J Surfact Deterg (2018)

DOI 10.1002/jsde.12033

ORIGINAL ARTICLE

Physicochemical Characterization of Chrysin-Derivative-Loaded

Nanostructured Lipid Carriers with Special Reference to
Anticancer Activity
Gourab Karmakar1 · Prasant Nahak1 · Priyam Chettri2 · Biplab Roy1 · Pritam Guha1 · Koji Tsuchiya3 ·
Kanjiro Torigoe3 · Anoop Kumar2 · Ranendu K. Nath4 · Sukhen Bhowmik4 · Utpal C. De4 ·
Kaushik Nag5 · Amiya K. Panda6

Received: 25 April 2017 / Revised: 22 November 2017 / Accepted: 27 December 2017

© 2018 AOCS

Abstract Homologues long-chain chrysin derivatives
(LCD, Cn: 8–18) were synthesized and incorporated into
nanostructured lipid carriers (NLC) with the aim to treat
human neuroblastoma. Mutual miscibility and attractive
interactions among the NLC components, namely tripalmi-
tin (TP), cetyl palmitate (CP), oleic acid (OA), and the
chrysin (CHR) derivatives (LCD) at the air–water interface
were assessed by the Langmuir monolayer approach. Opti-
mum combination for the NLC formulations was found to
be 2:2:1 (M/M/M) for TP/CP/OA, respectively. NLC for-
mulations, both in the absence and presence of LCD, were
characterized by combined dynamic light scattering, elec-
tron microscopy, atomic force microscopy, and differential
scanning calorimetry. The size and zeta potential of the
NLC formulations were found in the range 200–350 nm
and −12 to −18 mV, respectively. Encapsulation efficiency
and release kinetics of CHR and LCD when loaded into
NLC were also evaluated. LCD exhibited maximum incor-
poration, drug-loading capacity, and sustained release
because of its enhanced hydrophobicity. Superior incorpo-
ration efficiency and sustained-release profile of LCD were
able to enhance their anticancer activity against human neu-
roblastoma cell lines, compared to CHR, making them
promising agents in combating cancer.
Keywords NLC
CHR
LCD
Drug loading
Drug
release
Neuroblastoma cell
J Surfact Deterg (2018).

Electronic supplementary material The online version of this article Introduction

(doi:10.1002/jsde.12033) contains supplementary material, which is
available to authorized users. Development of new drugs alone is not sufficient to
improve therapeutic efficacy (Das & Chaudhury, 2011;
* Amiya K. Panda Jenning & Gohla, 2000; Jenning, Thunemann, & Gohla,
akpanda@mail.vidyasagar.ac.in 2000; Yadav, Khatak, & Sara, 2013). One complicating
1
factor is insufficient bioavailability of certain drugs (Yadav
Department of Chemistry, University of North Bengal, Darjeeling
734013, West Bengal, India et al., 2013). In order to overcome those problems, nano-
2 structured lipid carriers (NLC) emerged in the beginning of
Department of Biotechnology, University of North Bengal,
Darjeeling 734013, West Bengal, India the 1990s (Müller, 2007; Müller, Radtke, & Wissing,
3 2002a, 2002b; Müller, Rűhl, & Runge, 1996). They are
Department of Pure and Applied Chemistry, Tokyo University of
Science, 2641 Yamazaki, Noda, Tokyo 278-8510, Japan also known as modified solid lipid nanoparticles (SLN).
4
Department of Chemistry, Tripura University, Suryamaninagar,
They are nanocolloidal suspensions in the size range
Agartala 799022, Tripura, India 10–1000 nm (Karmakar et al., 2016; Nahak et al., 2015;
5
Department of Biochemistry, Memorial University of Sapkota et al., 2015). The major difference between SLN
Newfoundland, St. John’s, Newfoundland and Labrador, Canada and NLC lies in the choice of the lipid blends. While sym-
6
Department of Chemistry and Chemical Technology, Vidyasagar metric hydrocarbon-chain-containing lipid components are
University, Midnapore 721102, West Bengal, India used for the preparation of SLN, two or more structurally

J Surfact Deterg (2018)

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3`
2`
8 1 Dry K2CO3, RBr
HO O RO O
1` 5` Dry acetone, reflux
7 6`
4 3
5 R = CH3(CH2)7 - (octyl)
CH3(CH2)9 - (decyl)
OH O OH O
CH3(CH2)15 - (hexadecyl)
CH3(CH2)17 - (octadecyl) LCDs of Chrysin
Chrysin (1)
2a: R = CH3(CH2)7 -
2b: CH3(CH2)9 -
2c: CH3(CH2)15 -
2d: CH3(CH2)17 -

