NH HCl
2.
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A
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OH OH
R'=
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OH
OH OH
Scheme 1. Reagents and conditions: (a) SOCl , MeOH, rt, 45 h; (b) (COCl) , 15 °C → rt, 3 h; (c)
2
2
A, TEA, DCM 0 °C → rt, 12 h; (d) N-methyl-D-glucamine, MeOH, reflux, 24 h.
Dimethyl-L-aspartate hydrochloride (A). In a typical reaction procedure, 280 mmol of
thionyl chloride was added dropwise via addition funnel in a nitrogen atmosphere to a
suspension of 200 mmol of L-aspartic acid in methanol (150 mL) at 0 °C equipped with a
magnetic stirrer. The mixture was stirred for 45 h at room temperature and then concentrated.
The crude product was recrystallized thrice from the cold ether, and dried to give A as a white
powder.
General procedure for dimethyl-N-acyl-L-aspartate. Under the solvent-free condition,
fatty acid (60 mmol) was dissolved in neat oxalyl chloride (85 mmol) and was stirred for 3 h
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until gas evolution ceased. The excess oxalyl chloride was evaporated to get acyl chloride. To a
suspension of L-aspartic acid dimethyl ester hydrochloride (50 mmol) in 137 mL CH Cl , acyl
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chloride (60 mmol), and triethylamine (70 mmol) were added successively at 0 °C. The reaction
mixture was allowed slowly to come to the room temperature and the reaction was continued for
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2 h. The reaction was quenched by addition of saturated aqueous NaHCO and extracted thrice
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with CHCl . The combined organic layer was washed with brine, dried over anhydrous Na SO ,
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and concentrated under reduced pressure. The resulting material was purified by column
chromatography (CHCl /MeOH).
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General procedure for ammonolysis. To a 500 mL round-bottomed flask equipped with a
magnetic stirring bar, 30 mmol of Dimethyl-N-acyl-L-aspartate, 45 mmol of N-methyl-D-
glucamine, and 150 mL of methanol were added under an inert atmosphere. The reaction mixture
was heated to reflux and stirred for about 24 h. Then the solvent was removed from the reaction
mixture and the crude product was purified by chromatography on silica gel (CHCl MeOH) to
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afford the desired products.
Preparation and Characterization of Gels:
Preparation of the gel: A weighed amount of glycolipid gelator and a known volume of
solvent were taken in a vial. The gelation test was employed by heating in an oil bath ( 90 C)
and spontaneous cooling to room temperature, by vigorous shaking on Vortex Shaker (Spinix,
Tarsons) or sometimes both heating and shaking. In case of water immiscible solvents a minute
quantity of water is added to dissolve the gelator before solvent addition to the vial containing
gelator.
Gel melting temperature (T ): A homeopathy vial having gel sample was placed in a
gel
temperature controlled oil bath at 25 C and the temperature of the bath was increased stepwise.
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The visual observation of gel flowing inside the vial was noted per every degree rise in
temperature. Thermal stability of the gels was again determined by using a DSC 6000
calorimeter (Perkin Elmer). A 20 mg of gel sample was kept in a closed standard aluminum pan
at 25 °C and heated at a rate of 10 °C/min.
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Rheological properties: Rheology experiments were performed by using Bohlin Instruments
(C-VOR), UK, at 1 mm gap distance with parallel-plate geometry (20 mm diameter). Initially,
the frequency sweep experiments from 1 to 100 Hz were performed at constant 1 % strain. Later,
the strain sweep experiments from 1 % strain were executed at a constant frequency of 1 Hz.
Microscopy Studies: The HRTEM studies were performed on Talos 200X high resolution
transmission electron microscope. In sample preparation; firstly, the diluted gel sample was drop
casted on a carbon coated 400 mesh copper grids. After 30 sec excess material was removed and
grids were stained with 1% uranyl acetate solution. Before recording micrographs, the grids were
air dried. A Hitachi S3000N microscope was used to perform SEM measurements. Before going
to study gel morphology by SEM, the gel sample was prepared in xerogel form by using freeze
drying method. For SEM studies, prior to imaging the sample was sputtered with metal (gold).
