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ISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, Early Online: 1–8

! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2013.829092

ORIGINAL RESEARCH ARTICLE

Development and characterization of lutein-loaded SNEDDS for

enhanced absorption in Caco-2 cells
Pattravee Niamprem, Soravoot Rujivipat, and Waree Tiyaboonchai
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Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand

Abstract Keywords
A self-nanoemulsifying drug delivery system (SNEDDS) has been developed for enhanced oral Absorption, Caco-2 cells, dissolution, lutein,
bioavailability of lutein. Its permeation enhancement has been evaluated using monolayers self-nanoemulsifying drug delivery system,
of Caco-2 cells. SNEDDS is composed of a mixture of LexolÕ and EmulmetikÕ 900, LabrasolÕ , solubility
and Tween 80 as oil, surfactant and co-surfactant, respectively. Upon dilution of lutein-
loaded SNEDDS with water, a nanoemulsion was obtained in 510 s with spherical droplets of History
40–150 nm in diameter. The zeta potential was in the range of 19 to 32 mV. Increasing the
ratio of surfactant to co-surfactant decreased the mean droplet size. Dissolution studies showed Received 12 March 2013
that lutein was released rapidly (55 min) from SNEDDS into 0.1 N HCl and pH 6.8 phosphate Revised 6 June 2013
buffer solution without any aggregation. In vitro studies using Caco-2 cells revealed that lutein- Accepted 11 July 2013
loaded SNEDDS showed shorter lag time and greater (2-fold) cellular accumulation compared Published online 28 August 2013
with the lutein dispersion.
For personal use only.

Introduction delivery systems16–18, the effects of different formulation factors,

