Synthesis of luteolin loaded zein nanoparticles for targeted cancer therapy improving bioavailability and efficacy

Article abstract of DOI:10.1016/j.jddst.2019.04.044

A naturally occurring flavonoid, Luteolin possess wide range of pharmaceutical activities from anti-inflammatory to anticancer effect. Luteolin inhibits cell proliferation by arresting cell cycle pathways and triggers apoptotic cell death. Poor aqueous solubility of luteolin limits its oral bioavailability. Therefore, to improve the oral bioavailability of luteolin by use of biodegradable protein zein and sodium caseinate as nanocarrier. The nanoparticles were prepared by simple precipitation process. The resulting nanoparticles had mean size of 200–300 nm with negative zeta potential with 92% encapsulation efficiency and luteolin release at intestinal pH. Anti-oxidant activity of loaded molecule with respect to parent molecule significantly indicate the free radical scavenging potential of luteolin increases after encapsulation in zein nanoparticles. Luteolin loaded zein nanoparticles showed enhanced cytotoxicity against SW480 colon cancer cells and induces apoptosis. In this paper, we have shown the potential of luteolin loaded zein nanoparticles for treating colon cancer which could serve as promising strategy to develop oral formulation of this water insoluble drug enhancing its bioavailability.

Full text of DOI:10.1016/j.jddst.2019.04.044

Accepted Manuscript
Synthesis of luteolin loaded zein nanoparticles for targeted cancer therapy improving
bioavailability and efficacy
Priyanka Shinde, Hina Agraval, Ajeet Singh, Umesh C.S. Yadav, Umesh Kumar
PII:
S1773-2247(19)30459-9
https://doi.org/10.1016/j.jddst.2019.04.044
JDDST 1050
DOI:
Reference:
To appear in: Journal of Drug Delivery Science and Technology
Received Date: 2 April 2019
Revised Date: 18 April 2019
Accepted Date: 25 April 2019
Please cite this article as: P. Shinde, H. Agraval, A. Singh, U.C.S. Yadav, U. Kumar, Synthesis
of luteolin loaded zein nanoparticles for targeted cancer therapy improving bioavailability and
efficacy, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/
j.jddst.2019.04.044.
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Title:
Synthesis of luteolin loaded zein nanoparticles for targeted cancer therapy Improving
bioavailability and efficacy.
Authors:
a
b
c
b
a*
Priyanka Shinde , Hina Agraval , Ajeet Singh , Umesh C.S.Yadav , Umesh Kumar
Affiliation:
a
School of Nano Sciences, Central University of Gujarat, Gandhinagar-382030, Gujarat,
India
b
School of Life Science, Central University of Gujarat, Gandhinagar-382030, Gujarat, India.
c
CAMS Venture Center, 100, NCL Innovation Park, Pune 411008, Maharashtra, India.
*
Corresponding Author:
Umesh Kumar
Assistant Professor,
School of Nano Sciences,
Central University of Gujarat,
Gandhinagar-382030, Gujarat, India.
E-mail ID:
Mobile No: +918376802055
1

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Highlights
•
Green synthesis of plant derived zein-caseinate based biocompatible nanocarrier with
92 % encapsulation of luteolin.
>
•
•
These nanocarrier showed specific intestinal pH dependent drug release.
The encapsulated luteolin indicated improved anti-oxidant and apoptotic activity
against cancer cells.
•
The present nanocarrier could become a potential drug delivery and cancer targeting
system that increase the efficacy of anticancer drugs.
2

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Graphical Abstract
3

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Abstract
A naturally occurring flavonoid, Luteolin possess wide range of pharmaceutical activities
from anti-inflammatory to anticancer effect. Luteolin inhibits cell proliferation by arresting
cell cycle pathways and triggers apoptotic cell death. Poor aqueous solubility of luteolin
limits its oral bioavailability. Therefore, to improve the oral bioavailability of luteolin by use
of biodegradable protein zein and sodium caseinate as nanocarrier. The nanoparticles were
prepared by simple precipitation process. The resulting nanoparticles had mean size of 200-
3
00 nm with negative zeta potential with 92% encapsulation efficiency and luteolin release at
intestinal pH. Anti-oxidant activity of loaded molecule with respect to parent molecule
significantly indicate the free radical scavenging potential of luteolin increases after
encapsulation in zein nanoparticles. Luteolin loaded zein nanoparticles showed enhanced
cytotoxicity against SW480 colon cancer cells and induces apoptosis. In this paper, we have
shown the potential of luteolin loaded zein nanoparticles for treating colon cancer which
could serve as promising strategy to develop oral formulation of this water insoluble drug
enhancing its bioavailability.
