Artificial Cells, Nanomedicine, and Biotechnology
An International Journal
ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: http://www.tandfonline.com/loi/ianb20
Silymarin nanoemulsion against human
hepatocellular carcinoma: development and
optimization
Usama Ahmad, Juber Akhtar, Satya Prakash Singh, Badruddeen, Farhan
Jalees Ahmad, Sahabjada Siddiqui & Wahajuddin
To cite this article: Usama Ahmad, Juber Akhtar, Satya Prakash Singh, Badruddeen, Farhan
Jalees Ahmad, Sahabjada Siddiqui & Wahajuddin (2017): Silymarin nanoemulsion against human
hepatocellular carcinoma: development and optimization, Artificial Cells, Nanomedicine, and
Biotechnology, DOI: 10.1080/21691401.2017.1324465
To link to this article: http://dx.doi.org/10.1080/21691401.2017.1324465
Published online: 14 May 2017.
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Date: 14 May 2017, At: 11:05
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2017
https://doi.org/10.1080/21691401.2017.1324465
Silymarin nanoemulsion against human hepatocellular carcinoma: development
and optimization
Usama Ahmada, Juber Akhtara , Satya
Prakash Singha, Badruddeena, Farhan Jalees Ahmadb,
d
c
Sahabjada Siddiqui and Wahajuddin
a
Department of Pharmaceutics, Faculty of Pharmacy, Integral University, Lucknow, India; bDepartment of Pharmaceutics, Faculty of Pharmacy,
Jamia Hamdard, New Delhi, India; cMolecular Endocrinology Laboratory, Department of Zoology, University of Lucknow, Lucknow, India;
d
Department of Pharmacokinetics, Central Drug Research Institute, Lucknow, India
ABSTRACT
ARTICLE HISTORY
Objective: Nanoemulsion of silymarin was developed and optimized.
Materials and methods: Nanoemulsion was made by aqueous titration method. Sefsol 218 (5.8% v/v),
Kolliphor RH40 and polyethylene glycol 400 (Smix; 2:1; 28.99% v/v) were used as oil phase, surfactant
and co-surfactant while distilled water (65.22% v/v) acted as an aqueous phase. Nanoemulsion was
characterized on the basis of particle size, viscosity, electrical conductivity and refractive index. Further,
in vitro release, in vivo pharmacokinetic study, stability study and cancer cell line studies were also
performed.
Results and discussion: The optimized formulation (NE9) with mean particle size of 21.24 nm showed
a minimum viscosity of 9.59 cps, maximum drug release (97.75%) in 24 h. The NE9 formulation also
showed higher AUC (p < .01) and Cmax (p < .01) and shorter Tmax (p < .05) compared with conventional
and standard suspensions of silymarin. The stability study also showed considerably stable formulations
at refrigerator temperature as compared with room temperature (p > .05). The cancer cell line studies
also confirmed that silymarin nanoemulsion reduced the cell viability and increased ROS intensity and
chromatin condensation (p < .05).
Conclusion: Our results concluded that nanoemulsion may be an efficient carrier for oral delivery of
silymarin against human hepatocellular carcinoma without damaging normal cells.
Received 14 March 2017
Revised 19 April 2017
Accepted 20 April 2017
Introduction
Silymarin is obtained from the purified extract of seeds and
fruits of Silybum marianum (commonly known as milk thistle
plant). It has been used for centuries as an herbal medicine
and food supplement for the treatment of liver and gallbladder disorders, including hepatitis, cirrhosis, jaundice, and to
protect the liver against poisoning from chemical and environmental toxins, including snake bites, insect stings, mushroom poisoning, and alcohol. Most of the medicinal
compounds found in milk thistle are present in high concentrations in the seeds. These compounds include silymarin,
which is composed of three isomer flavonolignans: silybin,
silydianin, and silychristin [1]. Silybin is considered the major
and most active component of silymarin [2,3]. It inhibit the
hepatotoxin binding to receptor sites on the hepatocyte
membrane; reduction of glutathione oxidation to enhance its
level in the liver and intestine; antioxidant activity; and stimulation of ribosomal RNA polymerase and subsequent protein
synthesis, leading to enhanced hepatocyte regeneration [4].
However, despite such potential benefits, silymarin is able to
produce little effect in vivo both in humans and in animals
due to its poor bioavailability. The four major causes of
CONTACT Juber Akhtar
Pradesh, India
juberakhtar@gmail.com
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
Chang liver cell line; human
hepatoma; nanoemulsion;
sefsol 218; silymarin
limited silymarin bioavailability are extensive phase II metabolism, low permeability across intestinal epithelial cells, low
aqueous solubility, and rapid excretion in bile and urine.
These factors necessitate the incorporation of silymarin into a
form that can augment its bioavailability [5].