Scheme 1 Synthesis of C-7(O) modified derivatives (LCD) of CHR

different lipids (solid and/or liquid) are used for NLC. NLC
have superiority over the other drug delivery systems such
as liposomes, nanoemulsions, microemulsions, nanocap-
sules, nanosponges, and polymeric nanoparticles
(Karmakar et al., 2016; Nahak et al., 2015; Sapkota et al.,
2015; Yadav et al., 2013). Superior pharmaceutical loading
capacity, possibility of specific targeting, and sustained-
release characteristics over other drug delivery systems
make NLC superior. Additionally, NLC are capable of pro-
viding chemical stability and steadiness of the labile incor-
porated pharmaceutical. In spite of the mentioned
advantages, NLC formulations suffer from serious limita-
tions (Das & Chaudhury, 2011; Müller, 2007; Müller et al.,
1996, 2002a, 2002b), such as aggregation during storage,
which lead to stability problems (Karmakar et al., 2016;
Nahak et al., 2015). Therefore, the formulation of stable
NLC by judiciously choosing lipidic components with suit-
able compositions is a challenging task.
Usually, biocompatible solid lipids such as wax, phos-
pholipid, monoglyceride, diglyceride, and triglyceride in
combination with fatty acid (FA) are used in preparing
NLC (Karmakar et al., 2016; Müller, 2007; Müller et al.,
1996, 2002a, 2002b; Nahak et al., 2015; Ranpise, Kor-
abu, & Ghodake, 2014; Sapkota et al., 2015). However,
NLC formulations comprising cetyl palmitate (CP), tripal-
mitin (TP), and oleic acid (OA) have not yet been explored,
to the best of our knowledge. Use of a suitable lipid com-
position by exploring the nature of the interaction among
the lipid components and FA are also important in develop-
ing stable NLC formulations. However, detailed investiga-
tions of lipid interactions in formulating stable NLC
systems are lacking, or fragmented where available
(Karmakar et al., 2016).
Chrysin (CHR) (5,7-dihydroxy flavone), as a natural fla-
vonoid, is found in many wild and edible plants, honey,
and propolis (Sinha, Joshi, Srivastava, & Govil, 2014;
Zhang et al., 2004). It is known to have anti-inflammatory,
antioxidant, and anticancer activities. Some recent studies
have proven that it can markedly decrease the malondialde-
hyde level and elevate antioxidant enzyme activity
(Aishwarya, Surekha, & Sumathi, 2015; Bilia, Isacchi,
Righeschi, Clizia, & Bergonzi, 2014; Fonseca et al., 2015;
Sinha et al., 2014; Wang, Fan, Li, & Wang, 2015; Zhang
et al., 2004; Zheng, Cao, Meng, & Qing, 2003; Zheng,
Meng, Xu, Cao, & Qing, 2003). However, the low bio-
availability of CHR limits its use as a potential drug.
Because of its amphiphilic nature, CHR have unsatisfactory
loading and the encapsulation efficiency (Aishwarya et al.,
2015; Bilia et al., 2014). In order to overcome the afore-
mentioned limitations, efforts are being made to synthesize
CHR derivatives with long hydrocarbon chains so that the
lipid solubility and encapsulation efficacy of these deriva-
tives could be enhanced (Zheng, Meng, et al., 2003). In the
present study, 7-O-modified four long-chain hydrocarbon
CHR derivatives, namely octyl (C8), decyl (C10), hexadecyl
(C16), and octadecyl (C18), were synthesized (Scheme 1).
CHR and its derivatives were incorporated into NLC for-
mulation for evaluating the effectiveness of long-chain
chrysin derivatives (LCD) over the parent compound
(CHR) from the view point of drug incorporation, drug
loading (DL), and the sustained-release behavior.
In the present work, NLC formulations with CP, TP,
and OA in combination with CHR and its LCD were
assessed for mutual miscibility among the components
by Langmuir monolayer studies with the aim to develop
such NLC formulations. The prepared NLC formula-
tion, in the absence and presence of CHR and LCD,
were then characterized for size, polydispersity index
(PDI), and zeta potential (ZP) analysis by dynamic light
scattering (DLS). Differential scanning calorimetry
(DSC) was employed for the evaluation of the thermal
properties of the NLC formulations and determination

J Surfact Deterg (2018)

J Surfact Deterg
of the thermodynamic parameters, namely the phase
transition temperature (Tm), enthalpy change of the
phase transition (ΔH), and the heat capacity changes
(ΔCp), as well as the peak width of the chain melting
at half-maximum (ΔT1/2). Morphology of the NLC was
studied by transmission electron microscopy (TEM),
freeze-fractured transmission electron microscopy (FF-
TEM), and atomic force microscopy (AFM). CHR and
LCD were incorporated in the optimized NLC formula-
tion, and the drug-loaded NLC formulations were sub-