Keywords: Luteolin, Solubility, Zein, Anti-oxidant, Bioavailability.
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1
. Introduction
Flavonoids are plant derived biologically active chemical compounds possessing
pharmacological effects. Luteolin (3’, 4’, 5, 7-tetrahydroxyflavone) is a polyphenolic
compound which is an important flavonoid naturally occurring in various plant species.
Luteolin usually occurs in glycosylated form and chemically has C6-C3-C6 structure that
possess two benzene rings and one oxygen containing ring with 2-3 double bond. The
presence of 2-3 double bond and hydroxyl groups at carbon 5, 7, 3’ and 4’ positions play
crucial role in its multiple pharmacological effects [1]. Studies have reported that luteolin
possess anticancer activity against head and neck cancer, lung, breast, prostate, colon, liver
and skin cancer via cell cycle arrest, inducing apoptosis and also associated with supressing
metastasis and angiogenesis [2, 3]. However, the bioavailability of luteolin is critical matter
due to its poor water solubility. It is rapidly excreted via kidney and its extent of systemic
absorption is also low, this limits its medicinal use even though luteolin has high efficacy and
potency [4]. Nanotechnology provides a way to overcome this problem by entrapping this
molecule into nanoparticles or by developing smart materials to deliver two or more drugs at
the desired site. Nanomaterial not only encapsulate various drugs but also provides strategies
to efficiently deliver the agents to target sites, cross endothelial and epithelial barrier and
release in desired ratio which further increases treatment efficacy [5].
In past years, micro and nanoparticle of proteins have been of interest over biomolecules
like lipids, carbohydrates and other polymers for pharmaceutical application due to their
biocompatibility, high drug binding capacity, ease of availability and high surface area for
entrapped molecule [6, 7]. Also the multiple functional groups of protein allow them to create
diverse interaction with molecules of different polarities based on electrostatic interactions
and hydrophobic bonding [8, 9]. Previous literature reports that protein nanoparticles have
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enhanced mucoadhesive properties and capacity to cross blood brain barrier[10]. Also they
showed higher potential for targeted delivery of anticancer molecules to tumour cells [11].
The targeted delivery is mediated through various mechanisms exploiting inherent
characteristics of tumours e.g wide gaps between endothelial cells, acidic micro environment,
hyperthermic effect, over expression of receptors in tumour tissue and markers on cancer cell
surfaces [12]. People have also used albumin as targeting protein molecule since it binds to
membrane associated gp60 on endothelial cells of tumour vessels enhancing its uptake in
solid tumours. Similarly, Elastin like protein (ELP) gets accumulated passively in
hyperthermic tumour margins which are used for thermo targeted chemotherapy. Besides
this, lectins binds to glycosylated components of intestinal mucosal surface causing
glycotargeting of anticancer drugs [13].
Active research is undergoing on biomedical application of animal and plant proteins,
however hydrophobic plant proteins offer the capability of sustained drug release compared
to animal proteins. Also plant proteins require no chemical cross linkers or physical treatment
to form stable structure [13, 14]. Moreover plant proteins reduce the risk of transmission of
zoonotic disease compared to animal proteins [15]. In this respect zein, a proline-rich protein
have demonstrated capability to encapsulate and protect the active molecules [13, 16]. The
higher percentage of hydrophobic amino acid show property to extend plasma level of drug,
hence can also overcome the drawback of hydrophilic polymer to achieve sustained release
[
15, 17]. The structural features of zein make it capable of protecting loaded compound from
harsh stomach conditions and offer mechanism for controlled release [18]. Owing to
hydrophobic surface and low net charge at pH above 5 (as zein is close to its isoelectric point
6
.2), zein nanoparticles formulations are not physically stable and leads to aggregation [19].
This is a major challenge of zein nanocarrier for intestinal delivery having physiological pH
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around neutral. Several surfactants and biopolymers form stable complex with zein managing
hydrophobic precipitation and steric repulsion [20-22].
Sodium caseinate Generally Regarded as Safe (GRAS) is a soluble mixture of various
caseins. It contains various hydrophobic and hydrophilic groups and has been proposed to act
as natural stabilizer and emulsifier [23]. Studies have shown that casein can change its
molecular arrangement according to ionic strength and can adsorb on variety of surfaces [24].
Also casein stabilized emulsion are thermally stable as its confirmation is not affected by heat
as observed in other globular proteins [25, 26]. Nevertheless, the combination of zein-sodium
caseinate nanoparticles encapsulating luteolin has never been investigated so far.
