2013

http://informahealthcare.com/drdISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, 2013; 20(1): 10–18! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2012.742937

Potent enhancement of transdermal absorption and stability of humantyrosinase plasmid (pAH7/Tyr) by Tat peptide and an entrapment inelastic cationic niosomes

Jiradej Manosroi1,2, Narinthorn Khositsuntiwong1, Worapaka Manosroi3, Friedrich Gotz4, Rolf G. Werner5, andAranya Manosroi1,2

1Faculty of Pharmacy, 2Faculty of Pharmacy, Natural Product Research and Development Center (NPRDC), Science and Technology Research

Institute (STRI), 3Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 4Department of Microbial Genetics, Faculty of Biology, University

of Tubingen, Tubingen, Germany, and 5Boehringer Ingelheim Company, Ingelheim am Rhein, Germany

Abstract

Enhancement of transdermal absorption through rat skin and stability of the human tyrosinaseplasmid (P) using Tat (T) and an entrapment in elastic cationic niosomes (E) were described.E (Tween61:cholesterol:DDAB at 1:1:0.5 molar ratio) were prepared by the freeze-dried emptyliposomes (FDELs) method using 25% ethanol. TP was prepared by a simple mixing method.TPE was prepared by loading T and P in E at the T:P:E ratio of 0.5:1:160 w/w/w. For gelformulations, P, TP, PE and TPE were incorporated into Carbopol 980 gel (30 mg of plasmid per1 g of gel). For the transdermal absorption studies, the highest cumulative amounts andfluxes of the plasmid in viable epidermis and dermis (VED) were observed from the TPEof 0.31� 0.04 mg/cm2 and 1.86� 0.24mg/cm2/h (TPE solution); and 4.29� 0.40 mg/cm2 and25.73� 2.40mg/cm2/h (TPE gel), respectively. Only plasmid from the PE and TPE could be foundin the receiving solution with the cumulative amounts and fluxes at 6 h of 0.07� 0.01 mg/cm2

and 0.40� 0.08 mg/cm2/h (PE solution); 0.10� 0.01 mg/cm2 and 0.60� 0.06 mg/cm2/h (TPEsolution); 0.88� 0.03mg/cm2 and 5.30� 0.15 mg/cm2/h (PE gel); and 1.02� 0.05mg/cm2 and6.13� 0.28 mg/cm2/h (TPE gel), respectively. In stability studies, the plasmid still remained at4� 2 �C and 25� 2 �C of about 48.00–65.20% and 27.40–51.10% (solution); and 12.34–38.31%and 8.63–36.10% (gel), respectively, whereas at 45� 2 �C, almost all the plasmid was degraded.These studies indicated the high potential application of Tat and an entrapment in elasticcationic niosomes for the development of transdermal gene delivery system.

Keywords

Elastic cationic niosomes, human tyrosinaseplasmid, stability, Tat peptide, transdermalabsorption

History

Received 18 June 2012Revised 10 October 2012Accepted 19 October 2012

Introduction

Currently, transdermal drug delivery is one of the most

promising methods for drug administration. Increasing num-

bers of drugs are being added to the list of therapeutic agents

that can be delivered to the systemic circulation via skin

(Prausnitz et al., 2004). Currently available transdermal

delivery systems are scopolamine (hyoscine) for motion

sickness, clonidine and nitroglycerin for cardiovascular dis-

ease, fentanyl for chronic pain, nicotin to aid smoking

cessation, oestradiol (alone or in combination with levonor-

gestrel or norethisterone) for hormone replacement and tes-

tosterone for hypogonadism (Patel et al., 2011). The efficient

permeability of therapeutically active molecules through the

biological membranes remains the most important hurdle for

drug delivery. Elastic niosome is a non-ionic surfactant-based

nanovesicle composes of nanovesicular fluidized compounds

such as deoxycholate and ethanol (Choi & Maibach, 2005).

