PHARMACEUTICAL NANOTECHNOLOGY
Transdermal Absorption Enhancement of N-Terminal Tat GFP
Fusion Protein (TG) Loaded in Novel Low-Toxic Elastic Anionic
Niosomes
JIRADEJ MANOSROI,1,2 WARANGKANA LOHCHAROENKAL,1 FRIEDRICH GÖTZ,3 ROLF G. WERNER,4
WORAPAKA MANOSROI,5 ARANYA MANOSROI1,2
1
Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand
2
Natural Product Research and Development Center, Science and Technology Research Institute, Chiang Mai University,
Chiang Mai 50200, Thailand
3
Department of Microbial Genetics, Faculty of Biology, University of Tuebingen, Germany
4
Boehringer Ingelheim Company, Ingelheim am Rhein, Germany
5
Faculty of Medicines, Chiang Mai University, Chiang Mai 50200, Thailand
Received 14 May 2010; revised 13 July 2010; accepted 25 August 2010
Published online 1 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22355
ABSTRACT: Elastic anionic niosomes (Tween 61/cholesterol/dicetyl phosphate at 1:1:0.05
molar ratio of 20 mM) with various concentrations of ethanol and edge activators sodium
cholate (NaC) and sodium deoxycholate (NaDC) showed larger vesicular size (171.94 Ä… 63.52 
683.17 Ä… 331.47 nm) and higher negative zeta potential (-6.45 Ä… 2.76 to - 17.40 Ä… 2.51 mV)
than the nonelastic anionic niosomes. The elasticity (deformability index) and entrapment
efficiency of all elastic vesicles except the NaDC vesicles were higher than the nonelastic vesicles.
The morphology, under transmission electron microscope, of elastic and nonelastic niosomes
loaded and not loaded with Tat green fluorescent protein fusion protein (TG) were in large
unilamellar structure. TG loaded in elastic (1 mol% NaC) anionic niosomes gave the highest cell
viability both in HT-29 (92.32 Ä… 3.82%) and KB cells (96.62 Ä… 5.96%), the highest cumulative
amounts (62.75 Ä… 2.68 :g/cm2) and fluxes (10.46 Ä… 3.45 :g/cm2h) in receiving chamber in
rat skin transdermal study by Franz diffusion cells. This study has not only indicated the
synergistic enhancement effects of the Tat peptide and the niosomal delivery system on the
cellular uptake and transdermal absorption of TG but also 1 mol% NaC as an edge activator to
obtain a novel low-toxic elastic anionic niosomes for topical use of therapeutic macromolecules
such as proteins, as well. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association
J Pharm Sci 100:1525 1534, 2011
Keywords: Tat GFP; Nanoparticles; Cytotoxicity; Transdermal; Cell culture; Protein
delivery; Peptide
INTRODUCTION veloped to increase the entrapment efficiency of the
ionized substances. However, charged bilayer vesicles
Niosomes are nonionic surfactant-based vesicles,
showed significantly higher toxicity in comparing to
which have the same bilayer structures as phospho-
neutral vesicles with the toxicity order of cationic vesi-
lipid vesicles or liposomes. They have low cost, high
cles > anionic vesicles > neutral vesicles.2,3 The most
stability, and are able to encapsulate both water-
recent vesicle design is elastic liquid-state vesicles,
and oil-soluble substances to serve as drug and cos-
which differ from the conventional vesicles by their
metic carriers.1 The charged bilayer vesicles were de-
membrane characteristics with high fluid and elastic-
ity. High membrane elasticity of this elastic vesicle
Correspondence to: Aranya Manosroi (Telephone: +665-389-
was suggested to be crucial to push them through
4806; Fax: [phone]; E-mail: [e-mail])
a pore smaller than their average diameters. This
Journal of Pharmaceutical Sciences, Vol. 100, 1525 1534 (2011)
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association will be important for the success of the noninvasive
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011 1525
1526 MANOSROI ET AL.
carrier-mediated material to be transported across modified Eagle s medium with 10% fetal bovine
the skin.4 6 Ethanol is known as an efficient per- serum, 100 :/mL penicillin, and 100 :g/mL strep-
meation enhancer, which has been widely used in tomycin. All cell lines were grown at 37ć%C under
niosomal formulation for the enhancement of drug 5% CO2.