The main objective of the present study is to evaluate the potential of sodium caseinate
stabilised zein nanoparticle as a carrier to improve the bioavailability of luteolin, enhancing
its antitumor activity.
2
. Materials and methods
2
.1. Materials
Zein (ZN, having majority of α and β fraction, which includes 2 prominent bands of 22
and 24 kDa), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), Phosphate buffer salts, DMSO
molecular grade, Acridine orange and Ethidium bromide were purchased from Sigma Aldrich
(
St. Louis, MO, USA). Luteolin, sodium caseinate, Coumarin-6 and Potassium Bromide
KBr) were purchased from TCI (Japan). Trypsin EDTA (0.25%), Penicillin-streptomycin-
(
amphotericin B (PSA) cocktail and Fetal Bovine Serum (FBS) and [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Gibco, Thermofisher
scientific (Waltham, MA, USA). RPMI-1640 medium was purchased from Himedia
(Mumbai, India). Ethanol, methanol and acetone were obtained from Rankem. All aqueous
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solutions were prepared in water purified with Milli Q system (Merck Millipore, Milli-Q IQ
7
003/7005).
2
.2. Synthesis of nanoparticles
Powdered zein was weighed and dissolved in 80% (v/v) of aqueous ethanol to a final
concentration of 5mg/ml, under continuous stirring for overnight. This stock solution was
filtered using 0.22 micron filter. Sodium caseinate solution was prepared by adding 1.0 g of
sodium caseinate into 100 ml Milli Q water. Zein nanoparticles (ZNPs) were synthesized
using anti-solvent method [27]. Briefly, sodium caseinate solution was added slowly into a
glass vial containing zein solution under continuous stirring at 500 rpm using magnetic
stirrer. Different concentration of sodium caseinate was used to obtain various zein-sodium
caseinate ratios i.e 1:0, 1:0.75, 1:1, 1:1.25. The final solution was stored at 4°C until used for
further analysis. To obtain solid powder samples, the solution was freeze-dried for 24 h.
For synthesis of luteolin loaded zein nanoparticles (LNPs), 5mg luteolin were dissolved
in 1 ml of 80% aqueous ethanol. Luteolin and zein solution were mixed in different ratios
under stirring condition. The mixture was precipitated and purified as described for zein
nanoparticles.
2
2
.3. Characterization of nanoparticles
.3.1 Ultraviolet-visible spectroscopy
UV-visible spectra were recorded on ultraviolet visible spectrophotometer (UV-1800,
Schimadzu, Japan). Sample solutions were prepared to desired concentration and the solvent
was chosen as blank. 1cm optical path quartz cuvette were used during the experiment and
baseline was set using blank from 800 to 200 nm. Once the baseline was set, samples were
scanned, and spectra is recorded.
2
.3.2 Dynamic Light Scattering studies (DLS):
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The hydrodynamic size and zeta potential of zein nanoparticles and luteolin loaded zein
nanoparticles were determined using dynamic light scattering (DLS, Microtrac Zetatrac,
U2771, USA). Samples for DLS studies were prepared by dispersing the synthesized
nanoparticles in distilled water (1:10) at 25°C with 15 scans per run. Both size and zeta
potential measurements were performed in triplicates (3 different batch). The particle size
distribution is reported on scattering intensity basis.
2
.3.3 Microscopy studies:
The shape and morphology of nanoparticle was analyzed using field emission scanning
electron microscope (FE-SEM, Merlin Compact VP, ZEISS). The freshly prepared samples
were well dispersed and gently spread on cleaned silica sheet. These samples were allowed to
dry at room temperature and particles were sputter coated with gold using Quorum Q150
RES. SEM images were captured at 25°C at an accelerating voltage of 5 kV.
2
.3.4 Fourier transform infrared spectroscopy (FT-IR) studies:
The chemical structure of commercially available zein and its formulation were
investigated with Perkin Elmer Spectrophotometer. FT-IR samples were prepared by mixing
mg of lyophilized nanoparticles with 200 mg of KBr powder and compressed to form
2
-1
pellet. The spectra were scanned in transmittance mode from 400 to 4000 cm wavenumber
-1
in 64 scans with step size of 4 cm .
2
.3.5 Differential Scanning Calorimetry (DSC) thermal analysis:
The thermal characteristics were studied using differential scanning calorimetry (DSC
6
000, Perkin Elmer). Approximately 3mg of each, freeze dried sample were weighed in
aluminum pan and compress sealed. Empty sealed pan was applied for baseline and samples
were heated from 30 to 350°C with the rate of 10°C/min under constant nitrogen purging
(20ml/min).