jected to physicochemical characterization. CHR- and
LCD-loaded formulations were also studied with respect
to entrapment efficiency (EE), DL capacity, and release
kinetics. For the evaluation of their biological activity,
CHR- and the LCD-loaded formulations were subjected
to in vitro cytotoxicity studies through MTT-based
assay against the human neuroblastoma cell line
SHSY5Y. We believe that such a combined set of stud-
ies would eventually help in formulating a suitable drug
delivery system in the treatment of cancer.
Experimental
Materials and Synthesis
CP, TP, OA, and CHR were purchased from Sigma-Aldrich
Chemicals (St. Louis, MO,USA). AR-grade Tween® 60 was
procured from Sisco Research Laboratory (Mumbai, India).
All the chemicals were obtained as received and stated to be
>99.5% pure. HPLC-grade solvents and double-distilled
water with specific conductance of 2–4 μS (at 25  C) were
used in preparing the solutions.
General Method for the Synthesis of CHR
(1) Derivatives (2a–d)
To a solution of 0.1 CHR in 10 mL of anhydrous acetone
(93 mM) was added alkyl bromide (0.076 g, 0.394 mM)
and anhydrous K2CO3 (0.0815 g, 59.1 mM). The mixture
was refluxed for 24 h until the starting material disap-
peared, as shown by thin-layer chromatography (TLC). The
reaction mixture was filtered, and the filtrate was dried
under reduced pressure in a rotary evaporator. The solid
residue was then suspended in water and extracted three
times with ethyl acetate (3 × 10 mL). The ethyl acetate
extracts were mixed together, dried over Na2SO4, and con-
centrated by distillation under reduced pressure. The resi-
due thus obtained was subjected to silica gel column
chromatography and eluted with petroleum ether/ethyl ace-
tate in the ratio 1:4 to obtain the desired product in
pure form.
J Surfact Deterg (2018)
Characterization of the Derivatives
LCD, after purification through silica-gel column chroma-
tography, were characterized by physicochemical methods
such as melting point and solubility as well as spectro-
scopic studies such as Fourier transform infrared (FTIR),
1
H, and 13C NMR spectroscopy, the results of which are as
follows:
2a: Yield 98%, yellow solid, soluble in CHCl3, molecu-
lar weight 366.18 corresponding to the molecular formula
C23H26O4, mp. 105  C. IR (ν cm−1): 3457 (hydroxyl),
1662 (α,β-unsaturated carbonyl), 1620, 1590, 816 (aro-
matic), 2961, 2927, 2856 (C H). 1H NMR (CDCl3,
400 MHz): δ 12.70 (1H, s, 5-OH), 7.89 and 7.86 (2H, d,
J = 2.8 Hz, C-20 , C-60 ), 7.51 (3H, m, C-30 , C-40 , C-50 ), 6.65
(1H, s, H-3), 6.65 (1H, d, J = 2.8 Hz, H-6), 6.35 (1H, d,
J = 2.8 Hz, H-8), 4.02 (2H, t, J = 8.8 Hz, OCH2),
1.30–1.87 (12H, m, 6 × CH2) and 0.89 (3H, t,
J = 9.2 Hz, CH3); 13C NMR (CDCl3, 100 MHz): δ 182.4
(C-4), 165.2 (C-7), 163.9 (C-3), 162 (C-5), 157.8 (C-9),
131.8 (C-10 ), 131.3 (C-40 ), 129 (C-30 , C-50 ), 126.3 (C-20 , C-
60 ), 105.8 (C-3), 105.5 (C-10), 98.6 (C-6), 93.0 (C-8), 68.7
( OCH2), 22.6–31.8 (6 × CH2), and 14.05 ( CH3).
2b: Yield 97%, yellow solid, soluble in CHCl3, molecu-
lar weight 394.21 corresponding to the molecular formula
C25H30O4, mp. 90  C. IR (ν cm−1): 3415 (hydroxyl), 1666
(α,β-unsaturated carbonyl), 1624, 1580, 829 (aromatic),
2960, 2919, 2852 (C H). 1H NMR (CDCl3, 400 MHz): δ
12.80 (5-OH), 7.89 (2H, C-20 , C-60 ), 7.55 (3H, m, C-30 , C-
40 , C-50 ), 6.68 (1H, s, H-3), 6.47 (1H, brs, H-6), 6.31 (1H,
brs, H-8), 3.70 (2H, brs, OCH2), 0.86–1.56 (16H, m,
8 × CH2) and 0.83 (3H,m, CH3); 13C NMR (CDCl3,
100 MHz): δ 182.4 (C-4), 165.2 (C-7), 163.9 (C-3),
162 (C-5), 157.7 (C-9), 131.8 (C-10 ), 131.3 (C-40 ), 129 (C-
30 , C-50 ), 126.3 (C-20 , C-60 ), 105.7 (C-3), 105.5 (C-10),
98.6 (C-6), 93.0 (C-8), 68.7 ( OCH2), 22.6–31.9
(8 × CH2), and 14.06 ( CH3).
2c: Yield 97%, yellow solid, soluble in CHCl3, molecu-
lar weight 478.30 corresponding to the molecular formula
C31H42O4, mp. 80  C. IR (ν cm−1): 3409 (hydroxyl), 1666
(α,β-unsaturated carbonyl), 1615, 1586, 825 (aromatic),
2940, 2918, 2850 (C H). 1H NMR (CDCl3, 400 MHz): δ
12.70 (5-OH), 7.90 (2H, C-20 , C-60 ), 7.55 (3H, m, C-30 , C-
40 , C-50 ), 6.67 (1H, s, H-3), 6.50 (1H, brs, H-6), 6.36 (1H,
brs, H-8), 4.05 (2H, brs, OCH2), 0.88–1.82 (28H, m,
14 × CH2) and 0.86 (3H, brs, CH3); 13C NMR (CDCl3,
100 MHz): δ 182.5 (C-4), 165.2 (C-7), 163.9 (C-3),
162 (C-5), 157.7 (C-9), 131.8 (C-10 ), 131.3 (C-40 ), 129 (C-
30 , C-50 ), 126.3 (C-20 , C-60 ), 105.7 (C-3, C-10), 98.6 (C-6),
93.0 (C-8), 68.7 ( OCH2), 22.6–31.9 (14 × CH2), and
14.06 ( CH3).
2d: Yield 96%, yellow solid, soluble in CHCl3, molecu-
lar weight 506.33 corresponding to the molecular formula