transport across the skin.7,8 However, because of the
Expression of TG Fusion Protein
interdigitation effect of ethanol on the lipid bilayers,
vesicles cannot coexist with high concentrations of The TG fusion protein was expressed from pTG in
ethanol. Also, high content of ethanol could affect the Escherichia coli (E. coli). Briefly, E. coli BL21 was
membrane structure and increase the cytotoxicity.9 transformed with plasmids encoding TG fusion pro-
Recently, edge activators such as sodium cholate tein by calcium chloride method. The transformed
(NaC) and sodium deoxycholate (NaDC) have been cells were cultured in Luria Bertani broth supple-
used to prepare elastic vesicles instead of ethanol.10 12 mented with 30:g/mL kanamycin (Carl Roth GmbH,
These edge activators are often single-chain surfac- Karlsruhe, Germany). Protein expression was in-
tants that can destabilize the lipid bilayers of the duced by adding isopropyl $-D-1-thiogalactopyrano-
vesicles and increase the elasticity of the vesicles.13 side (Sigma) to a final concentration of 0.1 mM. The
Elastic vesicles have been shown to penetrate intact induced cells were harvested, lysed by lysozyme, and
skin via transdermal osmotic gradients and hydra- sonicated. After the removal of the cell debris by
tion forces, which could be a useful tool for transder- centrifugation, the supernatant was loaded onto a
mal drug delivery.12 In this study, the novel low-toxic nickel nitrilotriacetic acid column (Qiagen GmbH,
elastic anionic niosomes containing two edge activa- Hilden, Germany). The protein was eluted by an
tors including NaC and NaDC were developed. N- eluting buffer (500 mM imidazole, 300 mM NaCl,
terminal Tat fusion green fluorescent protein (GFP) 50 mM NaH2PO4·H2O, pH 8.0) and determined for
(TG), a fusion protein between the GFP and Tat [a the protein contents by a Bradford protein assay
cell-penetrating peptide (CPP) from HIV type I] was kit (Bio-Rad, Milan, Italy) using bovine serum al-
used as a model loaded protein in the elastic and bumin (Carl Roth GmbH, Karlsruhe, Germany) as a
nonelastic anionic niosomes in order to evaluate the standard.15 Fluorescent intensity was determined by
potential synergistic effects of the Tat peptide and the a multimode reader (Beckman, California) with exci-
niosomal delivery systems by the evaluation of cellu- tation and emission wavelength at 485 and 535 nm,
lar internalization assay in HT-29 (colon adenocarci- respectively.
noma) and KB (human mouth epidermal carcinoma)
Incubation Time and Cellular Uptake Efficiency of TG
cells, and rat skin permeation study by Franz diffu-
sion cells. The effect of incubation time on uptake efficiency of
TG in comparing to GFP was performed in HT-29
cells. The cells were grown to 90% confluence in 24-
MATERIALS AND METHODS
well plates and the culture medium was removed. The
cells were washed twice with phosphate buffer saline
Materials
(PBS) and then incubated with GFP or TG at 1 :M
pGFP and pTG were from our previous study.14 The
at various incubation times. The supernatant was re-
male Sprague Dawley rats weighing between 150
moved and the cells were washed twice with PBS.
and 200 g were from National Laboratory Animal
The cells were lysed by the ProteoJetTM mammalian
Centre, Mahidol University, Salaya, Nakhon Pathom,
cell lysis reagent (Fermentas, Ontario, Canada) and
Thailand. Cholesterol (CHL, 99.6%), polyoxyethylene
the fluorescent intensity of the supernatant was de-
sorbitan monostearate (Tween 61), dicetylphosphate
termined by a multimode reader with excitation and
(DP), NaC, and NaDC were from Sigma Chemical
emission wavelength at 485 and 535 nm, respectively.
Co. (St. Louis, Missouri). Chloroform and methanol
The amount of the uptake proteins were calculated
(analytical grade) were from LabScan (Bangkok,
from the plot between the fusion proteins concentra-
Thailand). Absolute ethanol (analytical grade) and
tions and the fluorescent intensity.
sodium hydrogen phosphate anhydrous (Na2HPO4)
Preparation of Blank and TG-Loaded Elastic
were from Merck Company (Damstadt, Germany).
and Nonelastic Anionic Niosomes
Sodium dihydrogen orthophosphate (NaH2PO4) was
from VWR International Ltd., West Sussex, UK. All
Empty nonelastic anionic niosomes were prepared by
other reagents were of analytical grade and used
the freeze-dried empty liposome (FDEL) method.16
without further purification.
Briefly, Tween 61, CHL, and the anionic lipid (DP)
at 1:1:0.05 molar ratio were dissolved in 1:1 volume
Cell Culture
ratio of chloroform and methanol. The solvent was re-
KB (human mouth epidermal carcinoma) and HT-29 moved by a rotary evaporator (R-124 Buchi, Flacil1,
(colon adenocarcinoma) were cultured in Dulbecco s Switzerland) to get a thin film. The film was dried by
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011 DOI 10.1002/jps
TRANSDERMAL OF N-TERMINAL TAT GFP FUSION PROTEIN ELASTIC NIOSOMES 1527
evacuation in a desiccator under reduced pressure for formvar for 10 min. The remaining dispersion was
more than 12 h. Phosphate buffer (100 mM, pH 7.0) removed and a drop of 2% aqueous solution of ammo-
was added to the film, and the dispersion was swelled nium molybdate was applied for 5 min. The remaining
by swirling in a water bath at 45ć%C for 30 min. The re- solution was then removed. The sample was air dried
sulting dispersion was sonicated by a microtip probe and examined with a TEM.