2
.3.6 Luteolin encapsulation and loading studies:
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The amount of luteolin encapsulated was determined indirectly based on difference
between total amount of luteolin and free amount. To determine the free amount the loaded
nanoparticle suspension was centrifuged at 8000 rpm for 10 min at 4°C and the supernatant
was retained. The absorbance was determined using UV-VIS spectrophotometry at 340 nm.
The amount of luteolin was determined using a standard curve, constructed using standard
solutions 1-50 µg of luteolin. The EE and LE were as calculated as follows:
ꢁ
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EE% =ꢀ
LE% =ꢀ
xꢀ100
(1)
ꢁ
ꢂꢃꢄꢅꢆꢇꢈꢇꢉꢊꢀꢋꢌꢍꢎ
ꢁ
ꢂꢃꢄꢅꢆꢇꢈꢇꢉꢊꢀꢋꢌꢍꢎꢏꢁꢂꢃꢄꢅꢆꢐꢌꢑꢑꢀꢋꢌꢍꢎ
xꢀ100 (2)
ꢁ
ꢂꢃꢄꢅꢆꢋꢌꢍꢎꢒꢓꢉꢓꢈꢔꢉꢌꢌꢕꢑꢌ
2
.3.7 Release of luteolin from nanoparticles
The in vitro release study was carried out using dialysis membrane method. The
nanoparticles were loaded in dialysis membrane with molecular cut off 12-14 kDa. 0.1N HCl
was used as release medium for initial two hours and then in 6.8 pH PBS for 6 hours. The
medium was kept under stirring condition at 150 rpm, 37°C, subsequently 2ml of medium
was taken and replaced by new medium at regular time interval (0.5, 1, 2, 3, 4, 5, 6, 7 and 8
h). Luteolin concentration in the medium was determined using UV spectrophotometer.
2
.3.8 Antioxidant activity
The anti-oxidant activity of free Luteolin and luteolin encapsulated particle was
evaluated using DPPH radical scavenging assay. Different concentration of samples were
taken and mixed with DPPH in 1:3 ratio. The mixture was shaken vigorously and incubated
in dark. The absorbance was taken at 517 nm, the reduction of DPPH absorbance was
monitored at specific time interval. The absorbance of DPPH solution at 517 nm was taken as
control and calculations were made according to following formula:
ꢤꢥꢦ.ꢧꢨꢀꢩꢧꢪꢫꢬꢧꢭꢏꢤꢥꢦ.ꢧꢨꢀꢥꢭꢮꢪꢯ
ꢖꢗꢘꢙꢚꢗꢛꢀꢜꢚꢗꢝꢗꢞꢟꢙꢞꢟꢀꢗꢚꢠꢙꢝꢙꢠꢡꢀꢢ%ꢣ =
ꢰꢀ100 (3)
ꢤꢥꢦ.ꢧꢨꢀꢩꢧꢪꢫꢬꢧꢭꢀ
1
0

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2
.4 Animal Cell culture
SW-480 cells were obtained from American Type Culture Collection (ATCC) and
cultured in RPMI 1640 medium supplemented with 10% FBS, 1% PSA at 37°C in a humified
5
2
% CO incubator. Cells were trypsinized after achieving ~80% confluency.
2
.4.1 MTT assay
SW-480 cells were seeded in 96 well plate at 7000 cells/well density followed by
incubation for 24 h. After attachment, cells were treated with different concentrations of
crude drug (CD i.e luteolin) (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 75 µg/ml), luteolin
loaded zein nanoparticles (LNPs) (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 75 µg/ml) and
zein nanoparticles (ZNPs) (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 75 µg/ml) for 24 h and 48
h. After completion of incubation time-points, media was removed and 50 µl MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye (5 mg/mL) was added to each
°
well and incubated at 37 C in dark for 4 h. Formazan crystals were dissolved by adding 150
µL DMSO. Absorbance was then noted at 570 nm using microplate reader (Synergy H1
Hybrid Reader, Biotek, and Winooski, VT).