A number of methods including some of the nano-based
approaches have been used previously to enhance the oral
bioavailability of silymarin and provide them a robust
strength against physical, chemical and environmental degradation. Nanostructured biomaterials, featuring a nanoscale
morphology and size, exhibit a wide range of advantages
over the conventional biomaterials, such as high bioavailability, improved cellular interaction, and specific designed functions [6,7]. It offers a promising solution to many difficulties
in drug delivery and tissue engineering [8], a nano-sized drug
vehicle has made significant progress in the delivery of conventionally undeliverable molecules, such as compounds with
low water solubility and genetic biomolecules [9,10]. The
newer formulation design approaches for bioavailability
enhancement includes incorporation of the active component
into inert lipid vehicles [11], such as oils [12], surfactant
dispersions [13–15], self-emulsifying formulations [16,17],
emulsions [18–21], micro or nanoemulsions [22–24], and
Department of Pharmaceutics, Faculty of Pharmacy, Integral University, Kursi Road, Lucknow 226026, Uttar
2
U. AHMAD ET AL.
liposomes [25]. Nanoemulsion offers several advantages over
these drug delivery systems like higher solubilisation capacity,
rapid onset of action (no extra time for dispersion), reduced
intersubject variability in terms of gastrointestinal fluid volume and longer shelf life [26], and toxicological safety, a high
content of the lipid phase and the possibility of large-scale
production by high-pressure homogenization [27].
The aim of the present investigation was to formulate and
optimize a stable nanoformulation of silymarin by aqueous
titration method. The prepared nanoemulsion was characterized and evaluated on the basis of size, surface morphology,
viscosity, conductivity, refractive index, and in vitro drug
release study. The pharmacokinetic parameters also showed
considerable values than conventional and standard suspensions of silymarin. The optimized silymarin nanoemulsion was
also assessing against human liver carcinoma cells.
three components i.e. oil, Smix (surfactant-co-surfactant mixture)
and distilled water [29]. Surfactant and co-surfactant were
mixed in different volume ratios in stock of 50 ml to obtain
best result. For each phase diagram, oil and a specific Smix
ratio was mixed properly in different volume ratios from 1:9 to
9:1 in separate glass vials. Sixteen different combinations of oil
and Smix (1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 2:1, 3:1, 4:1, 5:1,
6:1, 7:1, 8:1, 9:1) were slowly titrated with aqueous phase and
visually inspected for transparency and flow ability [22]. The
physical state of the nanoemulsion was marked on the phase
diagrams with three axes representing an aqueous phase, oil
phase, and Smix phase. For each phase diagram, nanoemulsion
area was plotted and the wider region indicated the better
self-nanoemulsifying efficiency. From each phase diagram constructed, different formulations were selected from nanoemulsion region varying the proportion of oil (10–30%v/v) at a
minimum concentration of Smix. Selected formulations were
subjected to stability and dispersibility tests.
Materials and methods
Materials used
Thermodynamic stability tests
Silymarin was purchased from Sigma Aldrich (St. Louis, MO),
Sefsol 218 and Kolliphor RH40 were obtained as a gift sample
from Nikko Chemicals (Tokyo, Japan) and BASF (Mumbai,
India), PEG 400 was purchased from SD Fine Chem. (Mumbai,
India). Human hepatocellular carcinoma HepG2 and Chang
liver cell line was obtained from cell repository-National
Centre for Cell Sciences, Pune, India. All other chemicals were
of analytical grade and were used as and when required.
Selected formulations were subjected to thermodynamic stability stress tests as heating cooling cycle, centrifugation, and
freeze–thaw cycle:
Nanoformulation development and its characterization
Solubility studies in various oils, construction of pseudo ternary phase diagram and thermodynamic stability tests were
performed so that a stable and robust formulation was
developed.
Solubility studies for screening of oil phase
Heating–cooling cycle: in this study, the prepared formulations
were kept at 45 C and at 0 C temperature for not less than
48 h for each temperature cycle.
Centrifugation tests: formulations were centrifuged (REMI,
Mumbai, India) at 5000 rpm for 30 min and observed for
phase separation, creaming, or cracking.
Freeze–thaw cycle: the prepared formulations were exposed at
two different temperatures i.e. 20 C and 20 C for each
temperature cycles not than 24 h. For the better estimation
of accelerated stability studies, three such cycles were run for
each batch of formulation. The formulations which showed
the maximum stability were selected for further study [30].
Dispersibility tests
An important aspect for the selection of oils for nanoemulsion formulation is the solubility of silymarin in different oils.