sonicator (Vibra Cell TM, Sonics & Materials, Inc.,
Determination of Entrapment Efficiency
Newtown, Connecticut) at pulse on 3.0 and pulse off
2.0, 25% amplitude, for 10 min and centrifuged to
The amounts of the loaded TG were determined by
remove titanium particles. The dispersion was then
gel electrophoresis and gel documentation (Universal
lyophilized by a freeze dryer (Alpha 1 2 LD Model
Hood, Bio-Rad).17 The gel electrophoresis was carried
Christ, Osterode am Harz, Germany) at room temper-
on a 12% SDS-PAGE in Tris glycine system buffer at
ature (27ć%C) and pressure at 0.25 mbar for 12 h, and
a constant voltage (100 mV) for 150 min. The gel was
kept at 4ć%C until use. TG in phosphate buffer at 1:M
stained by coomassie brilliant blue R-250. The non-
was loaded in nonelastic anionic niosomes by recon-
loaded TG was determined from the band on the gel
stitution the lyophilized empty vesicular powder with
located at the same level as the purified TG, whereas
1 :M TG in phosphate buffer solution (100 mM, pH
the loaded TG remained at the site of application to-
7.0) for 10 min at room temperature (30 ą 2ć%C). Elas-
gether with the niosomes. The band densities were de-
tic anionic niosomes loaded with TG were prepared
termined by the quantity one program analysis, and
by reconstitution of the lyophilized niosomal powder
the entrapment efficiency was calculated as the fol-
with ethanol at 5% 25% in phosphate buffer instead
lowing equation:
of the phosphate buffer only. For edge activators, NaC

or NaDC at various concentrations (1 25 mol%) was
Itotal - Id
added in the step of film forming to the mixture of Entrapment efficiency (% ) = × 100
Itotal
Tween 61, CHL, and DP dissolved in 1:1 volume ra-
tio of chloroform and methanol and dehydrated with
where, Itotal was the band densities of the total pro-
phosphate buffer to obtain elastic niosomes contain-
teins initially loaded in niosomes and Id was the band
ing edge activators.
densities of the nonloaded protein.
Physical Characteristics of Vesicles
Determination of Deformability Index
The Vesicular Sizes and Å› (Zeta) Potential
The elasticity values in the term of deformabil-
Determination
ity index (DI) were determined by the extrusion
The sizes of the vesicles were measured at 25ć%C by
method.18,19 Briefly, an amount of 10 mL of the nio-
a dynamic light scattering (Zetasizer Nano Series
somal dispersion was extruded through a polycar-
Nano-S, Malvern instrument Ltd., Malvern, UK) us-
bonate membrane filter (50 nm pore size; Millipore,
ing the nonnegative constrained least squares algo-
Massachusetts) at a constant pressure (2.5 bars) for
rithm mode particle size distribution analysis. The
10 min. The DI was then calculated according to the
measurements were performed in triplicate with
following equation:
three cycles in each measurement. The measurement
conditions were set at 30 s with 10 s pulse between cy- 2
rv
cles. The .(zeta) potential values of all samples were
DI = j x
rp
obtained by the phase analysis light scattering soft-
ware (Malvern instrument Ltd., Malvern, UK). The
time-dependent correlation function on the scattered where j is the weight of the sample extruded in 10 min
light intensity was measured at a scattering angle of through a polycarbonate membrane filter, rv is the
90ć%. The measurements were performed in triplicate size of the vesicles after extrusion, and rp is the pore
with five cycles in each measurement. size of the membrane filter.
Visualization of Vesicles Sulforhodamine B Assay
TG and non-TG-loaded elastic anionic niosomes The cytotoxicity of TG-loaded elastic and nonelastic
containing NaC at 1 mol% were observed by a anionic niosomes on HT-29 and KB cells were deter-
transmission electron microscope (TEM; 80 kV, mined in a 96-well plate by sulforhodamine B (SRB)
TEM1200SJEOL, JEOL Ltd., Tokyo, Japan) using assay.20 Briefly, cells were seeded into 96-well-plate
negative staining technique employing 2% (w/v) of (1 × 104 cells/well) and left overnight. The adherent
ammonium molybdate solution. A drop of the disper- cells were exposed to the sample for 1 h. After the
sion was applied on a 300-mesh formvar copper grid samples were removed, the adherent cells were fixed
on paraffin and allowed the sample to adhere on the in situ by 50 :L of cold 50% (w/v) trichloroacetic acid
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
1528 MANOSROI ET AL.
(Merck) and kept for 60 min at 4ć%C. The supernatant buffer, TG in 100 mM phosphate buffer, TG loaded in
was then discarded, washed for three times with dis- nonelastic anionic niosomes, and TG loaded in elas-
tilled water, and air dried. SRB solution was added tic anionic niosomes. The GFP and TG concentration
and the cells were allowed to be stained for 30 min at used were at 10 :M.
room temperature (27 ą 2ć%C). The unbound SRB was The rat skin was mounted on the receptor com-
removed by washing three times with 1% acetic acid partment of the vertical Franz diffusion cells with
solution. The plates were then air dried and the cell- the stratum corneum (SC) side facing upwards to
bound stain was dissolved with the unbuffered Tris the donor compartment. The donor compartment was
base solution (Sigma Chemical Co.). The optical den- filled with 0.5 mL of the samples. An amount of 14 mL
sity was measured at 540 nm on a microplate reader of PBS, pH 7.4, was put in the receiver chamber. The
(Bio-Rad). The cytotoxicity was evaluated from the available diffusion area (rat skin) was 2.46 cm2. The
cell growth inhibition in the treated versus the un- receiver compartment was maintained at 37 ą 2ć%C
treated cells. The negative control for 100% of cell vi- and stirred by a magnetic bar. All experiments were
ability was the untreated cells. The viability (%) was performed in triplicate and carried out with nonoc-
calculated in comparing to the untreated cells. The de- cluded donor compartments. At 1, 3, and 6 h, the dif-
crease of viability (%) represented the cytotoxic effect fusion cells were withdrawn. The skin was removed
of the sample, whereas the increase of viability (%) from the cell and swung twice in 100 mL of distilled
represented the proliferation effect. Cell viability (%) water, and the rinsed water was discarded. For SC,
was calculated according to the following equation: the amount of the fluorescent protein in SC was col-
lected by stripping the skin with a 3M Scotch MagicTM
tape (1 × 1cm2, 3M, St. Paul, MN, USA).21 Nine tapes
Cell viability (% )
were used for each skin sample and pooled in a 5 mL
optical density at 540 nm of the treated cells
vial. The tapes were cut into small pieces and 3 mL of
=
100 mM phosphate buffer were added. The vial was
optical density at 540 nm of the untreated cells
vortexed for 5 min, allowed to stand for 10 min, and
× 100
vortexed again for 5 min. For the stripped skin [viable
epidermis and dermis (VED)], it was cut into small
Cellular Uptake of TG-Loaded Elastic and Nonelastic pieces and pooled in 1.5 mL microcentrifuge tube con-
Anionic Niosomes taining 1 mL of 100 mM phosphate buffer, vortexed
for 5 min, allowed to stand for 10 min, and vortexed
Cellular uptake of TG-loaded elastic and nonelastic
again for 5 min. All samples including the receiver so-
anionic niosomes was performed in HT-29 and KB
lution were centrifuged at 22,400 g for 10 min and the
cells as described in  Incubation Time and Cellular
supernatants were collected for the determination of
Uptake Efficiency of TG. . One hour incubation time
the fluorescent intensity. The amounts of GFP or TG
was used for all samples. The untreated cells were
in each sample at various time intervals were calcu-
used as a blank in order to determine the nonspecific
lated from the standard curve between the fluorescent
absorption.