Encapsulation and Release of Bioactive Compounds:
Solid dispersion method was employed for encapsulation of bioactives like curcumin and
riboflavin (vitamin B ). To the freshly prepared glycolipid gel, a known quantity of bioactive
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compound was added directly and then vigorously shaken for 1 min. The bioactive molecule was
continuously added to the gel until the phase separation was observed and by considering the
weight of the gelator, encapsulation capacity of bioactive (mg/mg) was determined. In order to
perform enzymatic release study, trypsin (1 mg/mg of gelator) was added to a bioactive loaded
formulation and shaken vigorously. At 37 °C, the kinetic release behavior of the bioactive from
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formulation was analyzed by measuring absorption at 550 nm on Lambda 35, Perkin-Elmer,
UV/visible spectrophotometer.
Metal Removal Protocol:
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Stock solutions of pyridinium chlorochromate or copper (II) acetate monohydrate were
prepared in methanol. Excess of chromium/copper stock solution was mixed in sunflower oil and
the methanol was evaporated to dryness by passing N gas. The resultant solution was stirred for
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h and centrifuged (5000 rpm) for 30 minutes at room temperature. To 1 mL of hydrogel at
MGC, 1 mL of metal ion containing oil solution was added. The resultant mixture was stirred for
h and centrifuged (5000 rpm) for 30 minutes at room temperature. The oil phase was subjected
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for atomic absorption spectrometer, ZEEnit 700 P, Analytik Jena, Germany, to determine the
percentage of metal removal before and after treatment.
RESULTS AND DISCUSSION
To understand the affect of un-saturation in hydrophobic chain and polar headgroup size on the
gelation behavior of glycolipid amphiphiles and their applications, systematic studies were
performed on the glycolipids amphiphiles shown in Figure 1. The glycolipids presented in Figure
1
are classified into two series: (i) asymmetrical branched glycolipids (18:XM) and (ii)
symmetrical branched glycolipids (18:XD). In each series, the number of carbon atoms present
in fatty acid of glycolipids is same, i.e. C fatty acids (stearic acid (octadecanoic acid), oleic acid
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((9Z)-octadec-9-enoicacid), linoleic acid ((9Z,12Z)-octadeca-9,12-dienoic acid), and γ-linolenic
acid ((6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid)). Further, the extent of un-saturation in
hydrophobic moiety of the glycolipids is increased with increase in number of double bonds (0-
3
) in their hydrophobic tail. These eight asymmetrical and symmetrical branched glycolipids are
used to explain the outcomes in two different ways. First, influence of headgroup size on
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gelation behavior at a fixed hydrophobic chain. Later, affect of un-saturation in hydrophobic
moiety on gelation behavior at a fixed headgroup size. Herein, the outcomes were discussed in
three parts: (i) gelation behavior, (ii) bioactive encapsulation and release, and (iii) metal-ion
scavenging.
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OH OH
OH OH
O
N
O
N
O
OH
O
OH
OH OH
OH OH
OH OH
R
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H
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H
OH
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OH OH
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18:XD, X=0,1,2,3
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18:1D
4
18:3D
Figure 1. Chemical structures of the glycolipids.
Gelation Behavior. The gelation ability of the glycolipids was examined in aliphatic
hydrocarbons, aromatic hydrocarbons, polar organic solvents, and water. Initially, glycolipids are
poorly soluble in water immiscible organic solvents like n-pentane, n-hexane, cyclohexane, n-
heptane, benzene, toluene, xylene, and chloroform even after heating near to their boiling points.
However, glycolipids at their maximum solubility in minimum volume of water exhibited semi
solid like material upon simple mixing. Further, the gelation ability of glycolipids in water
immiscible organic solvents shown in Table 1 were examined in presence of water (80 L/mg of
glycolipids) by simple mixing at room temperature. The gelation ability test of glycolipids in
remaining polar solvents was done by heating-cooling method; the mixture was heated near to
the solvent boiling point.
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2
2
2
2
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Table 1. Gelation behavior of glycolipids in various organic solvents and water.