such as ratio of oil phase to surfactant phase and ratio of surfactant
Age-related macular degeneration (AMD) is one of the leading
to co-surfactant on the in vitro release, transport, and accumu-
causes of blindness in people over the age of 60 in industrialized
lation in Caco-2 cells, are not known.
countries1. AMD affects 50% of people at age 75  5 years
The objectives of this study, therefore, were to characterize the
and is rarely found in people 550 years of age2. The etiology
physicochemical properties of the lutein-loaded SNEDDS under
of AMD is not completely understood, but oxidative stress is
various formulation factors with measurement of emulsification
known to play a role.
time, droplet size and zeta potential analysis, morphology, and
Lutein, a well-known antioxidant, is highly concentrated in
in vitro release of SNEDDS formulation. Moreover, the transport
the macula and lens of the eyes. It plays an important role in the
and accumulation of lutein have been investigated using mono-
prevention of AMD by blocking the high-energy wavelengths of
layers of Caco-2 cells.
blue light3–5. This effect is thought to reduce drusen formation4,6.
In addition, clinical and epidemiological studies have indicated
Materials and methods
that increased intake of lutein (6 mg/day) can reduce the risk of
AMD2,7,8. Materials
Unfortunately, lutein exhibits poor water solubility and as a
Lutein was purchased from KEB Biotechnology Co., Ltd.
result, it has a low oral bioavailability. To overcome this problem,
(Beijing, China). Standard lutein was purchased from Sigma
in recent years, much attention has been focused on developing
chemical (Lot No.070M4056, MO). LabrasolÕ (caprylocaproyl
lipid-based formulations for enhancing the oral bioavailability
macrogol-8-glycerides, LS) and TranscutolÕ HP (diethylene
of poorly water-soluble drugs9,10. Self-nanoemulsifying drug
glycol monoethyl ether) were purchased from Gattefosse
delivery system (SNEDDS) is one of the most popular approaches
(Cedex, France). EmulmetikÕ 900 (Soybean phospholipids,
that can increase interfacial area of the drugs to improve
E 900) was purchased from Lucas Meyer Cosmetic (Champlan,
solubilization as well as absorption across the mucosa11,12.
France). Tween 80 [Polyoxyethylene (20) sorbitan monooleate,
SNEDDS is an isotropic mixture of natural/synthetic oils,
T80] was purchased from Ajex Finechem Pty Ltd. (Seven Hills,
surfactants/co-surfactants and drug. Upon mild agitation followed
Australia). LexolÕ (medium chain triglyceride), propylene glycol,
by dilution with aqueous media, oil-in-water nanoemulsions
polyethylene glycol 400 (PEG 400) were purchased from
are instantaneously formed9,10,13–15. Although several studies
Namsiang Co., Ltd. (Bangkok, Thailand). Caco-2 cells were
have developed and characterized lutein in self-emulsifying drug
purchased from the American Type Culture Collection (VA).
Dulbecco’s modified Eagle’s medium (DMEM F-12) and all the
materials for cell culture were purchased from Sigma chemical
(MO). Cell culture plate and TranswellÕ plate (12 mm diameter,
Address for correspondence: Waree Tiyaboonchai, Department of
0.4 mm-pore-size polycarbonate membrane) were purchased
Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, from Corning Costar (MA). 3-[4,5-dimethylthiazol-2-yl]-2,5-
Naresuan University, Phitsanulok, Thailand. Tel: +66 55 261873. Fax: diphenyl-tetrazolium bromide (MTT) and dimethy sulfoxide
+66 55 261057. E-mail: wareet@nu.ac.th were purchased from AmrescoÕ (OH).
 2 P. Niamprem et al.
Development of lutein-loaded SNEDDS
Solubility study
The solubility of lutein in various vehicles, including oil,
surfactant and co-surfactant, were determined using shake flask
method19. An excess amount of lutein was added to 1 ml of each
vehicle. The mixture was shaken for 48 h at ambient temperature.
After equilibrium, the mixture was centrifuged at 14 000 rpm for
10 min (Mikro 120, Hettich, Burladingen, Germany). The super-
natants were diluted appropriately with a mobile phase, a mixture