For this study, three oils, Sefsol 218, Triacetin, and Isopropyl
meristate, were selected. An excess amount of drug was
added in 2 ml of each oil separately in 5 ml capacity stoppered vials, and mixed using a vortex mixer [28]. These vials
were then kept at 25 ± 1.0 C in an isothermal shaker (IKAV KS
400i, Staufen, Germany) for 72 h to reach equilibrium. The
equilibrated vials were removed from shaker and centrifuged
at 10,000 rpm for 15 min using centrifuge (REMI, Mumbai,
India). The supernatant was taken and filtered through a
0.45-lm membrane filter. The concentration of silymarin was
determined in different oils by using HPTLC spectrophotometer at wavelength of 288 nm.
R
Pseudo ternary phase diagram study
Construction of pseudo ternary phase diagrams was done by
in situ emulsification method (aqueous titration method) using
One millilitre of each formulation was added to 500 ml of
0.1 N HCl and in distilled water in a USP Dissolution apparatus Type II at 37 ± 0.5 C to assess its efficiency of self-emulsification. A standard stainless steel dissolution paddle
rotating at 75 rpm provided gentle agitation. The formulation
was visually assessed using the following grading system:
Grade A: Forming (within 1 min) nanoemulsion, having a clear
appearance.
Grade B: Rapidly forming, slightly less clear emulsion.
Grade C: Fine milky emulsion formed within 2 min.
Grade D: Dull white emulsion having slightly oily appearance
that is slow to emulsify (longer than 2 min).
Grade E: Formulation, exhibiting either poor or minimal emulsification with large oil globules present on the surface.
Among the formulations which passed the stability and also
dispersibility tests in Grades A and B were selected for preparing drug-loaded batches utilizing minimum concentration of Smix for each percentage of oil.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
Preparation of silymarin-loaded nanoemulsions (SLN) by
aqueous titration method
The SLN were prepared by dissolving 20 mg of silymarin in
oil (10%, 15%, 20%, and 25% v/v). Respective Smix ratio was
added to the oil, mixed using vortex mixer, and followed by
the addition of aqueous phase to obtain nanoemulsion.
Physicochemical characterization and evaluation of SLN
Visual observation
Visual observation was done to differentiate between SLN
and macroemulsion.
Dynamic light scattering (DLS) measurement
The average droplet size and polydispersity index (PDI) of
SLN were determined by DLS technique that analyzes the
fluctuations in light scattering due to Brownian motion of the
particles using a zetasizer ZS 90 (Malvern instruments,
Worcestershire, UK). Light scattering was monitored at 25 C
at a 90 angle. Samples were diluted with distilled water and
filtered through 0.45 lm membrane filter and then were directly placed into the module [30].
3
In vitro drug release study
In vitro release test was performed in 900 ml of simulated
intestinal fluid using dissolution apparatus # 2, at 50 rpm and
37 ± 0.5 C (Hanson Research SR8 plus, Chatsworth, CA). One
millilitre of silymarin nanoemulsion formulation was placed in
treated dialysis bag (MWCO 1200 g/mole, Sigma Aldrich, St.
Louis, MO). One millilitre of samples was withdrawn at regular
time intervals (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 24 h) and
aliquot amount of simulated intestinal fluid was replaced
[32]. The samples were analyzed for the drug content by
HPTLC spectrophotometer at 288 nm [31]. The release of the
drug from nanoemulsion formulations was compared with
the available LimarinV suspension.
R
Stability studies
The optimized SLN formulations were subjected to accelerated stability studies at 25 ± 2 C/60 ± 5% RH, 40 ± 2 C/
65 ± 5% RH and 60 ± 2 C/75 ± 5% RH. The humidity and temperature control stability chambers (Thermolab, Mumbai,
India) were used wherein samples were placed in glass bottles and observed at specified time intervals of 0, 30, 60, and
90 d. The droplet size, polydispersity index, refractive index,
viscosity, and percent transmittance were determined [32].
Viscosity determination
In vivo pharmacokinetic study
Brookfield DV III ultra V6.0 RV cone and plate rheometer
(Brookfield Engineering Laboratories Inc. Middleboro, MA)
with spindle # CPE40 at 25 ± 0.5 C was used for the determination of viscosity of the SLN.
Animal study protocol
In vivo pharmacokinetic study was carried out after obtaining
approval from Integral University, Institutional Animal Ethics
Committee, Lucknow, Uttar Pradesh (Registration no. IU/
Pharm/Ph.D./CPCSEA/15/06) and CPCSEA guidelines were followed for the whole study. The in vivo study was made to
accomplish oral administration of SLN formulations in male
Wistar rats (150–180 g). There were six rats in each group.