Behavior
Solvent
18:0M 18:1M 18:2M 18:3M 18:0D 18:1D 18:2D 18:3D
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
n-Pentanea
n-Hexanea
Cyclohexanea
n-Heptanea
Benzenea
I
I
G
G
G
G
E
E
E
E
S
G
G
G
G
G
G
G
E
S
E
E
E
E
E
E
E
E
S
S
S
S
S
E
E
E
E
E
E
E
E
S
S
S
S
S
G
G
G
G
E
E
E
E
S
G
G
G
G
E
E
E
E
S
I
E
I
I
I
E
E
E
E
E
S
S
S
S
G
I
Toluenea
I
Xylenea
I
Chloroforma
E
S
S
S
S
G
1
,4-Dioxaneb
Acetonitrileb
DMFb
S
S
S
S
S
S
S
S
DMSOb
Waterb
S
S
S
S
S
S
S
S
G: gel; E: emulsion; I: insoluble; S: soluble. The gelation test was performed by gradual addition
of solvent in steps to 1 mg of glycolipid with heating-cooling or agitation-rest method (solvent
a
volume varied between 0.045 mL to 4.3 mL). in presence of water (80 L/mg of glycolipids).
b
heating-cooling method.
The results obtained from the gelation test of the glycolipids in various non-polar and polar
solvents were presented in Table 1 and the maximum solvent used for each gel is presented in
supporting information Table S1. From Table 1, the gelation ability of glycolipids synthesized
with stearic acid and oleic acid (18:0 and 18:1) exhibited headgroup dependence. The less
hydrophilic asymmetrical branched glycolipids (18:0M and 18:1M) showed the hydrogel
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formation. Whereas the corresponding symmetrical branched glycolipids (18:0D and 18:1D)
are soluble in water due to their more hydrophilic nature. The gelation ability of glycolipids
synthesized with linoleic acid and γ-linolenic acid (18:2 and 18:3) exhibited almost similar type
of gelation behavior irrespective of size of the headgroup. In case of aromatic hydrocarbons,
glycolipid 18:3M exhibited formation of gel and its corresponding symmetrical branched
glycolipid (18:3D) exhibited formation of emulsions.
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0
An increase in degree of un-saturation of glycolipids induced excellent biocompatibility in the
4
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biological environments. Chemically, the un-saturated chains possesses strong interactions with
water molecules than the saturated chains and the water molecules will be localized at
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unsaturated site. As a result, the fluidity of membrane was increased with an increase in degree
46
of un-saturation. In the present study, the gelation behavior of the asymmetrical (18:XM) and
symmetrical (18:XD) branched glycolipids exhibited strong dependence on the degree un-
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saturation of the hydrophobic moiety of the glycolipids. In 18:XM glycolipid series, the
saturated (18:0M) and mono-ene (18:1M) form hydrogels, whereas, di- and tri-enes (18:2M and
1
8:3M) form organogels in presence of water (Table 1).In case of 18:XD glycolipid series, the
saturated (18:0D) and mono-ene (18:1D) do not form gels, whereas, di- and tri-enes (18:2D and
8:3D) form organogels in aliphatic solvents in the presence of water (Table 1). Overall, the
1
increase in un-saturation of hydrophobic tail of the glycolipids favored the formation of gel in
hydrocarbon solvents with small quantity of water. Herein, added water increase the fluidity of
network structures of the glycolipids with high degree of un-saturation (18:2M, 18:2D, 18:3M
and 18:3D) and induced the gelation in hydrocarbon solvents.
Glycolipids 18:2M, 18:3M, 18:2D, and 18:3D exhibited water induced spontaneous gelation
in hydrocarbon solvents like n-pentane, n-hexane, cyclohexane, and n-heptane (Table 1). In
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absence of water gelation was not noticed for the glycolipids in hydrocarbon solvents and the
glycolipid remained in solid state even after heating. Upon solubilizing the glycolipid in small
volume of water, high viscous and semisolid like material was noticed just by gentle agitation
with hydrocarbon solvents. Herein, the H-bondings between glycolipid molecules were induced
by addition of water and favored the formation of supramolecular self-assemblies.47
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Figure 2. The plot of maximum volume of solvent gelled per mg of glycolipid by glycolipid (i)
1
8:2M, (ii) 18:3M, (iii) 18:2D, and (iv) 18:3D as a function of volume of water.
To understand the effect of water volume on gelation of hydrocarbon solvent, maximum
volume of solvent gelled per mg of glycolipid was measured and presented in Figure 2. From
Figure 2, for all glycolipids (18:2M, 18:3M, 18:2D, and 18:3D) and hydrocarbon solvents (n-
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pentane, n-hexane, cyclohexane, and n-heptane) the maximum volume of solvent gelled by the
glycolipid increased with an increase in water volume and reaching maximum. Except glycolipid
18:2D (18:2M, 18:3M, and 18:3D), the increasing order of maximum volume of hydrocarbon
solvent gelled with respect to the solvent is n-heptane  cyclohexane  n-hexane  n-pentane
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(for glycolipid 18:2D, cyclohexane  n-heptane  n-hexane  n-pentane). All glycolipids
exhibited maximum solvent entrapment in n-pentane, a short chain hydrocarbon solvent. While
comparing the results with respect to the glycolipid, the increasing order of maximum volume of
hydrocarbon solvent gelled is 18:2M18:3M 18:2D18:3D. Hence, with increase in size of
headgroup and degree of un-saturation in the hydrophobic tail increased the volume of solvent
gelled per mg of glycolipid.