of 2-propanol/dichloromethane/methanol (20:10:70, v/v), before
applied to the high performance liquid chromatography (HPLC).
The dissolved lutein was quantified with a calibration curve
of standard lutein in a concentration range of 0.01–5 mg/ml.
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An HPLC apparatus (Model LD10A, Shimadzu, Kyoto, Japan)
equipped with a Vertisep bio C30 column (5 mm, 4.6  100 mm)
was used. A flow rate of 1.0 ml/min and detection wavelength of
446 nm were employed.
Construction of pseudo-ternary phase diagram
Pseudo-ternary phase diagrams were constructed (using CHEMIX
School software, version 3.51) in the absence of lutein by
water titration method at ambient temperature20–22. Based on the
solubility study, three SNEDDS formulation systems were
investigated. The mixture of LexolÕ and EmulmetikÕ 900 at 9:1
was used as oil phase in all systems. Surfactants and co-
surfactants (LabrasolÕ and Tween 80; LabrasolÕ and TranscutolÕ
HP; Tween 80 and TranscutolÕ HP) were used in system I, II and
III, respectively. Surfactant and co-surfactant were mixed in
For personal use only.
different volume ratios (7:3, 8:2 and 9:1). For each pseudo-ternary
phase diagram at specific surfactant/co-surfactant ratio, oil and
surfactant/co-surfactant mixtures were mixed thoroughly in
different volume ratios (1:9,1.5:8.5, 2:8, 2.5:7.5, 3:7, 3.5:6.5,
4:6, 5:5, 6:4, 7:3, 8:2 and 9:1). Each mixture was titrated with
distilled water and visually examined for transparency after
equilibrium. Then, the mixture was further titrated with aliquots
of distilled water until turbidity has been reached. Clear and
isotropic samples were deemed to be within the microemulsions
regions corresponding to the selected optimum ratios of combin-
ation vehicles for developing lutein-loaded SNEDDS.
Preparation of lutein-loaded SNEDDS formulations and
content determination
From pseudo-ternary phase diagram, lutein-loaded SNEDDS,
0.5% lutein, were prepared using the mixture of LexolÕ and
EmulmetikÕ 900 at 9:1 as oil phase and LabrasolÕ and Tween 80
as surfactant phase. All components were mixed using a magnetic
stirrer at 80  C for 10 min or until a clear solution was obtained.
Then, the formulation was equilibrated at ambient temperature
for 24 h to examine for turbidity or phase separation before
cm2 were used22,24.
characterization. All samples were prepared in triplicate. The
varying formulation factors were the ratios of oil phase to
surfactant phase at 3:7, 2.5:7.5 and 2:8 and the ratios of LabrasolÕ
to Tween 80 at 9:1, 8:2 and 7:3.
The lutein content in SNEDDS was determined. Each formu-
lations (20 ml) were diluted with 980 ml of 95% ethanol.
The mixture was then vortexed vigorously for 2 min and diluted
100-fold with the mobile phase before the content of lutein was
determined by HPLC as describe earlier.
Physicochemical characterization of lutein-loaded
SNEDDS formulations
The morphology of lutein-loaded SNEDDS was investigated by
transmission electron microscope (TEM, Tecnai 12, Philips, OR).
Pharm Dev Technol, Early Online: 1–8
Micro/nano-emulsions was obtained by introducing 50 ml of
formulation in 50 ml deionized water and mixed for 10 min.
On carbon-coated 300 mesh copper grid, 20 ml of the micro/
nano-emulsions were directly deposited and stained with 10 ml
of uranyl acetate solution (0.5% w/v). Excessive solvent was
removed by Whatman filter paper (11 mm). The dried sample was
kept in desiccator overnight before observation by TEM.
The mean droplet size and size distribution of SNEDDS were
determined by dynamic light scattering with a ZetaPALSÕ
analyzer (ZetaPALS, Brookhaven Corp., NY). The micro/nano-
emulsions were transferred to polystyrene cuvette. Measurements
were then carried out at 90 to the incident light, the wavelength
of 659 nm in 10 mm diameter cell. The measurement was
repeated 10 times. The mean droplet size and size distribution
were obtained from the method of cumulants. In addition, the zeta
potential of micro/nano-emulsions were determined by measuring