The animals were anesthetized by diethyl ether. The blood
samples were withdrawn from the tail vein at 0 (pre-dose),
0.5, 1, 2, 3, 4, 6, 8, 24, and 48 h, centrifuged at 5000 rpm for
20 min in centrifuge tubes restraining 8 mg of EDTA as an
anticoagulant. The plasma was separated, stored at 20 C
and analysed by HPTLC method [31]. A treatment schedule is
specified in Table 1.
Electro conductivity studies
The conductivity (r) of SLN was determined by using conductometer, CDM 230 (Radiometer, Copenhagen, Denmark). The
reading was taken at the frequency of 94 Hz, having a cell
constant of 0.11 cm1. The measurements were performed at
25 ± 1 C.
Refractive index and percent transmittance
The refractive index of the SLN was measured by an Abbe
refractometer (Bausch and Lomb optical Company, Garden
City, NY) by placing one drop of nanoemulsion on the slide
at 25 C. The percent transmittance of the system was measured at 288 nm using HPTLC spectrophotometer [31].
Transmission electron microscopy (TEM)
Surface morphology of SLN was studied by TEM TOPCON
002B (Topcon, Oakland, NJ) [30]. A drop of nanoemulsion was
diluted with distilled water (1:100), filtered (0.22 lm), and
applied on carbon coated grid with 2% phosphotungestic
acid and kept it for 30 s. The dried coated grid was taken on
a slide and covered with a cover slip. The slide was observed
under the light microscope operating at 200 KV.
Cell line and culture
Human hepatocellular carcinoma HepG2 cell line and Chang
liver cells (non-tumor cells) were obtained from cell repository – National Centre for Cell Sciences, Pune, India. Both cell
lines were maintained in Eagle’s MEM with 2.0 mM L-glutamine, 1.5 g/l NaHCO3, antibiotic solution (100 U/ml penicillin,
and 100 lg/ml streptomycin) and supplemented with 10%
(v/v) foetal bovine serum. Cells were grown at 37 C, 5% CO2
humidified atmosphere.
Cell viability assay
This assay was used to detect the cell viability of silymarin
nanoemulsion using following protocol [33]. Approximately
4
U. AHMAD ET AL.
Table 1. Treatment schedule of silymarin formulations in male Wistar rats.
Groups
Drugs
Treatment schedule
Standard suspension
Conventional suspension
Silymarin nanoformulation
Silymarin 42 mg/kg p.o. single dose given to male Wistar rats
Conventional formulation of silymarin 35 mg/kg p.o. single dose given to male Wistar rats
Optimized nanoemulsion of silymarin 20 mg/kg p.o. single dose given to male Wistar rats
Solubility (mg/mL)
I
II
III
160
140
120
100
80
60
40
20
0
158.5
36
SEFSOL 218
TRIACETIN
21
ISOPROPYL MERISTATE
Name of Oil
Figure 1. Silymarin showing maximum solubility in sefsol 218.
1 104 cells/well of HepG2 were seeded in 100 ll complete
culture medium in 96-well culture plate and incubated overnight in humidified air. Stock was prepared in phosphate
buffer saline (PBS) and diluted into culture medium to the
desired concentrations 0.2, 0.5, 1, 2.5, and 5 lg/ml added to
the wells. After 24 h of incubation period, 10 ll of MTT
(5 mg/ml in PBS) reagent was added and re-incubated at
37 C until purple formazan crystals developed. Formazan
blue crystals were dissolved in 100 ll of DMSO and read at
540 nm using microplate ELISA reader (BIORAD 680,
Hercules, CA). The plot of percent cell viability versus SLN
concentrations was used to calculate the concentration
lethal to 50% of the cells (IC50). The cellular morphological
changes were observed under inverted phase contrast
microscopy (Nikon ECLIPSE Ti-S, Tokyo, Japan). Similar test
of nanoformulation was also performed on Chang liver cells
to examine if the treatment had a distinguishable effect
between normal and cancer cells.
528 nm. Data were expressed as percentage of fluorescence
intensity relative to the control wells.
Intracellular reactive oxygen species (ROS) activity
Statistical analysis
Intracellular ROS generation was analyzed by using fluorescence microscopic imaging technique as per previous protocol [34]. Cells (1 104 per well) were exposed at three
effective concentrations i.e. 0.5, 1 and 2.5 lg/ml of silymarin
nanoemulsion for 12 h. Subsequently, cells were incubated
with DCFH-DA (10 mM) at 37 C for 30 min and washed
with PBS. Intracellular fluorescence intensity of cells was
visualized by inverted fluorescent microscope (Nikon
ECLIPSE Ti-S, Tokyo, Japan). For quantitative fluorometric
analysis, cells (1 104 per well) were seeded and treated
with SLN in 96-well black bottom culture plate.