Even though glycolipids exhibit good gelation ability in n-pentane, n-pentane is volatile
(boiling point, 36.1 C) and hence difficult to handle for the further studies. For further studies,
n-hexane, the next best gelling solvent was taken into the consideration. Hereafter, gel samples
are labeled using their glycolipid gelator and solvent gelled, for example, hydrogel of glycolipid
1
8:0M with water as 18:0M-W and organogel of glycolipid 18:2M with n-hexane as 18:2M-H.
Two hydrogels (18:0M-W and 18:1M-W) and four organogels (18:2M-H, 18:3M-H, 18:2D-H,
and 18:3D-H) of the glycolipids (Figure S2) were subjected for the further studies. The hydrogel
of saturated asymmetrical branched glycolipid (18:0M-W) produced translucent gel and a
transparent hydrogel was observed for the corresponding mono-ene (18:1M-W). All organogels
(
18:2M-H, 18:3M-H, 18:2D-H, and 18:3D-H) formed in hexane were translucent (Figure S2).
Gelation properties of glycolipids hydro/organogels like minimum gelation concentration
MGC), gelation number (GN), maximum independent frequency values (f), critical strain (c),
and T are presented in Table 2.
(
gel
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The gelation ability of the gelator can be quantitatively expressed in terms of MGC (minimum
quantity (mg) of gelator required per mL of the solvent). From Table 2, The MGC values of are
less in organogels as compared to those of hydrogels. While comparing the MGC values of
organogels of asymmetrical branched glycolipids (18:2M-H and 18:3M-H) with symmetrical
branched glycolipids (18:2D-H and 18:3D-H), in the later series the values are low compared
with the earlier series. These results shown an increase in headgroup size decreased the MGC
values and increased the gelation ability. Gelation number (GN, moles of solvent gelled per one
mole of glycolipid) is another important parameter to describe the gelation ability of the
glycolipid. The effect of water volume on GN of hydrocarbon solvent by the glycolipid
amphiphiles were calculated and presented in Figure S1. The GN at MGC of the gels 18:0M-W,
0
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1
8:1M-W, 18:2M-H, 18:3M-H, 18:2D-H, and 18:3D-H were presented in Table 2 and they
exhibited similar gelation capacity as observed in case of MGC. The organogels of symmetrical
branched glycolipids exhibited good gelation ability with high GN values.
Table 2. Gelation properties of glycolipids hydro/organogels.
MGC
mg/mL)
18:0M-W 15.38
Tgel (C)
TI
Gel
GN
f (Hz)
c (Pa)
(
DSC
2133
12.2 ± 0.3
5.1 ± 0.2
16.2 ± 0.5
9.2 ± 0.3
14.2 ± 0.6
8.2 ± 0.3
4.4 ± 0.1
83 ± 1
52 ± 1
81 ± 1
79 ± 1
80 ± 1
78 ± 1
84.8 ± 0.4
53.7 ± 0.2
83.3 ± 0.5
80.3 ± 0.2
82.7 ± 0.4
79.9 ± 0.3
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8:1M-W 22.22
8:2M-H 0.44
1472
519.3 ± 20.2
36.5 ± 0.8
22.2 ± 0.4
23.9 ± 0.5
15.7 ± 1.1
10258
10667
21659
20464
18:3M-H 0.42
1
1
8:2D-H 0.26
8:3D-H 0.28
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The rheology experiments were performed to determine the mechanical strength of gels, and
the obtained results were presented in Figure 3 (i. Frequency sweep, ii & iii. Strain sweep). In the
typical frequency sweep experiment at constant strain, the tolerance to the external pressure can
be determined by measuring the maximum frequency keeping constant storage modulus (G′) and
loss modulus (G″) values. The maximum independent frequency values (f) of the glycolipid gels
from Figure 3i were presented in Table 2. From Table 2, the tolerance to the external force of
hydrogels (18:0M-W and 18:1M-W) decreased with an increase in un-saturation in the
hydrophobic tail. Similarly, organogels (18:2M-H, 18:3M-H, 18:2D-H, and 18:3D-H) also
exhibited decrease in tolerance to the external force with an increase in un-saturation in the
hydrophobic tail. While comparing headgroup effect at fixed hydrophobic tail (18:2M-H/18:2D-
H and 18:3M-H/18:3D-H), the tolerance to the external force decreased with increase in
headgroup size of the glycolipid.