the droplet electrophoretic mobility using ZetaPALSÕ . The
measurement was repeated 5 times. The zeta potential was
calculated based on the Smoluchowski equation23.
Emulsification time was determined by adding 0.5 ml
SNEDDS formulation in a paddle apparatus (USP XXXII)
(VankelÕ VK 700, Vankel Industries, Edison, NJ) [50 rpm,
37  C, 900 ml, 0.1 N HCl and pH 6.8 phosphate buffer solution,
n ¼ 3]18. Time for the appearance of micro/nano-emulsions was
visually monitored.
In vitro dissolution studies
Dissolution studies were performed using a basket apparatus (USP
XXXII) (UDT-804, Logan Instrument Corp., NJ) [100 rpm, 37  C,
900 ml, 0.1 N HCl and pH 6.8 phosphate buffer solution, n ¼ 3].
Lutein-loaded SNEDDS or lutein powder equivalent to 5 mg
of lutein was filled into hard gelation capsule No. 0 prior to
introduction into the dissolution tester. An aliquot of 5 ml was
withdrawn at 0, 5, 10, 15 and 30 min and then replaced with fresh
medium. Samples were filtered and subjected to lutein analysis
using UV spectrophotometer at 446 nm.
Determination of lutein SNEDDS absorption in
Caco-2 cells
Cell culture
Caco-2 cells were maintained in DMEM/F-12 supplemented with
10% fetal bovine serum (heat-inactivated at a temperature of
56  C for 30 min) and 1% penicillin/streptomycin. Cells were
grown at 37  C, 5% CO2, and cultured for 14–21 days to obtain a
differentiated cell monolayer prior to use. All studies were
performed between passage 35 and 45. The integrity of the
monolayer was confirmed by measuring the transepithelial
electrical resistance (TEER) using a volt-ohm meter (MillicellÕ
ERS-2, Millipore Corporation, MA). Only cell monolayer with
TEER value of 500  100
Cell viability
Cell viability of lutein-loaded SNEDDS on the Caco-2 cells was
evaluated by MTT assay. Cells were seeded in 96-well plate at a
density of 1  104 cells/well in 100 ml of cell culture medium.
Immediately prior to each experiment, the medium was removed.
Then, the cells were incubated with the 100 ml of lutein-loaded
SNEDDS dilutions for 4 h. After that, 10 ml of MTT (5 mg/ml in
phosphate buffer saline) was added and incubated at 37  C, 5%
CO2, for 2 h. The formazan crystals were dissolved in 100 ml of a
mixture of 0.1 M glycine:DMSO (1:9). The absorbance of
formazan at 595 nm was measured using microplate reader (DTX
880, Multimode Detector, Beckmancoulter, CA). Cell viability
was expressed as a percentage of the control group.
 DOI: 10.3109/10837450.2013.829092
Lutein transport and cellular accumulation
Õ 5
Cells were seeded in Transwell plate at density of 1  10 cells/
well for 14–21 days. The culture medium was changed every 2
days. After differentiation, cells were incubated with serum-free,
non-phenol red medium overnight before use. At the beginning of
each experiment, 0.5 ml of lutein-enrich medium (containing
lutein-loaded SNEDDS with serum free medium) was added
to the apical chamber, and 1.5 ml of serum free medium was
added to the basolateral chamber. The samples were incubated
at 37  C, 5% CO2. In this experiment, the content of lutein
in three parts, apical chamber, basolateral chamber and cells,
were collected at 1, 2, 3, 4, 5 and 6 h. Medium from apical
and basolateral chamber were harvested. Cell monolayer was
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washed twice with 0.5 ml of ice-cold pH 7.4 phosphate buffer
saline to eliminate lutein on monolayer surface. Then, the
cell monolayer was scraped and collected in 1 ml of phosphate
buffer saline. All the samples were stored at 80  C under
nitrogen for a maximum 2 days before lutein extraction and
quantitation. The transport of lutein from the apical to the
basolateral chamber was expressed as permeation amount of
lutein versus time. The accumulation of lutein in Caco-2 cells
monolayer was expressed as accumulation amount of lutein versus
time. To investigate the effect of different SNEDDS formulations,
the content of lutein in three parts was examined after incubated
for 6 h.
For lutein extraction, 100 ml of aqueous sample was mixed
with 500 ml of dichloromethane and vortexed vigorously for 2 min.