C33H46O4, mp. 97  C. IR (ν cm−1): 3453 (hydroxyl), 1662
(α,β-unsaturated carbonyl), 1615, 1586, 820 (aromatic),
2919, 2856 (C H); 1H NMR (CDCl3, 400 MHz): δ 12.70
(5-OH), 7.90 (2H, C-20 , C-60 ), 7.54 (3H, m, C-30 , C-40 , C-
50 ), 6.67 (1H, s, H-3), 6.50 (1H, d, J = 2.8 Hz, H-6), 6.36
(1H, d, J = 2.8 Hz, H-8), 4.05 (2H, t, J = 8.8 Hz, OCH2),
1.26–1.82 (32H, m, 16 × CH2) and 0.85 (3H, t,
J = 8.8 Hz, CH3); 13C NMR (CDCl3, 100 MHz): δ 182.4
(C-4), 165.2 (C-7), 163.9 (C-3), 162 (C-5), 157.7 (C-9),
131.8 (C-10 ), 131.3 (C-40 ), 129 (C-30 , C-50 ), 126.3 (C-20 , C-
60 ), 105.7 (C-3), 105.5 (C-10), 98.6 (C-6), 93.0 (C-8), 68.7
( OCH2), 22.6–31.9 (16 × CH2), and 14.06 ( CH3).
Solubility of CHR and LCD in the Physical Mixture of
Lipids
Ten milligrams each of CHR and LCD was taken sepa-
rately in different test tubes. The physical mixture of the
lipids CP, TP, and OA in the molar ratio 2:2:1 was heated
above their melting points to obtain a lipid melt, which was
then gradually added to the test tubes containing CHR and
LCD with constant stirring using a cyclomixer. The
amounts of molten lipid required to solubilize the CHR and
LCD were noted. The observed ratios of lipid/CHR and
lipid/LCD were found to be approximately 100:1 and 50:1,
respectively.
Preparation of NLC
NLC were prepared using hot homogenization followed by
ultrasonication according to our previous works (Karmakar
et al., 2016; Nahak et al., 2015; Sapkota et al., 2015). In
the NLC formulation, CP and TP were used in equimolar
ratio, whereas the quantity of OA was varied from 10 to
30 mol% in steps of 10 mol%. The overall concentration of
NLC formulations was fixed at 1 mM, while 2 mM aque-
ous Tween® 60 solution was used as the dispersion
medium for testing the NLC formulation. OA formulations
comprising 20 mol% of OA were used for the incorpora-
tion of CHR and LCD.
Instrumentation
Surface pressure versus area (π–A) isotherms of the lipids
in combination with CHR/LCD were measured on a Lang-
muir surface balance (Micro Trough X, Kibron, Finland).
Monolayers were generated by spreading quantitative vol-
umes of 1 mM solution of the lipid mixture dissolved in a
chloroform/methanol mixed solvent system (3:1, v/v) at the
air–liquid interface with a Hamilton (Sigma-Aldrich)
microsyringe. The solvent was allowed to evaporate for
20 min, after which the film was compressed at a barrier
J Surfact Deterg
speed of 5 mm min−1. The surface pressure versus area iso-
therms were constructed with a 2 mM aqueous Tween®
60 solution as the subphase. The hydrodynamic diameter
(dh), PDI, and the ZP of the NLC formulations were deter-
mined by a DLS spectrophotometer (Nano ZS 90, Malvern,
UK). Conventional TEM and FF-TEM studies were made
by H-600 and H-7650 (Hitachi Science Systems Ltd.,
Tokyo, Japan), respectively. Surface topology and three-
dimensional analyses were performed by an AFM instru-
ment (Bruker Nanoscope V Multimode SPM, Santa Bar-
bara, CA, USA). DSC studies of the base and drug-loaded
NLC were carried out using a DSC apparatus (Mettler
Toledo, Switzerland) with a scan rate of 2  C min−1. A 40-
μL aluminum pan was used for the study. An identical pan
containing the dispersion medium (2 mM aqueous Tween®
60) was used as the reference.
EE and DL Capacity Studies
The centrifugation method was adopted for the evaluation
of the EE and the corresponding loading capacity. CHR-
and LCD-loaded NLC formulations were subjected to cen-
trifugation for 25 min at 20,000 rpm at 4  C to separate the
lipid phase containing the drug from the dispersion
medium. The supernatant was collected and analyzed color-
imetrically for the estimation of the free drug. EE and DL
were calculated as (Karmakar et al., 2016; Nahak et al.,
2015; Sapkota et al., 2015) follows:
Wtotal CHR − Wfree CHR
EE% = × 100% ð1Þ
Wtotal CHR
Wtotal CHR − Wfree CHR
DL% = × 100% ð2Þ
Wtotal CHR − Wfree CHR + Wtotal lipid
where Wtotal CHR, Wfree CHR, and Wtotal lipid represent the
total amount of drug, free drug present in the dispersion
medium, and the total amount of lipid in the NLC formula-
tion, respectively.
In Vitro Release Kinetics Studies
The standard dialysis bag (MWCO 12 kDa) method was
adopted for the evaluation of the release kinetics of the
incorporated drugs (Karmakar et al., 2016; Koirala et al.,
2016; Nahak et al., 2015; Sapkota et al., 2015). Aqueous
Tween® 60 (2 mmol) solution was used as the release
medium. Ten milliliters of the NLC formulation was sus-
pended inside the dialysis bag surrounded by 20 mL of the
release medium. The released CHR and the LCD were
quantified colorimetrically.
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In Vitro Cell Viability Test
Cytotoxicity of CHR and LCD loaded in the NLC formula-
tions on the human neuroblastoma cancer cell line
(SHSY5Y, obtained from National Center for Cell Science,
Pune, India) was determined by the MTT assay (Koirala
et al., 2016). The cells (5 × 103 well−1) were plated in
200 μL Ham’s F 12 medium (Thermo Fisher Scientific,
Waltham, MA, US) per well in a 96-well plate and incu-
bated in an incubator containing 5% CO2 for 24 h. After
incubation, 10 μL of the studied NLC formulations were
added. The experiments were performed with three differ-
ent drug concentrations and repeated three times. After
treatment, the medium was replaced with the MTT solution
(10 μL of 5 mg mL−1 per well) prepared in PBS and incu-
bated further for 3 h at 37  C in a humidified incubator
with 5% CO2. Then, 50 μL of isopropanol was added to
each well, the plates were gently shaken for 1 min, and
absorbance was measured at 595 nm by a microtiter plate.
The effect of CHR- and LCD-loaded NLC formulations on
the studied cell lines was quantified by calculating the per-
centage of cytotoxicity (Koirala et al., 2016), as
%cytotoxicity
Abs: for the control cell − Abs: for the treated cell
= × 100%
Abs: for the control cell
ð3Þ
The minimum concentration of the drug required for
50% inhibition of the treated cell is called the inhibitory
concentration (IC50) of the drug. The IC50 values of CHR
and LCD in the NLC formulation were also determined
separately.
Results and Discussion
Langmuir Monolayer Studies
Mutual miscibility and interaction between the lipid mix-
tures with CHR/LCD were assessed by Langmuir surface
pressure versus area measurements. Representative surface
pressure versus area isotherms of LCD in water and in
2 mM Tween® 60 solution are shown in Fig. 1a. The lift-
off area (A0) for CHR and its derivatives C8 (CHR 8), C10
(CHR 10), C16 (CHR 16), and C18 (CHR 18) appeared at
0.64, 0.66, 0.61, 0.66, and 0.63 nm2 per molecule, respec-
tively, in water. In the case of Tween® 60 as the subphase,
the observed lift-off area for CHR, CHR 8, CHR 10, CHR
16, and CHR 18 appeared at 0.68, 0.73, 0.71, 0.75, and
0.74 nm2 per molecule, respectively. The surface pressure
versus area isotherms for CP, TP, and OA using water and
aqueous (2 mM) Tween® 60 as subphase are shown in
Fig. S1 (Supporting Information). The A0 values for CP,
J Surfact Deterg (2018)
Fig. 1 (a) Surface pressure (π) versus area (A) isotherm of CHR
10 (blue) and CHR 16 (red) using water (dotted line) and 2 mM aque-
ous Tween® 60 solution (solid line) as subphase. (b) π versus A iso-
therms of the mixed lipidic system (CP + TP + OA, 2:2:1, M/M/M) in
the absence (black) and presence of 50 mol% CHR 10 (blue) and
CHR 16 (red) using 2 mM aqueous Tween® 60 as subphase. Temper-
ature 25  C
TP, and OA were found to be 0.4, 0.63, and 0.5 nm2 per
molecule, respectively, in water. These values were found
in good to be agreement with the literature values (Mao
et al., 2013; Zdravkova & Eerden, 2007). In aqueous
Tween® 60 as the subphase, the A0 values were upshifted
to 0.42, 0.76, and 0.6 nm2 per molecule for CP, TP, and
OA, respectively. The surface activity of Tween® 60 was
mainly responsible for the increase in A0 values (Karmakar
et al., 2016).
In the case of the mixed lipidic system (CP + TP + OA),
an equimolar mixture of CP and TP was considered as
component 1 and OA as component 2. For component
1, the A0 value appeared at 0.6 nm2 per molecule. The addi-
tion of OA caused a downshift in the A0 values but it was
not systematic (Karmakar et al., 2016), suggesting attrac-
tive interaction. In order to understand the impact of CHR
and LCD on the lipid mixture (CP + TP + OA, 2:2:1, M/M/
M), π–A isotherms were constructed by considering the
lipid mixtures as component 1 and CHR/LCD as the com-
ponent 2, as shown in Fig. 1b, representatively. For both
CHR and LCD, the isotherms did not change significantly.
In order to evaluate extent of association and miscibility
among the components, the excess area (Aex) and changes
in the excess free energy of mixing (ΔG0ex ) were calculated.
The mean molecular area (Aid) for a mixed monolayer can
be calculated as (Guha et al., 2015; Karmakar et al., 2016;
Panda, Nag, Harbottle, Possmayer, & Petersen, 2004,
Panda, 2007):
Aid = x1 A1 + x2 A2 ð4Þ
where x and A represent the mole fraction and area of the
pure component, respectively. Aex was calculated as (Guha
et al., 2015; Karmakar et al., 2016; Panda et al.,
2004, 2007):