2
.4.2 Acridine Orange-Ethidium Bromide assay
SW-480 cells were seeded in 12 well plate at 50,000 cells/well density followed by
incubation for 24 h. Cells were then treated with CD (10 µg/ml and 25 µg/ml) LNPs (10
µg/ml and 25 µg/ml) and ZNPs (10 µg/ml and 25 µg/ml) for 24 h. After treatment, cells were
trypsinized and centrifuged at 1500 rpm for 5 minutes. The pellet was re-suspended in 50 µL
of PBS. From the suspension 10 µL of volume with additional 1 µL of Acridine orange
(1µg/mL) and 1 µL of Ethidium bromide (1µg/mL) were taken on glass slide. This was
followed by fluorescence microscopy (Olympus, Japan) at 200X. All the photographs were
procured within 20 minutes of sample processing.
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2
.4.3 Cellular uptake studies
Cellular uptake potential for 6-coumarin LNPs was studied using SW-480 cells. SW-
80 cells were seeded on cover slips in 12 well plate at 40,000 cells/well cell density. After
4 h, cells were treated with 100 ng/mL of native-coumarin-6 and 6-coumarin LNPs followed
4
2
by incubation for 30 min, 1 h, 4 h, and 8 h. After each incubation time-point, cells were
washed with cold PBS for two times followed by fluorescence microscopy at 200X
magnification [28].
2
.5 Statistical analysis
All the measurements were done in triplicates. The data were subjected for the calculation
of standard error of the mean (SEM). All the groups were analyzed by student’s t-test
GraphPad Prism 6.0 software, La Jolla, CA) where the p values of <0.05 were considered to
be statistically significant.
(
3
. Results and Discussion
To explore the optical properties of luteolin loaded zein nanoparticles ultraviolet-visible
spectroscopic studies were carried out for nanoparticles and luteolin as well. The absorption
spectra of luteolin possess two characteristic picks around 346 and 250 nm for Band I (300-
4
00 nm) and Band II (240-280 nm), associated with absorption of cinnamoyl and benzoyl
system [29]. The encapsulation of luteolin in zein nanoparticles caused the displacement of
spectral band to 339 nm (indicating hypsochromic shift (blue shift). The change in the spectra
states electronic redistribution or transition of π–π* conjugation system of luteolin upon
encapsulation [30].
Further to determine the size and dispersity of prepared nanoparticles Dynamic Light
Scattering studies was carried out. We used different mass ratio of zein to sodium caseinate to
synthesize zein nanoparticles which affected its stability. Our results indicate that in absence
1
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of sodium caseinate, nanoparticles were of moderate size with zeta potential of -11.11 mV
and had higher polydispersity index. As sodium caseinate percentage increases, particle size
decreases. At 1:1 and 1:1.25 mass ratio, decrease in particle size was observed and stable
dispersion were also formed. These samples also had polydispersity index of less than 0.2,
indicating narrow particle size distribution (Table-1). Although sodium caseinate imparted
only a small increment in negative zeta potential of nanoparticles, however casein can
provide sufficient steric barrier to prevent particle aggregation. Compared to unloaded
nanoparticle luteolin loaded nanoparticle showed a significant decrease in particle size which
is similar to previous reports [31].
In order to validate our DLS results and determine the shape of synthesized nanoparticles,
microscopical studies were carried out with scanning electron microscope. Scanning electron
micrograph of zein nanoparticles and luteolin loaded zein nanoparticles is shown in Fig. (2),
indicating the presence of spherical particles. It is apparent from the images (Fig. 2 A and B)
that compact spherical particles with an average diameter of 265 nm were obtained with zein
to sodium caseinate mass ratio of 1:1, for zein nanoparticles (ZNPs) whereas the average
particle size was reduced to 250 nm (Fig 2. C and D) in case of luteolin loaded (1 mg) zein
nanoparticles (LNPs) which is in accordance with our previous DLS results. Size of the
nanoparticles have large influence on pharmacokinetics and clearance mechanism. Particles
smaller than 20-30 nm get eliminated from the body via renal clearance very quickly whereas
larger particles of 150-300 nm are taken up by mononuclear phagocytic system. Reported
data suggests that nanoparticles developed for cancer treatment are between 70-280 nm for
targeting vital organs [32]. Our developed delivery system was within the acceptable limits
for cancer treatment.