intensity and the GFP or TG concentrations.
Transdermal Absorption Through Rat Skin by Vertical
Franz Diffusion Cells
Preparation of Rat Skin RESULTS AND DISCUSSION
Full-thickness abdominal skin was obtained from the The Effect of Incubation Time on Cellular Uptake
male Sprague Dawley rats (National Laboratory An- Efficiency
imal Centre, Mahidol University), weighing between
The effect of incubation time on cellular uptake effi-
150 and 200 g. Hair on abdominal area was shaved
ciency of TG in comparing to GFP in HT-29 cells was
off and left overnight. The rats were killed, and the
shown in Figure 1. The fusion protein TG showed
abdominal skin was removed. The subcutaneous fat
rapidly uptake into HT-29 cells with superior cellular
was trimmed off, and the prepared skin was freshly
uptake efficiency in comparison with GFP. The up-
used. The investigational protocol for all procedures
take efficiency of TG was almost the same when the
has adhered to the  Principles of Laboratory Animal
incubation times increased from 0 to 1 h. However,
Care.
the uptake efficiency of TG decreased when the incu-
bation time increased to 2 h. This might be due to the
Transdermal Absorption Experiment
less amount of viable cells at 2 h incubation than at
The samples used for the permeability study through 1 h incubation. Thus, 1 h incubation time was selected
the excised rat skin were GFP in 100 mM phosphate for further study.
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TRANSDERMAL OF N-TERMINAL TAT GFP FUSION PROTEIN ELASTIC NIOSOMES 1529
Figure 1. The amount of uptake proteins (:M) into HT-29 cells at various incubation time
(h). The data were presented in mean Ä… SD (n = 3).
Preparation and Characterization of Nonelastic ing ethanol or NaC or NaDC were comparable except
and Elastic Anionic Niosomes the niosomes containing 5% NaDC, which showed the
largest vesicular size (683.17 Ä… 331.47 nm). The ef-
After reconstituted in 100 mM phosphate buffer, fects of ethanol concentrations on the vesicular size
both elastic and nonelastic empty niosomal dispersion in this study were conformed to the previous study,22
showed physical stability with no sedimentation for which showed that the average vesicular size was
3 months at room temperature (30 ą 2ć%C). However, increased when the ethanol concentration was in-
niosomes containing NaDC at 20 and 25 mol% after creased from 3.3% to 20%. An increase vesicular sizes
3 months gave precipitation. The vesicle sizes and . of niosomes by adding NaC and NaDC were observed.