Novel non-ionic surfactant-based elastic niosomes, containing

ethanol as nanovesicular membrane fluidizer, was first

described by Manosroi et al. (2008). These nanovesicles can

squeeze themselves and pass through a small pore in stratum

corneum, which is smaller than their vesicular size. Hence,

these types of nanovesicles were more efficient in delivering

both low and high molecular weight drug in terms of quantity

and depth (Choi & Maibach, 2005). Elastic niosomes also

demonstrated prolonged release and better biological activ-

ity of the entrapped substances compared to conventional

niosomes (Bouwstra et al., 2003; Manosroi et al., 2010a).

Cell-penetrating peptides (CPPs), protein transduction

domains (PTDs) or membrane transduction peptides (MTPs)

consisting of 30 or less amino acids are classified as either

cationic or amphipathic and have the ability to cross the

cell membrane into cells. The ability of CPPs such as a

transcriptional activating (Tat) protein of human immunode-

ficiency virus type 1 and penetratin to translocate into the cell

is attributed to their amino acid sequence, which is mainly

contributed by basic amino acids (Desai et al., 2010).

Rothbard et al. (2000) were the first to report the applica-

tion of CPPs for the delivery of peptides into the skin.

Address for correspondence: Jiradej Manosroi, Faculty of Pharmacy,Chiang Mai University, Chiang Mai 50200, Thailand. Tel: þ66-53-894806. Fax: þ66-53-894169. E-mail: jiradej.manosroi8@gmail.com;pmpti006@chiangmai.ac.th

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The conjugate R7-CsA (polyarginine-7-cyclosporin A) can

release therapeutically-effective drug and its activity has been

shown in a dose-dependent manner both in vitro and in vivo.

Polylysine-9 (K9)- (Park et al., 2002) and Tat-coupled

(Kim et al., 2003; Eum et al., 2005) antioxidative enzymes

can translocate into epidermis and dermis. The Tat-linked

small peptide GKH (glycine-lysine-histidine) showed 36

times more absorption than the GKH (Lim et al., 2003).

This study investigated the transdermal absorption enhance-

ment of tyrosinase plasmid through rat skin by Tat peptide

and an entrapment in elastic cationic niosomes, and the

potential application for further development to vitiligo gene

therapy was anticipated.

Materials and methods

Materials

Human tyrosinase plasmid (pAH7/Tyr, P) was provided by

Boehringer Ingelheim Company, Germany. The map of the

pAH7/Tyr, containing 4986 base pairs with the CMV

promoter is shown in Figure 1. The Tat peptide

(GRKKRRQRRRPPQRKC) was purchased from Chengdu

KaiJie Bio-pharmaceutical Co., Ltd. (Chengdu, China).

Tween61 (polyoxyethelene sorbitan monostearate), choles-

terol and DDAB (dimethyl dioctadecyl ammonium bromide)

were from Sigma Chemicals, St. Louis, MO. Phenol/chloro-

form/isoamylalcohol and ethanol were analytical grade

reagents (Fluka, Buchs, Switzerland).

Preparation of elastic cationic niosomes

The 20 mM elastic cationic niosomes (E) were prepared by

the freeze-dried empty liposomes (FDELs) method (Kikuchi

et al., 1999). Briefly, Tween 61:cholesterol:DDAB at 1:1:0.5

molar ratio were mixed, placed in a clean, dry round-bottom

flask and dissolved in 10 ml of chloroform. The solvent was

removed by a rotary evaporator (R-124, Buchi, Flawil,

Switzerland) at 50� 2 �C. The resulting film was dried by

evacuation in a desiccator at room temperature (25� 2 �C)

under reduced pressure for over 12 h and rehydrated with

10 ml of distilled water at 50� 2 �C for 30 min. The

dispersion was sonicated using a microtip probe sonicator

(Vibra Cell�, Sonics & Materials, Inc., Newton, CT) at pulse

on 3.0, pulse off 1.0 with 33% amplitude for 15 min and

centrifuged at 2190g, 4 �C for 1 min. The dispersion was

further lyophilized overnight by a freeze-dryer (Alpha 1-2 LD

model, Christ, Osterode am Harz, Germany) and kept at 4 �Cuntil use. The lyophilized powder was reconstituted in 10 ml

of 25% ethanol and the resulting dispersion was further

sonicated at 4 �C in an ice bath for 15 min. The niosomal

dispersion was centrifuged at 2190g, 4 �C for 1 min, filtered

through a 0.45 mm membrane filter and kept at 4 �C until use.