Further we also used Infra-red spectroscopy to investigate the chemical changes in
luteolin, if any, while encapsulating it in prepared zein nanoparticles. The FTIR spectra of
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-1
zein protein shows four characteristic bands (Fig 3). The band between 2800 and 3500 cm ,
called amide A corresponding to N-H and O-H bonds of amino acids of protein. Other two
-1
characteristic bands at 1658.66 and 1535.6 cm were amide I and amide II respectively,
corresponding to stretching of carbonyl (C=O) of amide group and angular deformation of N-
-1
H bond. The band associated with vibration of C-N bond observed at 1234.4 cm [33]. The
-1
IR spectra of luteolin showed –OH vibration band at about 3431.6 cm due to the presence of
-1
phenolic group. The band appearing at 1613.6 cm was the result of C=O vibrations of
central heterocyclic ring of luteolin [34]. The O-H stretching peak luteolin and zein shifted
-1
from 3431.6 and 3307.2 to 3442 cm after formation of nanoparticles suggesting increased
hydrogen bonding activity during formation of nanoparticles [33]. The shift in the band of
-1
-1
amide I and amide II from 1658.66 and 1535.6 cm to 1655.2 and 1546 cm indicating
electrostatic interaction. This phenomenon suggest a stable ring was formed due to conjugate
-1
effect [35]. The peak at 1164 cm is of C-O-C stretching vibrations remained unchanged in
loaded nanoparticles indicating that the ring oxygen atom is not participating in bond
formation [36].
Thermal stability is a very important criteria for any nano-formulation. Therefore, we also
characterized these synthesized nanoparticles with differential scanning calorimetry (DSC).
The DSC curves of pure zein, luteolin, zein nanoparticles and luteolin loaded nanoparticles
are shown in Fig.4. The calorimetric analysis of luteolin showed sharp endothermic peak at
around 342°C which represents its melting point [37]. Zein had a characteristic endothermic
peak at 73.8°C which is consistent with the previous report that demonstrated zein
endothermic peak at 73.2°C [31]. The DSC curves of encapsulated nanoparticles showed that
characteristic endothermic peak of luteolin disappeared indicating only van der Waals force
or hydrogen bonds involved in interactions [37].
Determining actual drug dosage that is required for anticipated physiological effect is
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also very important for any nano-formulations. Hence, we investigated these parameters in
terms of encapsulation and loading efficiency (EE and LE). Luteolin loaded zein
nanoparticles with different dose of luteolin were evaluated using ultraviolet-visible
spectrophotometer. These studies indicate that with increased dose of luteolin from 0.5 to
1
.25 mg, the EE increased from 81.68 to 92.73% along with increase in loading efficiency
from 3.88 to 10.30 % (Fig 5). Luteolin interact hydrophobically with the protein molecules
during antisolvent addition which minimizes its escape from participating in particle
formation causing greater encapsulation efficiency. The encapsulation efficiency of 1 mg
dose which was further used for all studies was also calculated using LC-MS, which was
around 98.38% (Supplementary Information). This calculated efficiency was higher since
LC-MS is more sensitive than UV-visible.
Once loading and encapsulation efficiency has been determined under optimized
parameter, it becomes very essential to study the pharmacokinetics of developed nano-
formulation. Therefore, we studied the release of luteolin from synthesized zein nanoparticles
under physiological condition (acidic pH followed by alkaline pH), as depicted in Fig. 6.
Remarkably the release of luteolin from zein nanoparticles was found to be pH dependent.
The pH influences electrostatic interaction between luteolin and zein, if the charges are
opposite in sign then lead to electrostatic interaction lowering the rate of release. While for
similar net charges a rapid release of drug from nanoparticle is expected. There was about 6%
release of luteolin when nanoparticles were incubated in HCL solution (pH 2) for first 2 h.
After nanoparticles were moved to PBS with pH 6.8, luteolin appears to be released more
rapidly. Thus, at the end of experiment around 79% of luteolin encapsulated in nanoparticles
was released within 8 h. These results indicate that luteolin release from zein nanoparticles
follows Hixson–Crowell model [38] which is well accepted for drugs intended for Oral
delivery [39]. This model explains controlled diffusion of drug from a particulate system.
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Also, drug release is affected by particle size as for smaller particles most of drug is
associated with surface causing faster drug release while larger particles show steady release
due to presence of drug in core.
Further we were interested in determining the bio efficacy of these developed nano
formulations, since luteolin is known to exhibit many activities like antioxidant, anti-cancer,
wound healing etc. [40, 41]. Luteolin play an important role as free radical scavenger in
prevention of cancer as cancer cells reveals higher level of oxidative stress. The antioxidant
activity of luteolin decreases the expression of mitochondrial SOD (superoxide dismutase)
with increased amount of superoxide radicals in certain colorectal carcinomas. This lead to
change in redox potential of transcriptional factors and cell cycle regulatory protein causing
tumor suppression [42]. Therefore, we investigated the antioxidant activity of these
developed nano-formulations in terms of free radical (DPPH) scavenging activity. DPPH is
an organic free radical with absorption maxima at 517 nm. This absorption decreases upon
reduction with antioxidant compound. The antioxidant compound/material have the potential
to donate hydrogen atom to radical and convert it to DPPH-H, which gives a visible color
change from purple to yellow. Our study showed concentration dependent high DPPH radical
scavenging activity of encapsulated luteolin compared to parent compound (Fig 7).