(zeta) potential values of all niosomes were shown in This may be due to the electrostatic repulsion between
Table 1. Sizes of elastic anionic niosomes were larger the negative charges of DP and NaC or NaDC.23 The
than nonelastic niosomes (50.96 Ä… 10.93 nm) with zeta potential values of all elastic anionic niosomes
the size range between 171.94 Ä… 63.52 and 683.17 Ä… were more negative than the nonelastic anionic nio-
331.47 nm. Sizes of elastic anionic niosomes contain- somes owing to ethanol or NaC or NaDC, which may
Table 1. Vesicle Sizes, Zeta Potential and Deformability Index (DI) of Blank Elastic and Nonelastic Anionic Niosomes (Tween 61/CHL/DP
at 1:1:0.05 Molar Ratio) Containing Ethanol, NaC, or NaDC at Various Concentrations
Niosomal Formulations Size (nm) Zeta Potential (mV) DI
Nonelastic anionic niosomes 50.96 Ä… 10.93 -4.73 Ä… 0.94 4.81 Ä… 0.76
Elastic anionic niosomes containing
5% (v/v) ethanol 221.50 Ä… 6.48 -8.73 Ä… 0.45 2.01 Ä… 0.35
10% (v/v) ethanol 307.20 Ä… 83.84 -7.79 Ä… 0.17 5.59 Ä… 0.67
15% (v/v) ethanol 339.40 Ä… 12.46 -17.4 Ä… 2.51 7.25 Ä… 0.03
20% (v/v) ethanol 269.30 Ä… 39.08 -17.00 Ä… 3.42 10.53 Ä… 0.02
25% (v/v) ethanol 341.20 Ä… 64.35 -9.08 Ä… 0.28 10.92 Ä… 0.13
1 mol% NaC 221.57 Ä… 84.65 -6.45 Ä… 2.76 5.68 Ä… 5.73
2.5 mol% NaC 171.94 Ä… 63.52 -6.89 Ä… 4.71 8.09 Ä… 9.51
5 mol% NaC 336.87 Ä… 239.32 -8.58 Ä… 1.44 7.14 Ä… 9.66
10 mol% NaC 429.90 Ä… 294.09 -9.49 Ä… 2.95.96 Ä… 4.23
15 mol% NaC 307.60 Ä… 18.52 -9.93 Ä… 2.47 2.79 Ä… 2.69
20 mol% NaC 212.08 Ä… 114.5 -12.60 Ä… 1.54 0.69 Ä… 0.61
25 mol% NaC 273.40 Ä… 8.56 -9.32 Ä… 1.54 1.38 Ä… 0.81
5 mol% NaDC 683.17 Ä… 331.47 -10.80 Ä… 1.89 1.87 Ä… 2.03
10 mol% NaDC 258.13 Ä… 78.44 -10.90 Ä… 1.01 3.71 Ä… 4.41
15 mol% NaDC 349.90 Ä… 47.85 -11.10 Ä… 1.98 0.92 Ä… 1.28
20 mol% NaDC 258.87 Ä… 65.32 -11.10 Ä… 1.62.04 Ä… 2.84
25 mol% NaDC 278.5 Ä… 18.73 -11.70 Ä… 1.01 1.08 Ä… 0.94
The experiments were performed in triplicate.
CHL, cholesterol; DP, dicetylphosphate; NaC, sodium cholate; NaDC, sodium deoxycholate.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
1530 MANOSROI ET AL.
Figure 2. The negative staining TEM images of (a) blanck elastic anionic niosomes (Tween61/
CHL/DP/NaC at 1:1:0.02 molar ratio, 1 mol% NaC) (b) TG loaded elastic anionic niosomes.
impart the negative charges to the vesicles.12,24 The small vesicle sizes, which may be easily with higher
morphology of non-TG-loaded and TG-loaded elastic amount extruded through the 50 nm polycarbonate
anionic niosomes (with 1 mol% NaC) were shown in membrane, but also the elastic properties from the
Figure 2. Both TG-loaded and non-TG-loaded elas- component of Tween 61, a surfactant in polysorbate
tic anionic niosomes were in spherical shape with group, which can also act as an edge activator.26
large unilamellar vesicular structures. The flat con-
Entrapment Efficiency
tact was observed at the contact surface between the
vesicles. Fluorescent spots were detected in the TG-
Entrapment efficiencies of TG loaded in vesicles
loaded elastic anionic niosomes with high proportion
were in the range of 32.09 Ä… 0.09 88.14 Ä… 5.83%
at the vesicular membranes.
(Table 2). Higher entrapment efficiency (33.34 Ä…
12.78  47.02 Ä… 15.47%) in ethanol-containing
Elasticity of the Vesicles
Deformability is an important feature of elastic vesi- Table 2. Entrapment Efficiencies of TG Loaded in Different
cles that can be differentiated from other lipid dis- Elastic Vesicular Formulations
perse systems, which are typically nonelastic. The
Entrapment
DI of all formulations was shown in Table 1. They
Niosomal Formulations Efficiency (%)
were increased when the ethanol concentrations were
Nonelastic anionic niosomes 32.09 Ä… 0.09
increased from 5% to 25%. Ethanol might interact
(Tween 61/CHL/DP at 1:1:0.05 molar ratio)
with the polar head-group region of the surfactant
Elastic anionic niosomes containing
molecules in niosomes, resulting in the reduction of 5% (v/v) ethanol 33.34 Ä… 12.78
10% (v/v) ethanol 32.36 Ä… 5.63
melting point, thereby increasing the fluidity of the
15% (v/v) ethanol 47.02 Ä… 15.47
vesicles.7,25 Elastic anionic niosomes containing edge
20% (v/v) ethanol 44.98 Ä… 9.56
activators showed the highest DI values of 8.09 Ä…
25% (v/v) ethanol 32.76 Ä… 5.85
9.51 and 3.71 Ä… 4.41 for 2.5 mol% NaC and 10 mol%
1 mol% NaC 32.36 Ä… 42.72
NaDC, respectively, which were about 1.68 and 0.77 2.5 mol% NaC 54.29 Ä… 10.42
5 mol% NaC 66.83 Ä… 19.49
times of nonelastic anionic niosomes. These edge acti-
10 mol% NaC 62.58 Ä… 22.08
vators may decrease the transition temperature (Tm)
15 mol% NaC 81.81 Ä… 10.84
of the vesicles and induce the phase transition of the
20 mol% NaC 74.51 Ä… 13.49
vesicles to liquid crystalline phase resulting in the
25 mol% NaC 68.96 Ä… 18.99
increase of vesicle elasticity. However, after the max- 5mol%NaDC 78.39 Ä… 11.92
10 mol% NaDC 41.07 Ä… 7.76
imum DI value, the deformability decreased when
15 mol% NaDC 82.50 Ä… 9.43
the edge activator concentrations increased. This may
20 mol% NaDC 84.21 Ä… 9.18
be due to the abolishment of the pretransition en-
25 mol% NaDC 88.14 Ä… 5.83
dotherm of the vesicles resulting in the increase of
The experiments were performed in triplicate.
vesicular rigidity.26 Nonelastic anionic niosomes also
CHL, cholesterol; DP, dicetylphosphate; NaC, sodium cholate; NaDC,
showed some elastic property because of not only their sodium deoxycholate.