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Figure 3. (i) Variation of moduli (G and G) with frequency of gels at 1% strain. (ii & iii)
Variation of moduli (G and G) with strain of gels at 1Hz frequency. (iv) DSC heating runs of
gels.
Later, the strain sweep experiments at constant frequency were performed to obtain
oscillatory shear strain data of the glycolipid gels (Figure 3ii&iii). Above the critical strain (c)
value the gel starts to flow by breaking the gel networks, and the break points obtained from
Figure 3ii & 3iii are tabulated in Table 2. The  values almost followed the similar trend as
c
observed in frequency sweep experiment (Table 2). Surprisingly, the hydrogel 18:1M-W has
higher  value (519.3 %) than the rest of others. Thus, the thermoreversible hydrogel 18:1M-W
c
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shows high fracture toughness.48 Figure S3 shows how 18:1M-W gel undergoes tensile
elongation process. Further, gel samples were subjected to heat to determine the thermal
stability.
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Tube inversion (TI) method was employed to determine thermal stability of the glycolipid
hydro/organogels by measuring Tgel value at MGC and obtained values are listed in Table 2.
From Table 2, the thermal stability of hydrogels (18:0M-W and 18:1M-W) decreased with an
increase in un-saturation in the hydrophobic tail. Similarly, organogels (18:2M-H, 18:3M-H,
1
8:2D-H, and 18:3D-H) also exhibited small decrease in Tgel value with an increase in un-
saturation in the hydrophobic tail. While comparing headgroup effect at fixed hydrophobic tail
(18:2M-H/18:2D-H and 18:3M-H/18:3D-H), the Tgel value decreased with an increase in
headgroup size of the glycolipid. Upon heating, gel samples produced clear solutions by
complete breaking of the gel. Upon cooling, hydrogels (18:0M-W and 18:1M-W) exhibited
thermo reversibility, whereas organogels (18:2M-H, 18:3M-H, 18:2D-H, and 18:3D-H) did not
exhibit gelation either by cooling or agitation. Later, the DSC experiments were performed to
determine thermal stability of the glycolipid gels (Figure 3iv). The Tgel values obtained from
Figure 3iv are tabulated in Table 2. From Table 2, the Tgel values obtained from the DSC
experiments followed the similar trend as observed in TI method and almost in a similar range
(Table 2). In Figure 3iv, the additional endothermic peak of 18:0M-Wat 47 C is due to
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transformation of translucent gel to transparent gel. Finally, prepared hydro/organogels are
stable above 37 °C and may useful for gel-based delivery systems.
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Figure 4. (i) TEM and (ii) SEM images of gels.
In order to understand the morphology of gels, we performed TEM studies of the dried gel.
Figure 4i exhibits TEM images of the supramolecular structures in the gels produced by the
glycolipids. All the glycolipids reveal formation of fibrous aggregates. The pictures demonstrate
that the fibers are elongated for several micro meters. Further, to get better understanding on the
surface morphology of supramolecular structures produced by the glycolipid gelators SEM
studies were carried out on the freeze dried xerogels. Similar to TEM study, glycolipids form
long and continuous fibers in SEM imaging (Figure 4ii). However, the more extensive entangled
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fibrillar networks is a consequence of high thermal stability. Specially, the organogels 18:2M-
H, 18:3M-H, 18:2D-H, and 18:3D-H as shown in Table 2 (T ~80 C), which indicates efficient
gel
packing of glycolipid molecules in the fibers.
Bioactive Encapsulation and Release. The self-assembled network structures of the
hydro/organogels contain hydrophobic/hydrophilic pockets within the gel and they can be
utilized to encapsulate hydrophobic/hydrophilic bioactives towards controlled release of
bioactives.27,50 For encapsulation studies 1 mL of each hydro/organogel was prepared at MGC
concentration (Table S2). Hydrophobic/hydrophilic bioactives like curcumin/ riboflavin were
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encapsulated in glycolipid hydro/organogels by simple solid dispersion method at room-
temperature (25 °C). Encapsulation of bioactives at room-temperature has an advantage to
encapsulate thermo-degradable bioactives.