Preparation of tyrosinase plasmid-loaded elasticcationic niosomes, Tat/tyrosinase plasmid andTat/tyrosinase plasmid/elastic cationic niosomes

The tyrosinase plasmid-loaded elastic cationic niosomes (PE)

at the P:E ratio of 1:160 w/w were prepared by incubating P

with 20 mM E at room temperature (25� 2 �C) for 1 h using

100 mg of P per 16 mg of E. For Tat/tyrosinase plasmid (TP)

and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE),

Tat (T) was incubated with tyrosinase plasmid (P) at a T/P

ratio of 0.5:1 w/w at room temperature for 30 min. The TP

obtained was further incubated with E at the same condition

as of PE resulting in the TPE.

Preparation of P, TP, PE and TPE gel

Gel base was prepared by dispersing 0.6 g Carbopol 980 in

36.6 g distilled water with continuous stirring at 25� 2 �C for

1 h. The 0.3 g conc. paraben (2% propyl paraben and 18%

methyl paraben in propylene glycol) was mixed with 1.0 g

triethanolamine, added to the Carbopol 980 dispersion and

mixed until a clear gel was obtained. The P, TP, PE or TPE

solution was added into the gel base with the final concen-

tration of 30 mg tyrosinase plasmid per 1 g of gel. For

transmission electron microscopy, gel containing P, TP, PE

or TPE was dissolved in 500 ml of distilled water. A drop

of the dissolved gel was applied on a 300-mesh formvar

copper grid and allowed to adhere for 10 min. The remaining

dispersion was removed and a drop of 2% ammonium

molybdate was added for 4 min. The remaining solution

was then removed and the grid was air-dried overnight.

The sample was examined with a transmission electron

microscope (TEM, Philips Tecnai� 10, FEI Company,

Eindhoven, the Netherlands) with 80 kV acceleration voltage,

objective diaphragm 4 (20 mm) and 100 mm condenser

aperture.

Vesicular size and zeta potential

Vesicular size and zeta potential of TP, PE and TPE solution

and gel formulations were determined by the dynamic light

scattering technique using Zetasizer Nano ZS (Malvern

Instrument, Malvern, UK), with DTSv5.0 software (Malvern

Instrument, Malvern, UK). All samples were diluted for 30

folds with freshly distilled water prior to both measurements.

The measurement was performed at 25 �C for five individual

runs. The medium used in these measurements was water,

which has the viscosity, refractive index and dielectric

constant of 0.8872 cP, 1.330 and 78.5, respectively.Figure 1. Human tyrosinase plasmid (pAH7/Tyr) map.

DOI: 10.3109/10717544.2012.742937 Transdermal absorption of TPE 11

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Stability study

Five milliliters of P, TP, PE or TPE solution and 5 g of P, TP,

PE or TPE gel was transferred to a clear glass-vial and kept

at 4� 2, 25� 2 and 45� 2 �C in a dark chamber for three

months. Samples were withdrawn at the predetermined time

intervals (initial, one, two and three months). The viscosity of

the gel formulations was determined by using the rotational

viscometer (VR 3000 model, Myr, Tarragona, Spain). The

experiment was performed at 25� 2 �C. The rheology

behavior of each gel formulation was evaluated by a graph

plotted between shear rate and shear stress. The plasmid was

extracted from P and TPE by mixing 100 ml of PE or TPE

with 100 ml of phenol:chloroform:isoamylalcohol (P:C:IAA,

25:24:1), vortex for 1 min and centrifuged at 13 680g at 4 �Cfor 10 min. The TP or extracted TPE containing 2.5 mg of

DNA were separately mixed with 20 ml of trypsin solution

(5mg of trypsin in 50 mM HEPES pH 7.4) and incubated at

room temperature (25� 2 �C) for 24 h to hydrolyze the T from

TP or TPE. Trypsin can hydrolyze the Tat peptide which

cleaves the peptide chains mainly at the carboxyl side of

the amino acids lysine or arginine, except when either is

followed by proline (Adami et al., 1998). This hydrolyzation

resulted in the separation of the Tat peptide from the TP.