Fluorescence intensity was measured with a multiwell
microplate reader (Synergy H1 Hybrid Multi-Mode
Microplate Reader, BioTek, Winooski, VT) at an excitation
wavelength of 485 nm and at an emission wavelength of
The results were expressed as mean values ± SD The analysis
of variance (ANOVA) was applied to examine the significance
of differences in SLN properties (such as droplet size, polydispersity index, percent transmittance, refractive index, viscosity, conductivity, and drug content). In all cases, p < .05 was
considered to be significant.
Apoptotic effect of SLN using DAPI stain
Florescent nuclear dye DAPI was used to analyze the apoptotic effect of SLN as per previous method [35]. HepG2 cells
(1 105 cells per well) were seeded in 24-well culture plate
overnight and treated with silymarin nanoemulsion for 24 h.
Following incubation period, cells were washed and fixed in
4% paraformaldehyde for 15 min followed by permeabilization with permeabilizing buffer (3% paraformaldehyde and
0.5% Triton X-100) for 10 min. After staining with DAPI dye
(50 lg/ml), images of condensed nuclei undergoing apoptosis
were captured with an inverted fluorescent microscope
(Nikon ECLIPSE Ti-S, Tokyo, Japan). Apoptosis was quantitated
by morphological changes of nuclei wherein about 500 cells/
well representing one sample.
Result and discussion
Solubility studies
Solubility studies were aimed at identifying a suitable oil
phase for the development of silymarin nanoemulsion to
achieve optimum drug loading. The higher solubility of the
drug in the oil phase is important for the nanoemulsion to
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
F1
0
F2
100 Water
90
10
20
30
20
30
70
40
40
60
50
50
50
80
20
Oil 90
10
0
100
0.00 10.0020.0030.0040.0050.0060.0070.0080.0090.00100.00
Smix
80
20
10
90
0
100
0.00 10.0020.0030.0040.0050.0060.0070.0080.0090.00100.00
Smix
0
F3
10
100
30
40
50
60
70
80
Oil
Water
90
20
90
30
70
30
70
40
60
40
60
Oil
100
Water
90
80
70
60
50
0
10
80
5
0
F4
100
10
80
20
70
30
60
40
50
50
40
60
30
70
20
80
10
100
0
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Smix
Oil
80
70
60
50
40
30
20
10
90
100
0.00
Water
90
0
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Smix
Figure 2. Pseudoternary phase diagrams of silymarin nanoemulsions, F1 (Smix 1:0), F2 (Smix 1:1), F3 (Smix 2:1), and F4 (Smix 1:2).
maintain the drug in the solubilized form. In the oil phase
tested, the solubility of silymarin was found to be the highest
in sefsol 218 (158.5 mg/ml) followed by triacetin (36 mg/ml)
and iso-propyl meristate (21 mg/ml) (Figure 1). Thus, sefsol
218 was selected as the oil phase for the development of the
formulation.
Pseudo ternary phase diagram study
Phase diagram study of SLN containing sefsol 218 as the oil
phase and kolliphor RH40 as the surfactant showed the low
amount of oil (5.56% v/v) could be solubilized at higher
surfactant concentration (F1). It yielded a narrow range for
nanoemulsion formation. Generally, surfactant alone cannot
lower the oil interfacial tension sufficiently to yield a nanoemulsion; this necessitates the addition of an amphiphilic
molecule or co-surfactant to lower the surface tension close
to zero. Co-surfactants penetrate into the surfactant monolayer, providing additional fluidity to the interfacial film and
thus disrupting the liquid crystalline phases which are
formed when the surfactant film is too rigid. This could be
attributed to the fact that transient negative interfacial tension and fluid interfacial film is rarely achieved by the use
of single surfactant, usually necessitating the addition of a
cosurfactant. SLN containing sefsol 218 as the oil phase,
kolliphor RH40 as the surfactant, and PEG 400 as the cosurfactant showed the formation of broader nanoemulsion
region (F2). Equal Smix ratio (1:1) was used to obtain this
region. Nanoemulsions were transparent with slight bluish
tint and a maximum amount of oil was solubilized in this
region (18.8% v/v). It may be attributed to low interfacial
tension between oil and water phases since maximum
amount of oil and surfactant were solubilized in this area.
Moreover, a combination of a surfactant and co-surfactant
in optimal concentration leads to enormous solubilization of
oil phase. The presence of PEG 400 as co-surfactant
decreases the bending stress of interface and makes the
interfacial film sufficiently flexible to take up different curvatures required to form nanoemulsion over a wide range of
compositions. Results of F2 phase diagram depict larger
nanoemulsion formation in the surfactant-rich region, but
this region was comparatively lower than results obtained
from F3 (Smix, 2:1) diagram. Therefore, low amount of oil
was solubilized in this area; nanoemulsion region was confined to borders along the surfactant region. This may be
due to an increase in the concentration of kolliphor RH40
in the system. Phase diagram obtained from F4 indicates
nanoemulsion formation in the aqueous-rich region. Smix
ratio used for this system was 1:2 and due to increased
concentration of PEG 400 in the system interfacial tension
seems to have been reduced much lower as compared with
other systems.