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Figure 5. (i) Photograph of vials showing bioactive loaded hydro/organogels (1. 18:0M-W, 2.
1
8:1M-W, 3. 18:2M-H, 4. 18:3M-H, 5. 18:2D-H and 6. 18:3D-H). (ii) Time-dependent
enzymatic release of bioactive from bioactive loaded hydro/organogels.
The bioactive loaded glycolipid hydro/organogels were shown in Figure 5i and the
encapsulation efficiencies were tabulated in Table 3. As presented in Table 3, the encapsulation
capacity of hydrogels (18:0M-W and 18:1M-W) increased with an increase in un-saturation in
the hydrophobic tail. The encapsulation capacity of organogels (18:2M-H, 18:3M-H, 18:2D-H,
and 18:3D-H) decreased with an increase in un-saturation in the hydrophobic tail. While
comparing headgroup effect at fixed hydrophobic tail (18:2M-H/18:2D-H and 18:3M-H/18:3D-
H), the encapsulation capacity increased with an increase in headgroup size of the glycolipid.
Hence, the hydrophilicity of the glycolipid increased the loading ability of hydrophilic bioactive
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(
riboflavin). Later, we focused on enzymatic controlled release of bioactives from bioactive
loaded gel formulations.
Table 3. Encapsulation Capacity of Bioactive Compounds in Gels.
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Gel
18:0M-W 18:1M-W 18:2M-H 18:3M-H 18:2D-H 18:3D-H
Curcumin Riboflavin
Bioactive
Loading capacity* 0.32 ±0.02 0.47±0.04 1.51±0.08 0.54±0.02 1.72±0.07 0.72±0.03
*
mg/mg of gelator
Controlled drug-delivery applications of supramolecular self-assemblies by external
5
1
stimulienabled them an intelligent drug carrier system. The glycolipids shown in Figure 1 are
two (18:XM) or three (18:XD) amide linkages in their chemical structure. Amidases like trypsin
can destabilize the gel by cleaving amide bond present in the glycolipids. After enzymatic
hydrolysis of glycolipid, resultant degradation products are fatty acid, aspartic acid and
glucamine. This leads to the loss of amphiphilic activity and gelation ability of the glycolipids.
Thus, the bioactives entrapped inside hydrophobic/hydrophilic pockets of the gel get released.51
The saturated solution of trypsin was prepared in water and added to the glycolipid formulations
containing the bioactive at a concentration of 1 mg of trypsin per mg of glycolipid. Upon
addition of enzyme, the mixture was mixed vigorously and subjected for kinetic
52
spectrophotometric study at 37 °C. Herein, bioactive loaded gels are not 100% transparent, the
time dependent decrease in absorbance of the gel samples after addition of trypsin is a measure
of controlled delivery behavior. Hence, we determined the kinetic absorbance at 550 nm with
reference to solvent (water/hexane), where the glycolipids and bioactive (curcumin/riboflavin)
have zero absorbance. The time dependent absorbance of bioactive loaded hydro/organogels at
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50 nm was shown in Figure S4. The release percentage (R) of the bioactive was calculated
(Supporting information Equation S1) and the results obtained from the enzymatic release
studies were shown in Figure 5ii.
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As presented in Figure 5ii, the release rate of curcumin from hydrogels (18:0M-W and
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8:1M-W) increased with un-saturation in the hydrophobic tail. Similarly, the release rate of
riboflavin from organogels (18:2M-H, 18:3M-H, 18:2D-H, and 18:3D-H) increased with an
increase in un-saturation in the hydrophobic tail. While comparing headgroup effect at fixed
hydrophobic tail (18:2M-H/18:2D-H and 18:3M-H/18:3D-H), the release rate of riboflavin
from organogels increased with an increase in headgroup size of the glycolipid. Hence, the
hydrophilicity of the glycolipid and water content of the gel increased the release rate of
bioactive by trypsin which is a water-soluble enzyme.
Figure 6. Metal removal from oil by using hydrogels. (1, Cr containing oil; 2,/5, Cr containing
oil and 18:0M-W,/18:1M-W; 3 & 6, After stirring and centrifugation; 4 & 7, Oil after treatment.