Aex = A12 − Aid
where A12 is the experimentally obtained mean molecular
area. Changes in the excess free energy of mixing were cal-
culated using the following expression (Guha et al., 2015;
Karmakar et al., 2016; Panda et al., 2004, 2007):
ðπ
ΔG0ex = ½A− ðx1 A1 + x2 A2 Þdπ
0
The obtained Aex and ΔG0ex values, as shown in Fig. 2,
were found to be dependent on the composition of the
mixture.
Interactions among the lipidic components were found to
be associative in nature. The observed minima in the Aex
and ΔG0ex versus mol% of OA profiles at 20 mol% OA
indicated maximum association and the stability of that
lipid composition, respectively (Figs. 2a, b) (Karmakar
et al., 2016). Further increase in the mol% of OA reduced
the extent of association and ultimately showed a repulsive
type of interaction at 80 mol%. Hence systems with
20 mol% OA were considered to impart maximum stabil-
ity, and therefore NLC formulations were made using
20 mol% OA in combination with an equimolar ratio of the
other two components, TP and CP. To get a quantitative
idea of the nature of the interaction and the extent of misci-
bility of CHR and LCD with the lipid system, Aex and
ΔG0ex values were calculated and plotted against the mol%
of CHR and the LCD (Figs. 3a, b). The Aex and ΔG0ex data
at 30 mN m−1 pressure are presented as representative.
Positive Aex and ΔGex values for CHR and lipid mixture
indicated a repulsive interaction. The presence of the
hydroxyl group at the 5- and 7-positions of CHR makes the
molecule amphiphilic and restricts the interaction with the
hydrophobic part of the lipidic system, which causes the
repulsive interaction between CHR and lipid mixtures. On
the contrary, negative Aex and ΔG0ex values in case of LCD
indicate the association among the components. The extent
of association increased with increasing hydrocarbon chain
Fig. 2 Variation of the excess molecular area (Aex) and changes in the
excess free energy (ΔG0ex ) of mixing with the mol% OA (a and b,
respectively). Component 1: CP + TP (1:1, M/M), component 2:
OA. Surface pressure for (a) and (b) (mN m−1): ○, 5; Δ, 10; □, 15;
r, 20; ◊, 25; , 30. Temperature: 25  C
J Surfact Deterg
ð5Þ
ð6Þ
Fig. 3 Variation of the excess molecular area (Aex) and changes in the
excess free energy (ΔG0ex ) of mixing with the mol% CHR and LCD
(a and b, respectively). Component 1: CP + TP + OA (2:2:1, M/M/
M), component 2: CHR and LCD. Surface pressure for (a) and
(b) (mN m−1): ○, CHR; Δ, CHR 8; □, CHR 10; r, CHR 16; ◊,
CHR 18. Temperature: 25  C
length of the LCD, which could be rationalized on the basis
of increased hydrophobicity of the derivatives (Karmakar
et al., 2016). The observed results give a clear indication of
the enhanced miscibility of the LCD than pure CHR itself
with the lipids.
The elastic moduli (Cs− 1 ) of the studied monolayers were
calculated as (Guha et al., 2015; Karmakar et al., 2016;
Panda et al., 2004, 2007):
 