The sample was then centrifuged at 10 000 rpm for 10 min,
and the supernatant was collected. Lutein was extracted twice,
For personal use only.
and then the resultant supernatant was evaporated under nitrogen.
The dried extract was dissolved in 500 ml of mobile before
HPLC was used to determine the lutein content. In addition, the
cell samples were centrifuged at 3000 rpm for 5 min to remove
supernatant from cells. Then, lutein in cell samples was extracted
using the above method.
Statistical analysis
All data were expressed as mean  SD from at least three different
experiments. The data were analyzed by one-way analysis of
variance (ANOVA). Differences were considered to be significant
when the p value was 0.05.
Results
Development of lutein-loaded SNEDDS
Solubility studies
As shown in Table 1, among various oils examined, a mixing ratio
of LexolÕ to EmulmetikÕ 900 at 9:1 provided the highest
solubility of lutein, 2.12  0.08 mg/ml, and was chosen for further
investigations. In preliminary studies, ratios of 8:2, 7:3, 6:4 and
Table 1. Solubility of lutein in various vehicles.
Solubility
Vehicle Component (mg/ml)
Õ varying time intervals were examined. Lutein was rapidly released
Labrasol Surfactant 4.76  1.25
Tween 80 4.41  0.48
TranscutolÕ HP Co-surfactant 7.24  1.52
PEG 400 1.79  0.19
Propylene glycol 0.22  0.02
LexolÕ : EmulmetikÕ 900 (9:1) Oil 2.12  0.08
LexolÕ 1.82  0.24
Corn oil 0.93  0.09
Olive oil 0.75  0.11
Absorption of lutein SNEDDS in Caco-2 cells 3
5:5 produced turbid preparations, and hence were not employed.
Nonionic surfactants, LabrasolÕ (HLB 14) and Tween 80 (HLB
15), provided good solubility of lutein (4.76  1.25 and
4.41  0.48 mg/ml, respectively). Co-surfactant TranscutolÕ HP
was found to be an effective solubilizer for lutein with solubility
of 7.24  1.52 mg/ml, therefore it was used in the SNEDDS
development for drug loading improvement.
Construction of the pseudo-ternary phase diagram
Based on the results of solubility studies, pseudo-ternary
phase diagrams in the absence of lutein were constructed to
identify microemulsion and thus to determine the optimum
ratio of oil, surfactant and co-surfactant. The phase diagrams
were constructed for the following three systems; LabrasolÕ and
Tween 80, LabrasolÕ and TranscutolÕ HP, and Tween 80 and
TranscutolÕ HP.
The phase diagrams of the system containing LabrasolÕ
and Tween 80 showed the largest region of microemulsion
(Figure 1A) compared to preparations containing Tween 80 and
TranscutolÕ HP (Figure 1C) or LabrasolÕ and TranscutolÕ
HP (Figure 1B). TranscutolÕ HP enhanced the highest solubil-
ity of lutein but produced a narrower microemulsion region.
Therefore, the system containing LabrasolÕ and Tween 80, which
provide the largest microemulasion region, was chosen for further
investigations.
The effect of the ratios of LabrasolÕ to Tween 80 (7:3, 8:2 and
9:1) on the microemulsion region was investigated. The system
with LabrasolÕ to Tween 80 at a ratio of 7:3 showed the largest
microemulsion region followed by those containing a ratio of
8:2 and 9:1, respectively (data not shown). Increasing Tween 80
resulted in increased microemulsion region.
Physicochemical properties
Upon mixing lutein-loaded SNEDDS with water, oil-in-water
nanoemulsions formed rapidly without any precipitation even
after 1 day of storage. However, the system with LabrasolÕ and
Tween 80 at a ratio of 6:4 produced turbidity after 1 day of
storage. Regardless of formulation factors, TEM micrographs
of the nanoemulsions showed spherical droplets (Figure 2).
However, formulation factors affected the mean particle size.
It decreased from 154 to 111 nm upon an increase of surfactant
phase (Table 2). Increasing Tween 80 in LabrasolÕ and Tween 80
system decreased the droplet size from 139 to 36 nm (Table 2).
The zeta potential of all formulations was in the range of 19 and
32 mV.
Emulsification time of lutein-loaded SNEDDS was evaluated
as an important index for the assessment of the efficiency of self-