Encapsulation changes natural free radical scavenging ability of luteolin signifying better
inhibitory effect of encapsulated compound than free luteolin.
To analyze the effect of CD (Crude drug i.e Luteolin), LNPs and ZNPs on cell viability,
SW-480 cells were treated with different concentrations of CD (5 µg/ml, 10 µg/ml, 25 µg/ml,
5
0 µg/ml, 75 µg/ml), LNPs (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 75 µg/ml) and ZNPs (5
µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 75 µg/ml). Treatment of cells with CD at 24 and 48 h
resulted in decrease in cell viability in dose and time dependent manner. LNPs treatment to
cells was further able to increase the cytotoxic effects of the CD suggesting that the zein
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nanoparticles in combination with luteolin was able to enhance the cytotoxic potential of
luteolin (Fig.8). Also, ZNPs did not cause any significant cell death as observed. Cell
viability at 5 µg/mL, 10 µg/mL, 25 µg/mL LNPs treatment was reduced by 26.3 %, 47.1%
and 53.1% at 24 h, 23.31 %, 49.1% and 69.82% at 48 h respectively. All the differences at all
time points were statistically significant (P<0.05 - P<0.0001).
Acridine orange-ethidium bromide (AO/EB) double staining assay is used to observe the
changes in cellular morphology. Live cells are stained by acridine orange in green color while
dead cells are stained by ethidium bromide in red color (Fig.9) [43]. Cells when treated with
different concentrations of luteolin displayed significant cellular apoptosis. Cells further
exhibited typical characteristics of apoptosis like membrane blebbing, nuclear condensation
and apoptotic body formation when stained with AO/EB. Red fluorescence was also observed
in CD treated cells suggesting necrotic features. Cells treated with LNPs (10 µg/mL and 25
µg/mL) showed characteristics of apoptosis like nuclear condensation, nuclear fragmentation
and membrane blebbing. Significant necrosis was also observed in LNPs treated cells.
However, ZNPs did not cause any significant cell death at any concentration or at any time
point. ZNPs treated cells had intact nuclear organization with uniform green color. These
results indicated that luteolin loaded nanoparticles are able to induce cell death in dose and
time dependent manner. Also, they have the potential to enhance the anti-proliferative effect
of luteolin in SW-480 cells.
To determine the uptake potential of luteolin loaded zein nanoparticles (LNPs), SW-480
cells were treated with native 6-coumarin (100 ng/mL) and 6-coumarin-LNPs (100 ng/mL)
for different time intervals. Uptake studies were performed using fluorescence microscopy.
Cells treated with 6-coumarin-LNPs exhibited higher cellular uptake when compared to 6-
coumarin alone, at 30 min, 1 h, 4 h, and 8 h time points. 6-coumarin-LNPs treated SW-480
cells showed higher fluorescent intensity when compared to 6-coumarin alone. Highest
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uptake was observed after 8 h of treatment with 6-coumarin-LNPs. This data suggests that
LNPs have high cellular uptake potential. Also, LNPs effectively enhance the delivery of
luteolin in SW-480 cells and therefore, LNPs showed better cytotoxic effects in comparison
to Luteolin alone (Fig.10). Our results are in accordance with previous reports where people
suggested that negatively charged nanoparticle uptake occurs mainly through clathrin-
caveolin independent endocytotic pathway [44].
4
. Conclusion
Above findings from the present study showed size controlled, synthesis of spherical
shape zein nanoparticle under mild conditions. The synthesized particles exhibited
encapsulation efficiency of about 92% with improved release properties of luteolin under
physiological pH as demonstrated by our in vitro studies. In addition, encapsulation in zein
nanoparticles enhanced cytotoxic potential and cellular uptake compared to free luteolin.
These results prove that sodium caseinate stabilized zein nanoparticle could become a
promising nanocarrier for delivering small molecules (Luteolin) of nutraceutical importance
enhancing its bioavailability and efficacy. Further these types of developed nanocarrier have
the potential to get internalized within the cell and deliver its payload. This is a complete
green approach where both the carrier and active ingredient is derived from plant. Such type
of carrier could find application in agriculture also.
5
. Conflict of Interest
The authors of the manuscript have no conflict of Interest.