The remaining plasmid was analyzed in 1% agarose gel

electrophoresis at 100 V for 40 min and determined the

plasmid band by gel documentation (Universal Hood,

BioRad Laboratory, Milan, Italy) with Quantity One program

analysis. Quantitative determination of the remaining plasmid

was performed using Quant-iTTM dsDNA BR assay kit

(Invitrogen, Karlsruhe, Germany).

Transdermal absorption by vertical Franz diffusion cell

Transdermal absorption through rat skin of P, TP, PE and

TPE in solution and gel formulations were performed in

triplicate using vertical Franz diffusion cells. Sixteen male

Sprague–Dawley rats (150–200 g and 10–12 weeks old)

obtained from National Laboratory Animal Centre, Mahidol

University, Thailand, were used. The abdominal skin of

anesthetized rat was shaved and excised. The subcutaneous

fat was removed using a scalpel blade and mounted on the

receiving chamber with the stratum corneum (SC) side facing

upward. The area between the donor and the receiving

chamber of the diffusion cell was 2.46 cm2. The receiving

chamber was filled with 13 ml of phosphate-buffered saline

(PBS, pH 7.0), controlled at 37� 2 �C and stirred with a small

magnetic bar at 100 rpm throughout the experiment. One

milliliter of the solution or 1 g of gel formulation (containing

30 mg of tyrosinase plasmid) was added into the donor

chamber and covered with the parafilm. The experiments

were stopped at 30 min, 1, 3 and 6 h. The skin were removed

and swirled in 100 ml of distilled water of at least 1 min for

three times. The receiving solution was collected, lyophilized

and analyzed for P by gel electrophoresis and gel documen-

tation. The P in SC was assayed by the stripping method using

a Scotch Magic� tape (3M, St. Paul, MN; 1 cm� 1 cm)

(Dutkiewicz et al., 2000) and put into a glass bottle containing

3 ml of PBS. The stripped skin was cut into small pieces and

put into a glass bottle together with 3 ml of PBS. The P

contents in the stripped tapes and the viable epidermis and

dermis (VED) were extracted by adding 3 ml of P:C:IAA

(25:24:1), vortexed and centrifuged at 8780g, 4 �C for 10 min.

The aqueous phase was collected and assayed for the P

contents. For TP or TPE, the P in various parts of skin was

assayed by mixing with 20 ml of trypsin solution (5 mg of

trypsin in 50 mM HEPES pH 7.4) and incubated at room

temperature (25� 2 �C) for 24 h to hydrolyze the T from the

TP or TPE, respectively. The P concentration was determined

in 1% agarose gel electrophoresis using 10 ml of the sample

and run at 100 V for 40 min. The plasmid band density

was determined by gel documentation (ImageMaster� VDS,

Pharmacia Biotech, Freiburg, Germany) and the P concen-

tration was calculated from the calibration curve of the

plot between the band densities and tyrosinase plasmid

concentrations.

Results and discussion

Vesicular size and zeta potential of P, TP, PE and TPEsolution and gel formulations

Physical appearances of all gel formulations have been

observed. The gel base was clear and the gel appearance

Figure 2. TEM images of the PE (A) and TPE (B) in the gel formulations at the magnification of �20 000.

12 J. Manosroi et al. Drug Deliv, 2013; 20(1): 10–18

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was not changed when the P and TP solutions were