emulsification. Upon mixing with both medium (0.1 N HCl
and pH 6.8 phosphate buffer solution), all formulations exhibited
rapid self-nanoemulsification with emulsification time ranging
from 6 to 12 s (Table 2). No phase separation or aggregation was
observed in any of these formulations.
In vitro dissolution studies
The cumulative percent release of lutein form SNEDDS at
from SNEDDS, 90% within 5 min, and the release profile was
independent of formulation parameters. Moreover, similar drug
release pattern was observed for both dissolution mediums; 0.1 N
HCl and pH 6.8 phosphate buffer solution (Figure 3A and B).
On the other hand, lutein powder exhibited extremely poor
dissolution, 51% of lutein dissolved in 30 min in both medium.
As expected, the dissolution rate of SNEDDS was significantly
higher than that of lutein powder.
 4 P. Niamprem et al.
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For personal use only.
Figure 1. Pseudo-ternary phase diagrams of SNEDDS formulations. (A)
SNEDDS system I, (B) SNEDDS system II and (C) SNEDDS system III.
The gray area represents microemulsion regions.
Cytotoxicity and in vitro transport in Caco-2 cells
The surfactant in the SNEDDS formulations could damage the
intestinal epithelial barrier. Therefore, the potential cytotoxicity
of SNEDDS formulation on differentiated Caco-2 cells was
assessed by MTT assay. Cultures were incubated with varying
concentration of lutein-loaded SNEDDS for 6 h. Cell viability
Pharm Dev Technol, Early Online: 1–8
Figure 2. TEM micrograph of lutein-loaded SNEDDS OS8 at a
magnification of 120 000. Bar corresponds to 200 nm.
480% was considered as non-toxic25. At high lutein concentration
(30 mg/ml), the cell viability of SNEDDS LT1 and OS8 decreased
to 40% and 70%, respectively. On the contrary, SNEDDS tested
at lower lutein concentrations (10, 5, 3 and 1 mg/ml) possessed
cell viability 480% indicating no cytotoxicity. Thus, 3 mg/ml
of lutein-loaded SNEDDS was selected for transport studies (data
not shown).
The cellular accumulation of lutein-loaded SNEDDS OS8
was investigated comparing to lutein dispersion. As shown in
Figure 4, the cellular accumulations of lutein from SNEDDS and
lutein dispersion after 1-h incubation were 0.02 and 0.01 mg/
cm2, respectively; the difference was statistically significant
(p ¼ 0.002). Lag times of 3 and 4 h were observed with the
SNEDDS and lutein dispersion, respectively (Figure 5). However,
the permeation amount of lutein from SNEDDS was similar to
that of lutein dispersion (p ¼ 0.59). Therefore, the 6-h incubation
was chosen for further investigation of the effect of formulation
excipients on the transport. The lutein transport of different lutein-
loaded SNEDDS formulation was investigated using differentiated
Caco-2 cells. The lutein distribution of SNEDDS in apical
chamber, basolateral chamber and cell monolayer after 6-h
incubation was shown in Table 3. The percentage of lutein
recovery was 80% of the initial level. There were no significant
difference in percentage of lutein in apical and basolateral
chamber with varying formulation factors. However, the percent-
age of lutein in cell monolayer tended to increase with higher
ratio of surfactant phase and LabrasolÕ (Table 3). The TEER
value of cell monolayer before and after experiment was similar,
suggesting that the integrity of cell monolayer was maintained
during the in vitro absorption studies (data not shown).
Discussion
Lutein exhibits poor water solubility with a log octanol/water
partition coefficient (log Poct) of 14.8 resulting in low oral
bioavailability26. To overcome this problem, SNEDDS was
chosen as a delivery system as it could improve bioavailability
of lutein through surfactant-induced enhancement in its dissol-
ution. SNEDDS is an isotropic mixture of natural/synthesis oils,
surfactants/co-surfactants and drug. Upon mild agitation as the
 DOI: 10.3109/10837450.2013.829092 Absorption of lutein SNEDDS in Caco-2 cells 5
Table 2. Effect of formulation factors on physicochemical characterization of lutein-loaded SNEDDS.