−1 dπ
Cs = − A ð7Þ
dA T
Representative Cs− 1 versus the percentage of the com-
pressed area (%A) profiles are shown in Fig. S2. The Cs− 1
values passed through a maximum (in the range
41–67 mN m−1) (Guha et al., 2015; Karmakar et al., 2016).
In the case of 20 mol% OA, the Cs− 1 value was found to be
the maximum (67 mN m−1). The Cs− 1 value was compara-
tively low for CHR than its derivatives. The hydrocarbon
chain present in the LCD provided higher hydrophobic
attraction and assisted in the formation of a condensed
monolayer (Guha et al., 2015). In the present set of studies,
CP and TP were used as solid lipids and OA as the liquid
FA. The rationale behind the use of CP, TP, and OA with
structural dissimilarity was to create multicrystallinity in
the NLC formulation. The ratio of the lipids and the
amount of added FA were decided through comprehensive
investigation of their mutual interaction in the Langmuir
monolayer experiment. The optimized ratio of CP, TP, and
OA was found to be 2:2:1. To ensure the mutual miscibility
of the lipid components and the LCD, lipids were taken
after considering the similarity in the length of the hydro-
carbon chain with that of synthesized LCD.
DLS Studies
The hydrodynamic diameter (dh), PDI, and ZP are the rep-
resentative markers of NLC formulations (Karmakar et al.,
J Surfact Deterg (2018)
J Surfact Deterg
Fig. 4 Variation of dh, PDI, and ZP with time for 1 mM NLC formu-
lation (CP + TP + OA, 2:2:1, M/M/M) stabilized by 2 mM aqueous
Tween® 60 solution in the absence and presence of CHR and LCD at
25  C. Individual systems are mentioned in the figure. [CHR] and
[LCD]: 10 μM
2016; Müller, 2007; Müller et al., 2002a; Nahak et al.,
2015; Sapkota et al., 2015). The size of the NLC formula-
tion was in the range 200–350 nm, which is comparable
with the previously published values (Karmakar et al.,
2016; Nahak et al., 2015; Sapkota et al., 2015). The dh ver-
sus time profiles of some representative NLC formulations
are shown in Fig. 4. NLC were found to be stable for
90 days with no significant change in the dh value during
storage. OA (mol%) was varied from 10 to 30 mol% to
tune the rigidity and fluidity of the lipid matrix. NLC for-
mulation (without CHR or its derivatives) passed through a
minimum for systems comprising 20 mol% OA (data not
shown). The reduced dh value indicated the saturation limit
of OA in the NLC matrix, as also evidenced in case of the
mixed monolayer studies described earlier. Condensed
molecular packing at this composition was the main reason
for the observed size constriction (Karmakar et al., 2016).
For this reason, further formulations with CHR and its
derivatives were made using 20 mol% OA. The size of the
drug-loaded NLC formulations was in the range
300–350 nm, which was higher than that of the blank
NLC, indicating the incorporation of the drug into the
NLC. The stabilities of CHR- and LCD-loaded NLC were
also studied, which are shown in Fig. 4. The size of the
LCD-loaded NLC was found to decrease with increasing
hydrocarbon chain length of the LCD, which indicated an
important role of the hydrocarbon chains of the LCD in the
NLC matrix (Nahak et al., 2015; Sapkota et al., 2015). This
kind of size reduction might be due to the higher hydropho-
bicity of the LCD, which resulted in better organization of
the lipid systems inside the NLC. The observed associative
J Surfact Deterg (2018)
interaction in the Langmuir monolayer studies of the LCD
and the mixed lipidic system also corroborated this proposi-
tion. However, the presence of two hydroxyl groups at the
5- and 7-positions of CHR (1) makes the molecule amphi-
philic in nature and restricts its penetration inside, but a
shell-enriched NLC system was formed, resulting in size
enhancement of CHR-loaded NCL formulations. The larger
size of the CHR-loaded systems was due to repulsive inter-
action observed in the Langmuir monolayer studies. This
observation indicated the preferential surface accumulation
of CHR on the NCL, which was responsible for the size
enhancement. The formation of the shell-enriched structure
was also justified from the repulsive interaction of the pure
CHR and the mixed lipid system in the monolayer studies.
PDI is another marker in estimating the homogeneity of
colloidal dispersions. The PDI values of the studied NLC for-
mulations were found to in the range 0.2–0.3. The observed
PDI indicated the homogeneous dispersion of the lipid phase
in the dispersion medium during NLC preparation. The for-
mation of the homogeneous dispersion also signifies the
higher stability of the solution phase of the studied NLC sys-
tems (Das & Chaudhury, 2011; Müller, 2007; Müller et al.,
2002a, 2002b). The insignificant change in the PDI value
upon drug incorporation indicates that the drug molecules
did not change the surface homogeneity of NLC. Variation
in the PDI value with respect to the storage time is shown in
the middle panel of Fig. 4 (Sapkota et al., 2015).
ZP is another parameter for assessing the stability of col-
loids (Sapkota et al., 2015). The ZP values were found to lie
in the range −12 to −18 mV, which is not sufficiently high to
impart stability to the colloidal particles. However, the
enhanced stability of the NLC formulations, even with low
ZP values, could be rationalized on the basis of steric stabili-
zation assisted by the oxyethylene groups of Tween®
60 (Karmakar et al., 2016; Nahak et al., 2015; Sapkota et al.,
2015). The decrease in ZP with time (upper panel, Fig. 4)
was due to the desorption of FA from the NLC matrix, which
subsequently formed mixed micelles with Tween® 60. In
case of 20 mol% OA, no significant change in the magnitude
of ZP was found, indicating less desorption of FA. CHR
reduced the ZP, suggesting its accumulation onto the NLC
surface, which eventually masked the ZP.
In the case of LCD, further reduction in ZP values was
due to the increased lipophilicity of the LCD in the pres-
ence of the long hydrocarbon chain, which also suppressed
the dissociation of OA. The addition of the hydrocarbon
chain changes the pH of CHR because it is related to the
dissociation of the hydroxyl groups present in the LCD.
The presence of a long hydrocarbon chain enhances the
lipophilicity and consequently suppresses the dissociation
of the hydroxyl groups. Hence, the surface accumulation of
LCD was found to reduce the surface charge as well as ZP
of the LCD-loaded NLC formulations. CHR 18, having the