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TRANSDERMAL OF N-TERMINAL TAT GFP FUSION PROTEIN ELASTIC NIOSOMES 1531
niosomes than the nonelastic niosomes (32.09 Ä… TG loaded in various nanovesicles were performed in
0.09%) might be due to the solubility enhancement of HT-29 and KB cells again in this study to confirm the
ethanol on TG that may facilitate the entrapment.27 hypothesis. In addition, the cytotoxicity test in this
Niosomes containing NaC (32.36 Ä… 42.72  81.81 Ä… study has also aimed to detect the general toxicity on
10.84%) and NaDC (41.07 Ä… 7.76  88.14 Ä… 5.83%) the basal functions of cells, which can certainly be car-
also showed higher entrapment efficiency than the ried out on many cell types such as fibroblasts, HeLa,
nonelastic vesicles (32.09 Ä… 0.09%). This may be due hepatoma cell, KB, HT-29, and others cell lines.29
to the larger vesicular size from the incorporation of The TG concentration at 1 :M was selected to load
NaC and NaDC in the niosomal membranes result- in niosomal formulations as higher concentration of
ing in the higher entrapment amount of TG in the 2.5 10 :M gave less than 50% of cell viability in HT-
hydrophilic core.28 29 and KB cells. The concentrations of all niosomal
formulations used in this study were 20 mM because
the difference concentration of niosomes from 5 to
Cytotoxicity and Cellular Uptake of TG Loaded
20 mM showed no significant difference in cell via-
in Elastic and Nonelastic Niosomes in HT-29
bility (data not shown). The cell viability of free TG in
and KB Cell Lines
HT-29 and KB cells was 62.46 Ä… 5.45% and 65.86 Ä…
HT-29 and KB cells were used for cytotoxicity test in
7.43%, respectively. The loading of TG in nonelastic
this study. This was based on our previous study. GFP
anionic niosomes increased cell viability to 72.90 Ä…
and the fusion proteins including Tat-fusion GFP both
21.86 and 82.57 Ä… 2.02%. The effects of TG on cell
C-terminal (GFP-Tat, GT) and N-terminal (Tat-GFP,
viability by loading in elastic anionic niosomes ap-
TG) were produced and the cellular uptake of GFP
peared to depend on the concentration of ethanol and
and the fusion proteins in these cells (colon and mouth
edge activators. Viability of HT-29 and KB cells de-
carcinoma cells) were compared. We have found that
creased when the concentrations of ethanol and edge
TG gave the highest uptake efficiency, but still less
activators increased (Table 3). However, the elastic
than 6%. This may be from its cytotoxicity. So, the
anionic niosomes containing 1 mol% NaC showed the
SRB assay for cytotoxicity determination of these pro- highest cell viability of 92.32 Ä… 3.82 and 96.62 Ä…
teins was performed in these cells. The results showed
5.96% in HT-29 and KB cells, respectively, followed by
that after exposed to 1:M of TG, the viability of these
the ethanol and NaDC-containing elastic niosomes.
cells were about 60%.14 Hence, nanovesicular tech- NaC has been reported as the least toxic edge acti-
nology was used in this study in order to decrease
vator in comparison with NaDC and Tween 80. Sev-
cytotoxicity and enhance the cellular uptake of TG.
eral studies have revealed that NaDC could damage
Thus, the cellular uptake and cytotoxicity test of the
plasma and nuclear membranes, whereas NaC was
Table 3. Cytotoxicity and Cellular Uptake of TG Loaded in Elastic and Nonelastic Niosomes in HT-29 and KB Cells
Viability Uptake
Samples HT-29 KB HT-29 KB
Free TG 62.46 Ä… 5.45 65.86 Ä… 7.43 5.21 Ä… 0.24 5.40 Ä… 0.79
TG loaded nonelastic anionic niosomes 72.90 Ä… 21.86 82.57 Ä… 2.02 2.78 Ä… 0.67 3.37 Ä… 0.12
(Tween 61/CHL/DP at 1:1:0.05 molar ratio)
TG loaded elastic anionic niosomes containing
5% (v/v) ethanol 76.44 Ä… 6.09 84.33 Ä… 10.52 14.56 Ä… 0.99 15.55 Ä… 0.65