Emulsification time (s) (mean  SD)

Mean droplet size Polydispersity index Zeta potential Phosphate buffer
Formulation Formulation factor (nm) (mean  SD) (mean  SD) (mV) (mean  SD) solution 0.1 N HCl
Ratio of oil phase: surfactant phase
OS7 3:7 154  14 0.173  0.015 32.94  2.94 12  2 71
OS7.5 2.5:7.5 138  4 0.186  0.019 30.41  1.93 91 61
OS8 2:8 111  8 0.263  0.012 25.01  1.78 71 71
Ratio of LabrasolÕ :Tween 80
LT1 9:1 139  9 0.228  0.036 31.26  1.89 10  2 71
LT2 8:2 111  8 0.263  0.012 25.01  1.78 71 71
LT3 7:3 36  6 0.291  0.022 19.01  7.35 81 61
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For personal use only.

Figure 3. Cumulative percentage released of lutein from lutein powder and lutein-loaded SNEDDS in (A) 0.1 N HCl and (B) pH 6.8 phosphate buffer
solution. Each value represents means  SD (n ¼ 3).

digestive motility and dilution with aqueous medium, oil-in-water
micro/nano-emulsion could be instantaneously formed and spread
rapidly in gastrointestinal tract9,10,13,14,18.
The components of developed SNEDDS (oil, surfactant and
co-surfactant) were selected based on the solubility and pseudo-
ternary phase diagram studies. Oil is a necessary ingredient
for SNEDDS because it can solubilize the lipophilic drug in
a specific amount, facilitate self-emulsification, and increase
enterocyte and intestinal lymphatic uptake, thereby increasing
lipophilic drug absorption from the gastrointestinal tract.
 6 P. Niamprem et al. Pharm Dev Technol, Early Online: 1–8
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Figure 4. Accumulative amount of lutein in Caco-2 cells monolayer. Each value represents means  SD (n ¼ 3).