Fig. 5 TEM (a and b) and FF-TEM (c and d) images for NLC
(CP + TP + OA, 2:2:1, M/M/M) formulations in the absence (a and c)
and presence (b and d) of CHR 16
longest hydrocarbon chain among the synthesized deriva-
tives, was found to reduce the ZP value of the NLC formu-
lation to a considerable extent. The ZP versus time profiles
for the CHR/LCD loaded formulations were similar to that
of the base NLC formulations.
Morphology of the NLC Formulations
Morphological investigations on the NLC formulations
were carried out by combined TEM (both conventional and
freeze-fractured) and AFM. A spherical morphology of the
NLC systems with a smooth surface was observed, as
revealed from these studies (Fig. 5).
The observed size of the NLC formulations was in good
agreement with the DLS data. Insignificant change in the
surface morphology upon the incorporation of CHR and its
derivatives suggests their incorporation into the core of the
lipid matrices. Results of the AFM studies, shown in Fig. 5,
further established the spherical morphology with a smooth
surface. Figs. 6a, b present the two-dimensional and the
three-dimensional overview of the NLC, respectively. The
observed bright spots represent the NLC. The height of the
bright region in AFM analysis was in the range 40–45 nm
(Fig. 6c), which is in agreement with the reported values
(Hu et al., 2005; Muhlen, Muhlen, Niehus, & Mehnert,
1996; Shahgaldian, Quattrocchi, Gualbert, Coleman, &
Goreloff, 2003). No significant change in either the surface
morphology or topology was observed with the incorpora-
tion of CHR and LCD in the AFM studies.
J Surfact Deterg
Fig. 6 AFM image of NLC (CP + TP + OA, 2:2:1, M/M/M) formula-
tion where (a) and (b) represent two- and three-dimensional view,
respectively. (c) Roughness profile of the NLC formulation. Height
scale is given inside the figure. Scan area: 1 × 1 μm2
DSC Studies
To evaluate the extent of polymorphism and assess the
extent of crystallinity of the NLC formulation, DSC studies
were performed. The obtained results were correlated with
the encapsulation efficiency, release profile, and stability of
the NLC (Guha et al., 2015; Karmakar et al., 2016; Koirala
et al., 2016; Nahak et al., 2015; Sapkota et al., 2015;
Zdravkova & Eerden, 2007). DSC thermograms of the indi-
vidual lipids (CP, TP, and OA), CHR, and the synthesized
LCD were also recorded separately. The phase transition
temperatures (Tm) of the pure lipidic components were in
good agreement with the literature values (MacNaughtan,
Farhat, Himawan, Starov, & Stapley, 2006; Ruktanonchai
et al., 2008; Shaikh et al., 2004). The Tm value of CHR
was obtained as 286.07 and 282.8  C during heating and
cooling, respectively. The phase transition temperature of
CHR 8, CHR 10, CHR 16, and CHR 18 were 100.8, 91.80,
88.50, and 93.83  C, respectively, during the heating pro-
cess. The Tm values during cooling always were 2–3  C
lower temperature compared to those during heating, sug-
gesting their liquid-crystalline nature (Spicer, 2003, 2005a,
2005b). The Tm values of the NLC formulations were sub-
stantially lower than those of their physical mixtures, which
was mainly due to the enhanced polycrystallinity and
reduction in size with simultaneous increase in the surface
area (Karmakar et al., 2016; Nahak et al., 2015; Sapkota
et al., 2015). The cooling thermograms were found to be
more prominent than the heating thermograms, as shown in
Fig. S3. For this reason, the cooling thermograms were
considered for further calculation of the thermodynamic
parameters such as the full width at half-maximum (ΔT1/2),
change in enthalpy (ΔH), and specific heat capacity (ΔCp)
of the phase transition (Karmakar et al., 2016; Spicer,
2003, 2005a, 2005b). Fig. 7a shows the thermodynamic
parameters of the NLC formulation having 10, 20, and
J Surfact Deterg (2018)
J Surfact Deterg
Fig. 7 (a) Change in the thermodynamic parameters for NLC formu-
lations in the presence of different mol% of OA. (b) Change in the
thermodynamic parameters for the NLC (CP + TP + OA, 2:2:1, M/M/
M) formulation with the incorporation of CHR and LCD. The systems
are mentioned in the figure
30 mol% of OA. The Tm values associated with cooling of
the NLC formulations appeared at 22.3, 23.62, and 21.4  C
for NLC formulations containing 10, 20, and 30 mol% OA,
respectively. The Tm value for the formulation with
20 mol% OA was higher than those with 10 and 30 mol%,
suggesting formation of the compact molecular aggregates.
The ΔH and ΔCp values show a maximum at 20 mol%
OA. For 30 mol% OA, reduction in ΔH and ΔCp, indicates
enhanced polycrystallinity of the NLC. The observed
reduction in the ΔT1/2 value with increasing amount of OA
up to 20 mol% was indicative of the compact molecular
arrangement. Beyond 20 mol%, broadening of the thermo-
grams indicates the reduction in the crystallinity of the
NLC formulation. The result further supports the conclu-
sion that 20 mol% OA was the optimum in formulating the
NLC, as was also observed from the Langmuir monolayer
and DLS studies.
In order to obtain a quantitative idea on the crystallinity
of the NLC, the percentage crystallinity index (CI%) was
calculated as (Nahak et al., 2015; Sapkota et al., 2015)
ΔHNLC
CI% = × 100% ð8Þ
ΔHlipid mixture
where ΔHNLC and ΔHlipid mixture are the enthalpy of phase
transition of NLC and the corresponding lipid physical
mixture, respectively. The CI% versus OA mol% profile is
presented in Fig. S4a. The highest CI% was observed for
the system comprised of 20 mol% OA, which is in
good agreement with the calculated thermodynamic param-
eter for the NLC formulations comprising different
mol% of OA.
J Surfact Deterg (2018)
Incorporation of CHR and/or its derivatives did not sig-
nificantly alter the thermograms of the NLC; also, no sepa-
rate peak for CHR and LCD appeared. This suggests
complete adsolubilization of CHR and LCD into the core
of the NLC. However, an overall reduction in the Tm values
was observed upon the incorporation of CHR or LCD,
which indicates the enhancement in the microcrystallinity.
The Tm value for CHR was 21.32  C, whereas those for
CHR 8-, CHR 10-, CHR 16-, and CHR 18-loaded NLC
formulations were found to be 21.11, 22.07, 22.46, and
22.5  C, respectively. The progressive increase in the Tm
value with increasing hydrocarbon chain length of the LCD
suggests its enhanced association among the lipidic compo-
nents. The maximum value for the CHR 18-loaded NLC
formulation is attributed to the maximum association. The
similarity in the hydrocarbon chain of CHR 18 and OA also
influences the compact molecular arrangement (Karmakar
et al., 2016). Repulsive-type interaction between pure CHR
and the lipid made the system less organized, resulting in a
decrease in the ΔH values for CHR-loaded NLC. The pro-
gressive increase of ΔH and ΔCp with increasing hydrocar-
bon chain length of the CHR derivatives resulted in the
formation of compact molecular aggregates. The enhanced
hydrophobic interaction and the van der Waals attraction
with the increasing hydrocarbon chain length of the deriva-
tive assisted in the formation of a well-organized liquid-
crystalline system (Karmakar et al., 2016; Nahak et al.,
2015; Sapkota et al., 2015).The CI% values for NLC
loaded with CHR and its derivatives are graphically shown
in Fig. S4b. A greater increase in the CI% was noted for
LCD-loaded NLC than CHR. Progressive enhancement in
CI% was also noted with increasing hydrocarbon chain
length of the incorporated LCD. The enhanced molecular
association led to systematic molecular organization in the
lipid aggregated systems.
Drug Incorporation Efficiency and the DL Capacity
Studies
Drug incorporation (EE) efficiency and DL capacity are
important parameters for the development of a successful
drug-delivery system (Karmakar et al., 2016; Müller, 2007;
Müller et al., 2002a; Nahak et al., 2015; Ranpise et al.,
2014; Sapkota et al., 2015). In the present study, the incor-
poration efficiency and the loading capacity of CHR- and
LCD-loaded NLC were evaluated, as shown in Fig. 8a. EE
and DL capacity (Fig. 8a) for pure CHR were found to be
59 and 0.52%, respectively. The EE values for CHR 8-,
CHR 10-, CHR 16-, and CHR 18-loaded NLC were
75, 75.6, 89.2, and 92.5%, respectively. The corresponding
DL values were 1.15, 1.23, 1.70, and 1.85%, respectively.
The enhanced lipophilicity of the CHR derivatives led to