10% (v/v) ethanol 65.69 Ä… 2.64 82.46 Ä… 10.88 13.78 Ä… 0.44 15.76 Ä… 1.04
15% (v/v) ethanol 52.55 Ä… 17.73 79.89 Ä… 2.25 14.31 Ä… 0.26 15.43 Ä… 1.23
20% (v/v) ethanol 45.04 Ä… 5.02 70.55 Ä… 3.35 14.76 Ä… 0.51 16.16 Ä… 1.11
25% (v/v) ethanol 37.29 Ä… 0.67 62.48 Ä… 5.58 14.62 Ä… 0.07 15.32 Ä… 0.96
1 mol% NaC 92.32 Ä… 3.82 96.62 Ä… 5.96 14.99 Ä… 0.31 16.54 Ä… 0.52
2.5 mol% NaC 90.70 Ä… 4.66 89.59 Ä… 3.11 14.34 Ä… 0.18 16.42 Ä… 0.58
5 mol% NaC 85.44 Ä… 5.41 87.15 Ä… 14.46 14.15 Ä… 0.29 16.62 Ä… 1.53
10 mol% NaC 76.49 Ä… 10.04 82.57 Ä… 7.83 14.94 Ä… 0.49 16.47 Ä… 0.94
15 mol% NaC 34.41 Ä… 26.76 68.87 Ä… 10.50 15.14 Ä… 0.49 13.49 Ä… 1.08
20 mol% NaC 29.81 Ä… 1.77 54.62 Ä… 7.87 13.76 Ä… 0.38 14.98 Ä… 0.84
25 mol% NaC 23.51 Ä… 7.48 57.14 Ä… 5.11 13.66 Ä… 0.75 14.31 Ä… 0.64
5mol%NaDC 26.98 Ä… 3.55 36.14 Ä… 5.91 14.85 Ä… 0.12 12.74 Ä… 1.31
10 mol% NaDC 15.82 Ä… 13.65 39.04 Ä… 13.49 13.28 Ä… 0.48 15.89 Ä… 1.49
15 mol% NaDC 10.77 Ä… 9.69 28.99 Ä… 9.67 13.46 Ä… 0.57 16.21 Ä… 0.30
20 mol% NaDC 29.35 Ä… 10.95 40.06 Ä… 8.78 15.95 Ä… 0.78 14.21 Ä… 0.29
25 mol% NaDC 26.98 Ä… 12.21 45.07 Ä… 10.10 14.91 Ä… 0.27 15.45 Ä… 1.31
The experiments were performed in triplicate.
CHL, cholesterol; DP, dicetylphosphate; NaC, sodium cholate; NaDC, sodium deoxycholate.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
1532 MANOSROI ET AL.
less toxic.30,31 However, more than 90% of cell via- ferences of the uptake efficiency of the TG loaded in
bility in comparison with the negative control was various charged elastic niosomes and the differences
obtained from NaC and NaDC incorporated in the between the TG loaded in liposomes and niosomes. At
vesicles. However, the mechanism of higher toxicity 1 h incubation with TG loaded in neutral, cationic,
of NaDC was still unknown.11 For ethanol, its high and anionic elastic niosomes, the HT-29 cellular up-
contents may increase the leakage of cell membrane take efficiency of TG was 7.35%, 8.57%, and 14.62%,
leading to the decrease of cell viability.9 respectively. In comparing the uptake efficiency of TG
TG in buffered solution showed uptake efficiency of loaded in elastic anionic liposomes with TG loaded in
5.21 Ä… 0.24% and 5.40 Ä… 0.79% in HT-29 and KB cells, elastic anionic niosomes, twofold of TG uptake was
respectively. The entrapment of TG in elastic anionic observed in TG loaded in elastic anionic niosomes af-
niosomes but not in nonelastic anionic niosomes en- ter 1 h incubation.14. However, the uptake efficiencies
hanced the cellular uptake in both cell lines. The en- at 1 h in this present study were all nearly the same
hancement of cellular uptake of TG loaded in elastic at 12% 17%. This was due to the similarity of phys-
anionic niosomes in comparison with the nonelastic ical properties such as charge and lipid compositions
anionic niosomes in HT-29 and KB cells were demon- of all tested niosomal preparations (Tween 61/CHL/
strated in Table 3. All elastic vesicles showed similar DP = 1:1:0.05).
uptake efficiency of TG in the range of 13.28 Ä… 0.48%
Transdermal Absorption Through Rat Skin
 15.95 Ä… 0.78% in HT-29 cell and 12.74 Ä… 1.31%
 16.62 Ä… 1.53% in KB cells, whereas the nonelas- TG loaded in elastic anionic niosomes containing
tic anionic niosomes gave only 2.78 Ä… 0.67% and 1 mol% of NaC, which gave the highest cell viabil-
3.37 Ä… 0.12% in HT-29 and KB cells, respectively. ity, was selected for permeation study in rat skin by
The enhancement of cellular internalization of TG Franz diffusion cells in comparison with TG loaded
might be due to the ethanol or edge activators (NaC/ in nonelastic anionic niosomes, TG solution in phos-
NaDC) composition in the elastic niosomal formula- phate buffer, and GFP solution in phosphate buffer.