Thus, the phase diagram study (Figure 2) provides valuable
information on the role and concentration of surfactants, cosurfactants, and water in formulation of nanoemulsion. It is
observed from the diagram that an optimum concentration
of Smix ratio (2:1) provided better results.
6
U. AHMAD ET AL.
Table 2. Thermodynamic stability tests of prepared nanoformulation.
Thermodynamic stability tests
Formulation
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Dispersibility
Smix ratio
%Oil
%Smix
%Water
Heating/cooling
Centrifugation
Freeze–Thaw
0.1 N HCl
H2O
1:0
1.87
5
10
3
7.02
11.24
14.93
18.18
5.80
9.09
13.33
16.67
20.69
2.50
6
10.53
15
13.12
45
30
23
28.07
33.71
44.78
36.36
28.99
45.45
66.67
66.67
48.28
17.50
14
24.56
35
85
50
60
74
64.91
55.06
40.30
45.45
65.22
45.45
20
16.67
31.03
80
80
64.91
50
X
X
X
–
–
–
–
X
X
–
X
–
–
C
A
A
C
B
–
–
A
B
C
–
–
A
C
B
–
–
C
A
A
C
B
–
–
A
B
C
–
–
A
C
B
–
1:1
2:1
1:2
Thermodynamic stability tests
After optimization of nanoemulsion region, the prepared formulations were subjected to thermodynamic stability tests.
Nanoemulsions remain to be stable at stressed conditions.
Three tests, heating/cooling cycle, centrifugation, and freeze–
thaw cycles, were performed to evaluate the stability of
the formulations. Observations made during the tests are
given in Table 2.
Preparation of SLN
The optimized formulations that passed all the tests and possessed least concentrations of surfactants were used for incorporating silymarin.
Characterization and evaluation of nanoemulsion
Selected SLN formulations were characterized and evaluated
by following parameters.
Visual appearance
SLN was clear and transparent and free from any turbidity.
This test was done to differentiate it from macroemulsion
which is milky in appearance.
Dynamic light scattering (DLS) measurement
DLS technique was employed to measure the particle size
distribution, polydispersity index, and zeta potential
(Figure 3). Formulation NE9 showed the smallest particle size
(21.24 ± 0.291 nm) followed by NE3 (29.34 ± 0.634), NE4
(79.02 ± 1.651), and NE14 (31.31 ± 0.298). Polydispersity index
of formulations NE3, NE4, NE9, and NE14 were found to be
0.191 ± 0.008, 0.449 ± 0.022, 0.104 ± 0.016, and 0.168 ± 0.022,
respectively (Table 3). Lower value of polydispersity index
indicates that nanoformulations were uniform in their droplet
size. The selected combination of oil, Smix and water was able
Inference
FAILED
FAILED
PASSED
PASSED
FAILED
FAILED
FAILED
FAILED
PASSED
FAILED
FAILED
FAILED
FAILED
PASSED
FAILED
FAILED
FAILED
to produce small and stable droplets on nanoscale and the
results of particle size and polydispersity index produced
from selected composition of formulation was much better
than results reported earlier [36–38]. Therefore, the results
were indicative of the fact that selection of proper oil,
surfactant, and co-surfactant ratios were crucial for
obtaining smaller particle size while developing a stable
nanoemulsion.
TEM
TEM image of NE9 showed that SLN droplets were less than
50 nm (Figure 3).
In vitro drug release study
The four nanoformulations (NE3, NE4, NE14, and NE9) were
tested and compared with each other and with an available
conventional suspension of silymarin i.e. LimarinV. The concentration was calculated by extrapolation of calibration
curve and a graph was plotted between time and percent
cumulative release (Figure 4). The nanoformulation NE14
showed 94.99% drug release followed by NE3 (88.69%) and
NE4 (74.08%). The NE4 formulation exhibited slowest release
due to larger particle size of nanoemulsion. All above nanoformulation exhibited better drug release profile compared to
conventional silymarin suspension but only NE9 formulation
was selected for further study since it showed maximum
release (97.75%), finest globule size (21.24 nm), smallest polydispersity value (0.104), lesser viscosity (9.59 cps), and refractive index (1.316).
R
Accelerated stability studies
The stability studies revealed that during the storage
period of 3 months at 25 ± 2 C/60 ± 5% RH, 40 ± 2 C/
65 ± 5% RH, and 60 ± 2 C/75 ± 5% RH, optimized SLN
showed non-significant (p > .05) change in mean droplet
size, zeta potential, polydispersity index, refractive index,
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
7
Figure 3. Silymarin-loaded nanoemulsion: (A) droplet size, (B) transmission electron microscopy, and (C) zeta potential.