Fig. 8 EE%, DL% capacity (a), and release profile (b) of CHR and
different LCD form NLC formulation at 25  C. : ○, CHR; Δ, CHR 8;
□, CHR 10; r, CHR 16; ◊, CHR 18
EE and DL capacity, which also restrict the drug expulsion
during the preparation and storage (Sapkota et al., 2015).
In Vitro Release Kinetics Studies
The release profiles of CHR and individual LCD (CHR
8, CHR 10, CHR 16, and CHR 18) from NLC formulations
were studied using 2 mM aqueous Tween® 60 solution as
the release medium. The release profiles of CHR and the
LCD that have been studied are shown in Fig. 8b. Simple
diffusion of CHR and LCD through the dialysis membrane
in the Tween® 60 solution was also carried out as control
(data not shown). The release of CHR and LCD from the
NLC formulation was compared with the simple diffusion
of CHR and LCD through the dialysis membrane to elimi-
nate the effect of the dialysis membrane on the release
kinetics. The formulated NLC were found to sustain the
release processes. CHR and LCD followed almost similar
release profiles up to the time of 100 h. Faster release was
recorded for CHR, and relatively sustained release was
observed for the synthesized LCD. With increasing chain
length of the LCD, the release rate decreased. The lesser
miscibility and lipophilicity of CHR led to its faster release
from the NLC formulation. However, for the LCD,
enhanced lipophilicity and thus miscibility with the lipidic
aggregates took control and sustained the release process.
CHR 18, having the longest hydrocarbon chain, exhibited
the lowest release rate. With increasing hydrophobicity of
the LCD, the hydrophobic interaction with the lipid compo-
nents was augmented, resulting in the accumulation of the
loaded LCD into the NLC core. Thus, the release of the
LCD reduced with the progressive increase in the hydro-
phobicity of the LCD with the increase of the hydrocarbon
chain.
The obtained release profiles were analyzed with five dif-
ferent release models, namely the first-order, zero-order,
Korsemeyer–Peppas, Higuchi, and Weibull, with the help
of add-in software (DD Solver 0.1, Yong Zhang el. al.,
Nanjing, China) (Dash, Murthy, Nath, & Chowdhury,
J Surfact Deterg
2010; Karmakar et al., 2016; Nahak et al., 2015; Sapkota
et al., 2015; Zhang et al., 2010). The obtained release
kinetic data are presented in Table S1. All the systems fol-
lowed the Korsemeyer–Peppas release formalism (based on
the observed maximum R2 value). The calculated release
rate according to the Korsemeyer–Peppas formalism for
CHR was found to be 15.88 h−1. The rate constants were
found to be 10.28, 8.73, 4.52, and 2.07 h−1 for CHR
8, CHR 10, CHR 16, and CHR 18, respectively. The
release exponents (n) were in the range 0.4–0.5, suggesting
the classical Fick’s release formalism for CHR and
the LCD.
Anticancer Activity of the CHR- and LCD-Loaded
NLC Formulations
In order to assess the anticancer efficacy of CHR- and
LCD-loaded NLC, cytotoxicity studies were carried out by
measuring the percentage cell inhibition using the MTT
assay. Human neuroblastoma cell lines (SHSY5Y) were
used for the evaluation of the anticancer efficacy of the
studied formulations (Koirala et al., 2016). In the present
study, NLC without any active pharmaceutical component
was taken as control for the relative evaluation of the cyto-
toxicity of CHR- and LCD-loaded NLC formulations. The
cytotoxicity of the blank NLC formulation was separately
determined considering the dispersion medium as control,
and it was found to be 15% at the higher concentration.
Hence, the studied NLC was found to be almost nontoxic
in comparison to CHR- and LCD-loaded NLC toward the
human neuroblastoma cell line. The percentage of cell inhi-
bition was determined with three different concentrations
(5, 7.5, and 10 μM) of CHR and LCD. Although the work
is preliminary in nature regarding the anticancer activity of
the studied formulations, indications of cytotoxicity suggest
potential treatment systems. The percentages of cell inhibi-
tion (% cytotoxicity) obtained for different formulations at
three studied concentrations are presented graphically in
Fig. 9. The free drug was not separated from the NLC. In
the present work, a comparative evaluation of cytotoxicity
of CHR and LCD in the free form as well as NLC loaded
with CHR and LCD was performed. All the studied formu-
lations were found to be more cytotoxic than CHR and
LCD used alone against the studied cell line (data not
shown). Among the studied formulations, cytotoxicity was
higher for LCD. With increasing concentration of CHR and
LCD in the NLC formulation, an increase of the cell inhibi-
tion percentage was recorded. The calculated IC50 values
for CHR-, CHR 8-, CHR 10-, CHR 16-, and CHR 18-
loaded NLC were found to be 7.78, 6.51, 6.19, 5.83, and
5.58 μM, respectively. Hence, the presence of a long
hydrocarbon chain in the CHR enhanced the cytotoxicity
when loaded into NLC. The presence of the long
J Surfact Deterg (2018)
J Surfact Deterg
hydrocarbon chain in the LCD helped them to undergo
compact packing among the lipidic constituents inside the
NLC. Thus, LCD-loaded NLC formulations with smaller
size and higher effective surface area were achieved.
Because of the higher DL capacity, sustained release of the
loaded drug, and the reduced size of LCD-loaded NLC,
cytotoxicity was effectively enhanced. The reduced size of
the formulation enhanced the cell penetration of the LCD-
loaded NLC and the enhanced hydrophobicity of the LCD
effectively, increasing the cellular uptake (Nahak et al.,
2016). Further studies with different cancer cell lines and
in vivo evaluation of the anticancer efficacy of CHR and
LCD-loaded NLC formulations are warranted.
Conclusions
With an aim to formulate NLC using CP, TP, and OA in
combination with CHR and LCD, the interfacial miscibility
of the components was assessed through surface pressure
versus area measurements. The amphiphilic nature of CHR
lead to a repulsive-type interaction with the lipid system,
whereas the LCD showed as associative type of interaction
with the extent of association increasing with hydrophobic-
ity, i.e., increasing hydrocarbon chain length of the CHR
derivatives. CHR and LCD were successfully incorporated
into the NLC formulation comprising CP, TP, and OA with
the molar ratio of 2:2:1 using a hot homogenization fol-
lowed by ultrasonication. Base as well as CHR- and LCD-
loaded NLC formulations were characterized by a combina-
tion of DLS, DSC, TEM, FF-TEM, and AFM studies.
Their results indicated that NLC formulations comprising
20 mol% OA was the optimum.
Fig. 9 Percentage cytotoxicity at three different concentrations of
CHR and LCD loaded into the NLC formulation (CP + TP + OA,
2:2:1, M/M/M) against the human neuroblastoma cell line SHSY5Y.
Blank NLC formulation is taken as control. The individual systems
are mentioned in the figure
J Surfact Deterg (2018)
Experimental results also confirmed the penetration of
LCD into the NLC and surface accumulation of CHR on
NLC. The incorporation efficacy of the LCD was found to
be higher than that of CHR. CHR 18 showed the maximum
EE and DL ability. In vitro release showed that LCD exhib-
ited more sustained release than CHR from NLC. The
higher lipophilicity and the greater association of the LCD
with NLC led to the sustained release profile. A progressive
enhancement of the hydrocarbon chain length in LCD led
to a progressive reduction in the observed release rate.
CHR- and the LCD-loaded formulations were found to be
capable of showing cytotoxicity against a cancer cell line.
It could, therefore, be concluded that LCD-loaded NLC for-
mulations have advantages over the alternate CHR-loaded
NLC formulations for the development of promising anti-
cancer drug delivery agents.
Acknowledgements This work was supported by the Department of
Science and Technology, Government of India, New Delhi, in the
form of a research grant SR/S1/PC-32/2011. SB gratefully acknowl-
edges the Department of Biotechnology, Government of India, for the
award of a fellowship (under twining project) BT/526/NE/TBP/2013.
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