incorporated, while the transparency of the gel was decreased

when the PE and TPE complexes were added. The TEM

images of the PE and TPE in gel formulation are demonstrated

in Figure 2(A) and (B), respectively. The nanovesicles were

observed in irregular shape with the vesicular size of about

100–300 nm. The vesicular sizes and zeta potential values of P,

E, TP, PE and TPE solution and gel formulations are shown in

Table 1. TP, PE and TPE solutions exhibited larger vesicular

sizes (206.4� 10.9, 165.0� 2.8 and 123.5�8.0 nm, respec-

tively) than the E solution (100.2� 0.9 nm). The zeta potential

values of P and TP solutions were �38.6� 1.4 and

�9.7� 1.9 mV, respectively, whereas the E, PE and TPE

solutions demonstrated the positively charged of 42.6� 5.0,

37.9� 1.4 and 32.5� 7.2 mV, respectively. Since the nega-

tively charged plasmid could be located both inside the

aqueous phase between the vesicular bilayers and adsorbed

outside the vesicular membrane of the elastic cationic

niosomes, the charge repulsion between the plasmid molecules

might increase the vesicular size (Bouwstra & Honeywell-

Nguyen, 2002; Bouwstra et al., 2003). The plasmid might also

neutralize the positive charges of the vesicular surface resulting

in lowering of the zeta potential of the PE and TPE. The gel

formulations demonstrated larger vesicular size and lower zeta

potential values than the solution formulations. This might be

due to the network structure of the Carbopol gel that may

interfere with the light scattering intensity resulting in the

vesicular size in micrometer range (Manosroi et al., 2012).

Since Carbopol is a polymer of the acrylic acid cross-linked

with the polyalkenyl ethers or divinyl glycol providing

carboxyl groups by the acrylic acid backbone (Avinash et al.,

2006), the negative charge of the carboxyl groups in the

Carbopol polymer may neutralize the positive charges of the

cationic niosomes resulting in less negative zeta potential

values of PE and TPE gel (�44.1� 1.1 and �44.3� 2.3 mV,

respectively) than the P and TP gel (�62.2� 2.4 and

�51.9� 3.3 mV, respectively). The charge neutralization

may be beneficial for the loaded cationic niosomes in

Carbopol gel to have better physical stability (Jo et al., 2004).

Stability study

The viscosity and rheology of the gel containing either P, TP,

PE or TPE are shown in Table 2 and Figure 3, respectively.

All gel formulations exhibited the Non-Newtonian, pseudo-

plastic behavior. Table 2 presents the increases in vesicular

size observed in almost all gel formulations at all tempera-

tures after three months except in PE and TPE at 4� 2 �C.

For the solution formulations, only E, PE and TPE exhibited

the stable dispersion, whereas the P and TP solutions were

unstable when kept at 45� 2 �C for three months. The size

enlargement of the pAH7/Tyr loaded nanovesicles after three

months of storage might be due to the self-assembly between

the vesicular surface. Based on freeze-fracture electron

micrographs and X-ray diffraction studies, it was suggested

that DNA is sandwiched between many nanovesicles

(Sternberg et al., 1994; Radler et al., 1997). This structure

was in arrangement with the increase of vesicular size

(Almofti et al., 2003). Moreover, the negatively charged DNA

would neutralize cationic nanovesicles, resulting in aggrega-

tion and continuous fusion with time. All gel formulations

demonstrated the zeta potential values within the stable

dispersion range (out of �30 mV) at all temperatures after

three months of storage. The percentages remaining of the

Table 2. Vesicular sizes and zeta potential values of tyrosinase plasmid (pAH7/Tyr, P), Tat peptide (T), blank elastic cationic niosomes (E),Tat/tyrosinase complexes (TP), tyrosinase plasmid loaded elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomescomplexes (TPE) at initial and at 4� 2 �C, 25� 2 �C and 45� 2 �C after three months of storage.

Vesicular size (nm) Zeta potential (mV)

DosageAfter three months After three months

form Sample Initial 4� 2 �C 25� 2 �C 45� 2 �C Initial 4� 2 �C 25� 2 �C 45� 2 �C

Solution T NA NA NA NA 12.85� 4.0 NA NA NAP 799.7� 133.4 1163.0� 308.9 1234.7� 123.6 1584.0� 517.4 �38.6� 1.4 �54.0� 3.8 �45.7� 6.0 �27.8� 2.7E 100.2� 0.9 112.5� 11.7 164.4� 14.9 171.0� 8.9 42.6� 5.0 25.9� 1.4 33.6� 11.9 51.8� 3.7