Figure 5. Permeation amount of lutein across

Caco-2 cells monolayer. Each value repre-
sents means  SD (n ¼ 3).
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Table 3. The percentage of lutein in apical chamber, basolateral chamber and cell monolayer.

Lutein (%)
Formulation Formulation factor Apical Basolateral Cell
Lutein dispersion 64.92  2.51 11.70  0.33 0.94  0.10
Ratio of oil phase: surfactant phase
OS7 3:7 65.60  4.23 11.64  0.68 1.77  0.50
OS7.5 2.5:7.5 65.69  2.81 12.00  0.09 1.81  0.24
OS8 2:8 69.72  3.79 11.26  0.68 1.93  0.41
Ratio of LabrasolÕ :Tween 80
LT1 9:1 68.12  5.18 12.16  0.22 2.08  0.20
LT2 8:2 69.72  3.79 11.26  0.68 1.93  0.41
LT3 7:3 66.41  1.32 12.21  0.23 1.66  0.34

From the solubility study, the mixture of LexolÕ and
EmulmetikÕ 900 (9:1) was selected as an oil phase due to the
highest solubilizing effect (2.12  0.08 mg/ml). EmulmetikÕ 900
consists of 50% w/w phosphatidylcholine that could help
facilitate micelle formation16. In general, surfactant and co-
surfactant are preferentially absorbed at the interface, reducing
the interfacial energy to improve the thermodynamic stability of
microemulsions27. Upon dilution in aqueous solution, they assist
the immediate formation of oil in water droplets and rapid
spreading of the formulation in the medium. Based on pseudo-
ternary phase diagram studies, LabrasolÕ and Tween 80 were
selected as a surfactant phase. They are nonionic surfactants
 DOI: 10.3109/10837450.2013.829092
with HLB value of 14 and 15, respectively, which render them a
good affiance for self-emulsification. Even though, TranscutolÕ
HP, as a co-surfactant, showed the best solubilizing enhancement
effect for lutein, it provided comparatively less effective
emulsification at the narrowest microemulsion region than the
emulsification by the combined surfactants, LabrasolÕ and
Tween 80. The combined surfactant might have provided greater
HLB value and hence, the microemulsion region was greatly
increased in the phase diagram. A similar observation was
reported28,29. This result suggested that the selection of surfac-
tant should consider for both their emulsification and solubil-
ization efficiency. In addition, the microemulsion region
increased as the amount of Tween 80 was increased. This
might be explained by a greater HLB value of Tween 80 than
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LabrasolÕ , thus leading to a greater ability to produce water
soluble micelle.
The droplet size after micro/nano emulsification is one of the
most important property of SNEDDS formulation30. The smaller
droplet size (30 nm) leads to larger interfacial surface area for
drug absorption and provides a faster release rate of drug than
larger droplet size (150 nm)21. The mean droplet size prepared
here was in the range of 40–150 nm with a narrow distribution.
The ratio of oil phase to surfactant phase and the ratio of
LabrasolÕ to Tween 80 were found to have a major impact on the
mean droplet size. Increasing surfactant phase in SNEDDS
resulted in a decrease of mean droplet size from 154 to 111 nm.
This was attributed to a decrease of interfacial energy on the
surface of emulsion droplets12,18. However, when varying the ratio
of LabrasolÕ and Tween 80, a dramatic decrease in the mean
droplet size, from 139 to 40 nm, was observed with increasing
For personal use only.
amount of Tween 80. Although, LabrasolÕ has a lower molecular
weight than Tween 80, the latter showed higher HLB value that
may facilitate greater ability to reduce interfacial energy, resulting
in a smaller droplet size31. The zeta potential of the micro/nano-
emulsions of all formulations was in a range of j19j to j32j
mV, which are expected to inhibit aggregation because of charge
repulsion. In addition, lutein-loaded SNEDDS dispersed rapidly
and completely in 0.1 N HCl and pH 6.8 phosphate buffer solution
within 5 min, suggesting fast emulsification in the gastrointestinal
tract after intake of these formulations. The improvement of
lutein solubilization could be attributed to the high HLB value
of surfactant.
Even though SNEDDS appeared to be an effective approach
to increase lutein solubility as confirmed by the dissolution study,
the permeation study using Caco-2 cells model showed insignifi-
cant permeation profile between lutein dispersion and SNEDDS.
This could be attributed to two critical factors. Firstly, the
lipophilicity of lutein, shown by a high octanol/water partition
coefficient value of 14.8, resulted in high affinity of lutein to
the cell26. Even though lutein was able to rapidly dissolve
from SNEDDS and readily absorbed by the cell, it was slowly
partitioned to the medium, as evident from a lag time32. The
limitation of Caco-2 cells for very lipophilic drug has been
previously published32,33. Secondly, a limited number of recep-
tors/transporters capable of handling lutein may be responsible.
The transport process of lutein has been reported as passive
diffusion and facilitated process by a scavenger receptor class B
type I (SR-BI)26,34,35. Nevertheless, SNEDDS showed a shorter
lag time and a greater two-fold cellular accumulation than lutein
dispersion. Considering that, in human, there is a larger surface
area for absorption than in vitro study using Caco-2 cells, the
uptake of lutein SNEDDS would be faster and greater than lutein
dispersion32,36. Therefore, this study showed that the lag time and
cellular accumulation could be used as parameters to determine
the efficacy of lipophilic drug transport across Caco-2 cells.
In addition, Shanmugam et al. reported that SNEDDS could
Absorption of lutein SNEDDS in Caco-2 cells 7
improve lutein bioavailability compared to free drug in vivo
studies17.
Conclusion
In conclusion, the present study has clearly demonstrated the
potential of SNEDDS as a promising delivery system for lutein
to improve aqueous solubility and oral bioavailability by lutein
dissolution and absorption. The obtained lutein-loaded SNEDDS
performed rapid and complete self-nanoemulsification within
10 s, and lutein was completely dissolved in dissolution
medium within 5 min. Upon dilution in water, they possessed a
mean droplet size of 40–150 nm with a narrow distribution. The
improvement of lutein solubilization could be attributed to the
high HLB value of surfactant used in the formulation.
Additionally, the in vitro transport studies using Caco-2 cells
demonstrated that SNEDDS showed a shorter lag time and greater
two-fold cellular accumulation compared to lutein dispersion.
Therefore, lutein-loaded SNEDDS shows faster and greater
uptake than lutein dispersion, implying an increase in the oral
bioavailability of lutein. However, these assumptions may further
be confirmed by animal experimental followed by extensive
clinical evaluation.
Acknowledgements
The author would like to thank Associate Professor Srinivas SP for his
help in the manuscript preparation and discussion of the results.
Declaration of interest
This study was supported by Agricultural Research Development Agency
(Public organization) and Faculty of Pharmaceutical Sciences, Naresuan
University.
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