tion. Ethanol could increase membrane permeabil- Cumulative amounts (:g/cm2) and fluxes (:g/cm2h)
ity by interact with the hydrophobic core of Tween of GFP or TG from various systems in SC, VED, and
61 and CHL in the vesicular membrane,32 whereas receiver compartment solution at 6 h were shown in
the edge activators (NaC/NaDC) have the steroidal Table 4. The temperature in the receiving chamber,
backbone similar to CHL that can not only have an which represented the systemic circulation, was ther-
edge-activating effect but also a vesicular membrane- mostated at 37ć%C according to the body core temper-
stabilizing effect. These edge activators can interca- ature, which is about 37ć%C.34,35 Both unloaded and
late into the vesicular membrane resulting in the loaded TG were found in the receiving chamber so-
disordering of the bilayers, thereby producing the lution. TG loaded in 1 mol% NaC elastic anionic nio-
thinner and flexible vesicles that may facilitate somes gave the highest cumulative amounts (62.75 Ä…
the cellular uptake.33 2.68 :g/cm2) and fluxes (10.46 Ä… 3.45 :g/cm2h) in the
The effect of incubation time of more than 1 h in the receiving chamber, which were about twofold higher
cellular uptake study was not evaluated in this study than TG loaded in nonelastic anionic niosomes. This
because our preliminary study has shown that this has demonstrated the transdermal enhancement of
incubation time was long enough to obtain the dif- TG when loaded in elastic vesicles composed of 1 mol%
Table 4. The Cumulative Amounts (:g/cm2) and Fluxes (:g/cm2h) of GFP and TG from Various Systems in SC, VED, and Receiver
Compartment Following Transdermal Absorption Across Excised Rat Skin at 6 h by Vertical Franz Diffusion Cells
Cumulative Amounts (:g/cm2) Ä… SD Fluxes [:g/(cm2 ·h)] Ä… SD
Receiver Receiver
Systems SC VED Compartment SC VED Compartment
GFP solution in 100 mM phosphate buffer 24.15 Ä… 7.28 17.92 Ä… 0.78 22.61 Ä… 2.87 4.02 Ä… 1.21 2.99 Ä… 0.26 3.77 Ä… 0.48
(pH 7.0)
TG solution in 100 mM phosphate buffer 4.86 Ä… 0.55 6.06 Ä… 0.70 28.50 Ä… 12.07 0.81 Ä… 0.09 1.01 Ä… 0.70 4.75 Ä… 1.47
(pH 7.0)
TG loaded in non-elastic anionic niosomes 22.03 Ä… 4.01 0 30.24 Ä… 4.81 3.67 Ä… 2.33 0 5.04 Ä… 0.80
(Tween 61/CHL/DP at 1:1:0.05 molar
ratio)
TG loaded in elastic anionic niosomes 8.59 Ä… 1.33 0 62.75 Ä… 2.68 1.43 Ä… 0.22 0 10.46 Ä… 3.45
(Tween 61/CHL/DP at 1:1:0.05 molar
ratio) containing 1 mol% of NaC
Each value represented the mean Ä… SD (n = 3).
SC, stratum corneum; VED, viable epidermis and dermis; CHL, cholesterol; DP, dicetylphosphate; NaC, sodium cholate; NaDC, sodium deoxycholate.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011 DOI 10.1002/jps
TRANSDERMAL OF N-TERMINAL TAT GFP FUSION PROTEIN ELASTIC NIOSOMES 1533
NaC. In the receiver compartment, the cumulative ergistic effects of the Tat peptide and the niosomal
amounts and fluxes of TG in buffered solution were delivery in vitro have been demonstrated because the
similar to TG loaded in the nonelastic vesicles, but enhancement of cellular uptake of TG in both cells
higher than GFP in buffered solution. This may be was observed, when TG was loaded in elastic an-
due to the presence of the Tat peptide that contained ionic niosomes. Elastic anionic niosomes containing
highly positive charged amino acids, which can facil- NaC at 1 mol%, which gave the least cytotoxicity and
itate the endocytosis of TG resulting in the higher high cellular uptake of TG, were selected for the rat
amount of TG in the receiver compartment in com- skin transdermal experiment by Franz diffusion cells.
parison with SC and VED.36,37 However, only small The permeation through rat skin was compared be-
amount of TG was still detected in SC and VED due tween GFP in buffered solution, TG in buffered solu-
to the relative high molecular mass (<"30 kDa) of TG. tion, TG loaded in nonelastic niosomes, and elastic (1
When TG loaded in niosomes, the nanosize and the mol% NaC) anionic niosomes. TG loaded in elastic an-
phospholipid bilayer-like structure of niosomes could ionic niosomes gave the highest cumulative amounts
improve the partitioning of TG across the SC into the (62.75 Ä… 2.68 :g/cm2) and fluxes (10.46 Ä… 3.45 :g/
deeper layers of the skin without the accumulation in cm2h) in the receiving solution followed by TG loaded
VED, especially in NaC-containing niosomes, which in nonelastic niosomes, TG in buffered solution, and
has more influence on the lipid bilayer fluidity.8 GFP in buffered solution, respectively. This has sug-
gested the potential application of synergistic effects
of the niosomal delivery system and the Tat peptide
CONCLUSION
for an efficient systemic delivery of macromolecules
Various concentrations of ethanol and edge activa- such as therapeutic proteins.
tors (NaC and NaDC) were used to prepare the elas-
tic anionic niosomes composed of Tween 61/CHL/
DP at 1:1:0.05 molar ratio loaded with TG by the
ACKNOWLEDGMENTS
FDEL method. Sizes of all elastic anionic niosomes
This work was supported by the Thailand Research
were larger than the nonelastic niosomes with the
Fund (TRF) under the RGJ-PhD program, Natural
size range between 171.94 Ä… 63.52 and 683.17 Ä…
Product Research and Development Center (NPRDC),
331.47 nm. The zeta potential values of all nioso-
Science and Technology Research Institute (STRI),
mal formulations were negative, with the elastic nio-
and Nanoscience and Nanotechnology Research
somes showed higher negative zeta potential values
Center Project, Faculty of Sciences, Chiang Mai
[(-)17.00 Ä… 3.42 (-)6.45 Ä… 2.76] than the nonelastic
University, Thailand.
niosomes [(-)4.73 Ä… 0.94]. Elastic anionic niosomes
showed the increase DI values from 2.01 Ä… 0.35 to
10.92 Ä… 0.13 when the ethanol concentrations were
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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011 DOI 10.1002/jps