TP 206.4� 10.9 456.8� 56.8 614.5� 71.3 833.5� 232.2 �9.7� 1.9 �16.3� 1.8 �3.6� 0.5 �2.5� 0.2PE 165.0� 2.8 114.9� 1.1 132.0� 1.9 133.9� 2.3 37.9� 1.4 37.5� 8.9 36.9� 6.9 32.4� 1.6

TPE 123.5� 8.0 103.2� 3.0 116.4� 4.8 156.9� 1.5 32.5� 7.2 35.7� 0.9 36.2� 4.9 33.8� 0.9

Gel T NA NA NA NA �31.4� 1.8 NA NA NAP 2656.2� 291.5 4477.3� 586.0 6647.0� 991.5 23636.7� 704.0 �62.2� 2.4 �44.0� 3.1 �40.8� 3.0 �34.2� 1.7E 5444.0� 467.3 12928.7� 654.8 14740.0� 415.8 48794.7� 663.2 �46.9� 42.3 �54.7� 2.9 �52.2� 5.8 �55.5� 2.0

TP 2585.4� 548.1 6336.7� 878.1 17916.7� 592.1 21366.0� 953.8 �51.9� 3.3 �46.1� 5.9 �43.7� 0.7 �39.5� 1.0PE 3828.0� 873.2 2828.3� 953.8 5409.7� 898.1 6209.7� 196.6 �44.1� 1.1 �50.3� 3.8 �50.7� 3.4 �45.7� 0.5

TPE 2658.8� 657.2 1315.0� 271.9 2911.7� 728.0 5976.7� 1588.8 �44.3� 2.3 �44.7� 0.5 �51.4� 6.1 �39.5� 3.3

The values are represented as mean� S.D. (n¼ 3). NA: Not applicable.

Table 1. Viscosity values (cP) of tyrosinase plasmid (pAH7/Tyr, P),Tat/tyrosinase plasmid complexes (TP), tyrosinase plasmid-loadedelastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elasticcationic niosomes complexes (TPE) gel formulations.

Viscosity (cP)

RPM P gel TP gel PE gel TPE gel

10 7990 3670 4020 324012 6920 3310 3560 304020 4730 2410 2650 217030 3480 1810 1980 163050 2320 1240 1380 110060 2030 1140 1270 980

100 1450 840 960 730200 910 560 630 500

DOI: 10.3109/10717544.2012.742937 Transdermal absorption of TPE 13

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Fig

ure

3.

Rh

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beh

avio

ro

f(A

)gel

bas

e,(B

)E

gel

,(C

)P

gel

,(D

)T

Pgel

,(E

)P

Egel

and

(F)

TP

Eg

el.

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plasmid in various solution and gel formulations kept at 4� 2,

25� 2 and 45� 2 �C for three months are shown in Figure 4.

Comparing with the initial concentration (100%), significant

(p50.05) decreases in the percentages remaining of the

plasmid were observed from the P and TP solutions kept at all

temperatures, whereas the PE and TPE solutions kept at

4� 2 �C and 25� 2 �C for one month showed slight decreases

in the percentages remaining of the plasmid. The highest

percentages remaining of the plasmid in solution and gel

formulations were observed at 4� 2 �C followed by 25� 2 �Cand 45� 2 �C. At the first and second month, the remaining of

the plasmid from PE and TPE solutions and gel kept at all

temperatures were ranging from 67.60–97.10% to 61.70–

97.50% (solution); and 51.52–77.65% to 51.25–74.81% (gel),

respectively, which were higher than the remaining plasmid

from P and TP solution and gel formulations. In the third

month, the plasmid was still observed from all solution and

gel formulations kept at 4� 2 and 25� 2 �C of about 48.00–

65.20% and 27.40–51.10% (solution); and 12.34–38.31% and

8.63–36.10% (gel), respectively, whereas the plasmid in the

gel formulations kept at 45� 2 �C was almost degraded.