Figure 4. Drug release study of silymarin nanoemulsion and its comparison with marketed suspension.
viscosity, electrical conductivity, and transmittance. No
phase separation and flocculation were observed, proving
its stability. However, data obtained from temperatures
60 C/65RH for 90 d showed increased droplet size but
reduced transparency as compared with optimized SLN formulations at day zero.
In vivo pharmacokinetic study of optimized formulation
and its comparison with available commercial product
The Cmax of NE9 was found to be 38.62 ± 2.43 lg/ml. That
was higher than silymarin-marketed suspension (10.36 ± 1.06)
and standard suspension (5.24 ± 4.31). The AUC of
8
U. AHMAD ET AL.
Table 3. Droplet size, poydispersity index, zeta potential, viscosity, refractive index, conductivity, and percent transmittance of selected silymarin nanoemusion
formulations.
Formulation
NE 3
NE 4
NE 9
NE 14
Droplet
size ± SD (nm)
29.34 ± 0.634
79.02 ± 1.651
21.24 ± 0.291
31.31 ± 0.298
Polydispersity
index
0.191 ± 0.008
0.449 ± 0.022
0.104 ± 0.016
0.168 ± 0.022
Zeta potential (mV)
29.3 ± 0.345
12.1 ± 0.131
17.9 ± 0.527
22.6 ± 0.751
Viscosity ± SD (cps)
12.34 ± 0.927
11.83 ± 0.924
9.59 ± 0.764
10.9 ± 3.165
Refractive index ± SD
1.369 ± 0.0259
1.320 ± 0.0729
1.316 ± 0.0151
1.360 ± 0.0062
Conductivity ± SD (lS/cm)
430.340 ± 1.873
461.579 ± 1.214
480.432 ± 1.837
455.019 ± 2.045
Percent
transmittance
98.15 ± 0.91
98.47 ± 1.07
99.52 ± 0.54
99.02 ± 0.06
All data expressed as mean ± SD (n ¼ 3).
Table 4. Pharmacokinetic parameters obtained after oral administration of silymarin nanoemulsion and its comparison with
standard and conventional suspensions in male Wistar rats (n ¼ 6).
Formulation
Standard suspension
Conventional suspension
Silymarin nanoemulsion (NE9)
Cmaxa (lg/ml)
Tmaxb (h)
5.24 ± 4.31
10.36 ± 1.06#
38.62 ± 2.43##,
3.0 ± 0.31
2.0 ± 0.62#
0.5 ± 0.45##,
AUC
0 ! all
(lg h/ml)c
17.82 ± 7.32
37.43 ± 2.89#
308.51 ± 4.23##,
a
Peak of maximum concentration.
Time of peak concentration.
c
Area-under-the-concentration–time profile curve.
Mean ± SD (n ¼ 3).
##p < .01 and #p < .05 considered significant, when compare with standard suspension and conventional suspension.
p < .05 and p < .01 considered significant, when compared with conventional suspension.
b
nanoemulsion (NE9) was found to be 308.51 ± 4.23 lgh/mL
which was 8-fold higher than marketed suspension
(37.43 ± 2.89) and 17-fold higher than standard suspension
(17.82 ± 7.32) (p < .01) (Figure 5). The high value of AUC and
Cmax in the case of NE9 formulation ensured higher drug
absorption and availability at the site of action over a prolonged period of time. The quick onset of the drug action in
the body is attributed to the presence of a low Tmax value of
NE9 formulation (0.5 ± 0.45 h) as compared with conventional
suspension
(2.0 ± 0.62 h)
and
standard
suspension
(3.0 ± 0.31 h) of silymarin (p < .05). Although nanoemulsion of
silymarin showed improved oral absorption [36] but in present study, we were able to make better silymarin nanoemulsion which was more absorbed in less amount of time just by
choosing suitable proportions of oil, surfactant, co-surfactant,
and water (Table 4). The increase in the bioavailability of silymarin using a nanoemulsion might be due to the higher
solubilization of drug in oil. Moreover, the presence of a surfactant and cosurfactant in the nanoemulsion system might
have caused changes in the membrane permeability, and was
able to reach a maximum concentration in minimum possible
time [39,40]. Consequently, the present study showed rapid
onset and better absorption of orally administered silymarin
nanoemulsion than before.