9
, Cu containing oil; 10,/13, Cu containing oil and 18:0M-W,/18:1M-W; 11&14, After stirring
and centrifugation;12 & 15, Oil after treatment.
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Metal-Ion Scavenging. Hydrogels are interesting compounds to remove heavy metal ions/dyes
on the way to purification of water.31,36-41 Chromium and copper are heavy metal ions and widely
used for various industrials applications. Removal of chromium from water by glycolipids was
shown in our previous work and sugar based hydrogels can remove copper from water by
absorption phenomenon.40,42 In oil refining process, there is a lot of possibility for the corrosion
of oil with metallic components like chromium and copper. Hence, in the present study, we
attempted to remove chromium and copper from sunflower oil by using glycolipid hydrogels.
The metal removal process by using hydrogels was presented in Figure 6. Obtained results
after treatment of chromium and copper containing oil with glycolipid hydrogels (18:0M-W and
18:1M-W) were presented in Table S3. A parallel experiment was performed with water in place
of hydrogel under the similar experimental conditions to understand the role of water in metal
removal from oil (Table S3). From Table S3, pure water exhibited insignificant effect on metal
removal under similar experimental conditions. The interfacial tension between the water and oil
reduces the contact between the water and metal ions even after agitation of sample, leads to
easy phase separation with small time duration. As shown in Table S3, the glycolipid present in
the hydrogel successfully interacted with the metal ions present in oil and successfully reduced
the metal ion concentration in the oil phase. Glycolipids exhibited more efficiency in removal of
chromium over copper. Further, the metal removal capacity of hydrogels (18:0M-W and 18:1M-
W) from oil decreased with an increase in un-saturation in the hydrophobic tail.
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CONCLUSION
Gelation behavior of eight glycolipids with increase in un-saturation of hydrophobic tail and
polar headgroup size were studied. The gelation ability was examined in different nonpolar and
polar solvents by heating-cooling and agitation-rest methods. Glycolipids with small headgroup
size and less un-saturation in hydrophobic tail (18:0M and 18:1M) formed hydrogels by heating-
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cooling. At high un-saturation in hydrophobic tail (18:2M, 18:3M, 18:2D and 18:3D),
glycolipids exhibited water-induced organogelation by simple agitation. The organogelation
ability of glycolipids was increased with an increase in polar headgroup size. The mechanical
stability of glycolipids hydro/organogels decreased with an increase in un-saturation and
headgroup size. Surprisingly, the hydrogel 18:1M-W exhibited high fracture toughness with c
value (519.3 %). Further, the Tgel values of glycolipids organogels decreased slightly with an
increase in un-saturation in hydrophobic tail and headgroup size. Additionally, glycolipids
hydro/organogels are capable to encapsulate hydrophobic/hydrophilic bioactives like curcumin/
riboflavin and the encapsulation capacity increased with an increase in headgroup size of the
glycolipid. The obtained gel formulation shown controlled release of bioactives by an enzyme,
and the rate of bioactive release was increased with an increase in un-saturation in the
hydrophobic tail and headgroup size. Further, the glycolipid hydrogels exhibited chromium and
copper removal ability from the vegetable oil and the metal removal capacity decreased with an
increase in un-saturation in the hydrophobic tail. Finally, the outcomes on gelation behavior of
glycolipids with un-saturation in hydrophobic tail and variation in polar headgroup size are
useful in designing gel based delivery systems and metal removal in vegetable oil industry.
AUTHOR INFORMATION
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Corresponding Author
*E-mail: rrnayak@iict.res.in
ACKNOWLEDGMENTS
This work was supported financially by the project entitled “Vegetable oil based gels as trans-
free fat (oleogel) (MLP 0045) sponsored by Council of Scientific and Industrial Research
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(
CSIR)”.Authors acknowledge Director, CSIR-IICT (Manuscript Communication Number:
IICT/Pubs./2019/275) for providing all the required facilities to carry out the work. D.K.S.
acknowledges Department of Science and Technology, India, for Inspire Fellowship.
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ASSOCIATED CONTENT
Supporting Information
Spectral characterization of the glycolipids, gelation number, photographs of gels, tensile
elongation, gel composition, volume of solvent used for gelation, kinetic absorption study and
metal removal capacity of the glycolipid gels are available.
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