. Acknowledgements
6
Priyanka Shinde would like to acknowledge the generous support from Central University
of Gujarat, Gandhinagar for providing non net fellowship and facilities to carry out this work.
Hina Agraval acknowledges Department of Science and Technology, Govt. of India for
INSPIRE fellowship.
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7
. Tables
Table 1 Characterisation of zein nanoparticle with different zein to sodium mass ratio and
luteolin loaded zein nanoparticles (1mg luteolin loaded in 1:1 zein to sodium caseinate mass
ratio)
Zein to SC ratio
Particle size (nm)
294.07 ± 2.15
311.33 ± 4.93
280.83 ± 8.26
283.7 ± 5.02
Zeta Potential (mv)
-11.11 ± 0.75
-14.28 ± 0.34
-17.52 ± 0.91
-16.87 ± 0.77
-14.72 ± 3.22
PDI
1
1
1
1
:0
0.429 ± 0.60
0.117 ± 0.02
0.107 ± 0.05
0.126 ± 0.04
0.258 ± 0.07
:0.75
:1
:1.25
Loaded (1mg)
1:1 Z/SC)
215.4 ± 8.11
(
*values are expressed as mean ± SD (n=3).
Table 2 DPPH radical scavenging activity (percentage ± SD) of free luteolin, luteolin loaded
zein nanoparticles (LNPs) and zein nanoparticles (ZNPs) at 20 min for different
concentration of luteolin. The data illustrated free luteolin having maximum scavenging
activity of about 51% while encapsulated has 59%.
Luteolin Concentration
µg/ml)
Radical Scavenging Activity (%)
(
Free Luteolin
28.69 ± 0.57
35.33 ± 0.37
47.74 ± 9.7
LNPs
ZNPs
10
30
50
70
90
36.09 ± 3.92
40.60 ± 1.35
47.11 ± 1.08
54.38 ± 0.94
59.89 ± 0.86
11.27 ± 1.95
6.89 ± 1.20
6.64 ± 2.64
6.51 ± 1.32
14.66 ± 5.90
41.97 ± 2.07
51.37 ± 0.21
*values are expressed as mean ± SD (n=3).
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Figures Captions:
Fig. 1 Ultraviolet-visible absorption spectra of luteolin (solid line), zein nanoparticles (ZNPs,
dash line) and luteolin loaded zein nanoparticles (LNPs, dotted line) showing a subtle
hypsochromic shift of 7.0 nm in case of luteolin loaded nanoparticle.
Fig. 2 Scanning Electron Microscopy of ZNPs (A, B) and LNPs (C, D) for zein to SC mass
ratio of 1:1 at different resolutions.
Fig. 3 FTIR spectra of zein, luteolin, ZNPs and LNPs.
Fig. 4 Representative DSC thermograms of zein, luteolin, ZNPs and LNPs.
Fig. 5 Graphical representation of effect of luteolin concentration on encapsulation (A) and
loading (B) efficiency.
Fig. 6 Luteolin release profile from zein nanoparticles after incubation in HCl (0-2 h) and in
PBS (2-8 h). It follows Hixson–Crowell model. Data are expressed as the mean ± SD, n=3.
Fig. 7 DPPH radical scavenging activity (RSA %) of free luteolin (dash), ZNPs (dotted line)
and LNPs (solid black line) with increasing concentration of luteolin carried on 20 min (A).
The scavenging activity of respective samples at different time intervals for 10 µg/ml
concentration of luteolin (B). The plots show that higher activity against DPPH was observed
in encapsulated luteolin in concentration dependent manner at various time points. The data
are expressed as means ± SD with triplicates.
Fig. 8 Effect of luteolin on cell viability in SW480 cells treated with different concentration
of CD (5-75 µg/mL), LNPs (5-75 µg/mL) and ZNPs (5-75 µg/mL) for 24 h (A) and 48 h (B)
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time points followed by MTT assay. Control cells were left untreated. The cell growth data
shows mean ± SEM (n=3); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. respective
controls analysed.
Fig. 9 Acridine Orange-Ethidium Bromide double staining assay performed on SW-480 cells
treated with CD, LNPs and ZNPs at 10µg/mL and 25 µg/mL for 24h followed by
fluorescence microscopy. Control cells were untreated cells. Magnification of all the
photomicrographs are 200X.
Fig. 10 Cellular uptake studies for native 6-coumarin (100 ng/mL) and 6-coumarin- LNPs
(100 ng/mL) in SW-480 cells at different time intervals examined by fluorescence
microscopy.
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Fig. 4