Effects of silymarin nanoemulsion on percent cells
viability and cellular morphology
In vitro cytotoxicity studies
The unexposed cells remained smooth and healthy. However,
morphological changes were made in SLN treated cells as
revealed by the photomicrograph. The cells exposed to SLN
displayed cellular shrinkage and fragmented body as compared with the untreated cells (5 A). This result supports the
apoptotic features of the cells [35]. The cytotoxic data indicate that 0.2 lg/ml of SLN reduced the cell viability to
approximately 89.83% (p < .05) as compared with control.
The cell viability was drastically reduced to 76.25 and 51.41%
(p < .05) at 0.5 and 1 lg/ml of SLN, respectively. Further, SLN
at a concentrations 2.5 and 5 lg/ml reduced the viability
of cells to 28.45 and 15.89% (p < .05), respectively
(Figure 6(A,B)). The toxicity of oligonucleotide/cationic complexes reduced significantly by the SLN [34]. Although effects
of silymarin alone were given on HepG2 cells before [41,42]
but the present study showed better effects as SLN reduces
the cell viability of cancer cell line in a dose-dependent
manner without harming the surrounding Chang liver cells
(normal cells) (Figure 7).
Silymarin nanoemulsion induces intracellular ROS
generation
The HepG2 cells treated with nanoemulsion showed a significant increase in ROS intensity in a dose-dependent manner as
compared with untreated cells (Figure 6(C,D)). The results
of quantitative measurement of ROS level showed that 0.5 lg/
ml of nanoemulsion induced 121.43% (p < .05) enhancement
in ROS production as compared with control. Moreover, ROS
production was increased by 156.77 and 195.53% (p < .05) at 1
and 2.5 lg/ml of nanoemulsion when compared with
untreated cells (Figure 6(C)). During apoptosis, ROS are produced by mitochondria which increased the mitochondrial
membrane permeability and leads to the apoptotic phenotype
[41]. ROS are more reactive than molecular oxygen which may
be associated with the activation of signal molecules and
destabilization of mitochondrial membrane inducing the
release of apoptotic agents resulting in toxicity to cancer cells
[42,43]. Our results clearly stated that nanoemulsion provoked
the cells death by inducing ROS production.
Silymarin nanoemulsion induces nuclear condensation
As observed from photomicrograph (Figure 6(E)), HepG2 cells
treated with increasing concentrations of nanoemulsion
increased the chromatin condensation as compared with
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
9
Figure 5. Plasma concentration of various silymarin formulations in male Wistar rats after oral administration.
Figure 6. (A and B) In vitro cytotoxicity test. (C and D) Intracellular reactive oxygen species generation. (E and F) Chromatin condensation of optimized silymarin
nanoemulsion.
control cells. However, 1 and 2.5 lg/ml of SLN exhibited maximum condensation as observed under inverted fluorescence
microscope. Furthermore, approximately 13.33 and 20.33% of
apoptotic cells were observed at 0.5 and 1 lg/ml of SLN,
respectively (Figure 6(F)). Interestingly, 2.5 lg/ml of SLN
induced 36.33% apoptotic cells as compared with control.
Condensed and fragmented nuclei suggested that SLN
caused cell death by an apoptotic process.
Conclusions
Silymarin nanoemulsion was prepared effectively with an
optimized composition comprising oil phase (5.8% v/v, sefsol
218), 28.99% v/v of Smix (kolliphor RH40 and PEG 400, 2:1 as
a surfactant and co-surfactant, respectively) and 65.22% v/v
of distilled water as an aqueous phase. The above formulation was evaluated on the basis of particle size, viscosity, conductivity, and refractive index. That showed maximum drug
release in less amount of oil. The NE9 formulation also
showed higher Cmax and AUC and low Tmax than conventional
suspension and standard suspension of silymarin. Data
obtained from the stability studies also demonstrated that
the optimized silymarin nanoemulsion remains stable over
storage period of 3 months at 25± C/60 ± 5% RH, 40 ± 2 C/
65 ± 5% RH, and 60 ± 2 C/75 ± 5% as there was no creaming
or phase separation observed in the formulations (p > .05).
The optimized formulations also showed reduction in the cell
viability and increased ROS intensity and chromatin
10
U. AHMAD ET AL.
Figure 7. Effects of silymarin nanoemulsion on Chang liver cells (non-tumor cells).
condensation against human liver carcinoma cells without
harming normal cells. The nanosize particles of silymarin followed by higher surface area may permit quicker rate of drug
release and improved absorption pursued to enhanced bioactivity in lesser dose of drug.
[8]
Acknowledgement
[9]
Authors are thankful to Integral University Lucknow for providing the
necessary facilities required for successful completion of this research
work (IU/R&D/2017-MCN00043).
[6]
[7]
[10]
[11]
Disclosure statement
All authors have approved the final manuscript, and no potential conflict
of interest was reported by the authors.
[12]
ORCID
[13]
Juber Akhtar
http://orcid.org/0000-0002-2219-370X
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