These results indicated that the loading of the tyrosinase

plasmid in the elastic cationic niosomes in the form of free

plasmid or TP could enhance the thermal stability of the

Figure 4. Percentages remaining of the human tyrosinase plasmid (pAH7/Tyr) in free plasmid (P), Tat/tyrosinase plasmid (TP), tyrosinase plasmid-loaded elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) solution and gel formulations kept at 4� 2 �C,25� 2 �C and 45� 2 �C for 3 months. The * indicates significant (p50.05) difference of the percentages remaining of the plasmid compared to theinitial (100%).

Figure 5. Cumulative amounts (A) and fluxes (B) of the pAH7/Tyr in viable epidermis and dermis of free tyrosinase plasmid (P), Tat/tyrosinaseplasmid (TP), tyrosinase plasmid-loaded elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) solutionformulations.

DOI: 10.3109/10717544.2012.742937 Transdermal absorption of TPE 15

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Figure 8. Cumulative amounts (A) and fluxes (B) of the pAH7/Tyr in the receiving solution of free tyrosinase plasmid (P), Tat/tyrosinase plasmid (TP),tyrosinase plasmid-loaded elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) gel formulations.

Figure 7. Cumulative amounts (A) and fluxes (B) of the pAH7/Tyr in viable epidermis and dermis of free tyrosinase plasmid (P), Tat/tyrosinaseplasmid (TP), tyrosinase plasmid-loaded elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) gel formulations.

Figure 6. Cumulative amounts (A) and fluxes (B) of the pAH7/Tyr in the receiving solution of free tyrosinase plasmid (P), Tat/tyrosinase plasmid (TP),tyrosinase plasmid-loaded elastic cationic niosomes (PE) and Tat/tyrosinase plasmid/elastic cationic niosomes (TPE) solution formulations.

16 J. Manosroi et al. Drug Deliv, 2013; 20(1): 10–18

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loaded plasmid since the vesicular membrane might act as a

shield to protect the loaded plasmid from thermal and stress

of the environment (Varshosaz et al., 2003).

Transdermal absorption through rat skin

Transdermal absorption through rat skin of P from free P, TP,

PE and TPE were investigated by vertical Franz diffusion cell

for 6 h. In solution formulations, P was not observed from all

formulations in the SC layer, whereas P from all samples was

observed in VED at 0.5, 1, 3 and 6 h. Only P from PE and

TPE was found in the receiving solution at 3 and 6 h

(data not shown). The highest cumulative amount and flux

of the P in VED were observed from the TPE at 6 h of

0.31� 0.04 mg/cm2 and 1.86� 0.24 mg/cm2/h, respectively

(Figure 5A and B), which was 2.38 folds of P. TP and PE

demonstrated the cumulative amount and flux in VED of 1.38

and 2.23 folds of P, respectively. The maximum cumulative

amounts and fluxes of the P from PE and TPE in the receiving

solution were observed at 6 h of 0.07� 0.01 mg/cm2 and

0.40� 0.08 mg/cm2/h (PE) and 0.10� 0.01 mg/cm2 and

0.60� 0.06 mg/cm2/h (TPE), respectively (Figure 6A and B),

whereas the P from free P and TP could not be observed.

These might be due to the positive charge of the PE and TPE,

which could be attached to the negatively charged of the skin

via electrostatic interaction. In contrast, the negatively

charged P and TP could not pass through the skin due o the

charge repulsion effect between the negative charge of the P

or TP and the negative charge of the skin (Prausnitz et al.,

1993). Moreover, the high transdermal absorption through rat

skin of the PE and TPE might be due to the elasticity of the

elastic cationic niosomes that could squeeze through the small

pore in the stratum corneum, which is smaller than their

vesicular size into the deeper layer of the skin (Choi &

Maibach, 2005). Furthermore, the ethanol composition of the

elastic niosomal formulation might play an important role in

the enhancement of the transdermal absorption of the loaded

plasmid. Mechanisms of ethanol on skin permeation enhance-

ment have been reported, including an increase of drug

diffusion through the lipid pathway of the skin (Hatanaka

et al., 1993), the reduction of lipid polar head interactions or

disordering liquid–crystalline phases within the membrane

(Knutson et al., 1990) and an increase of drug solubility in

the SC (Megrab et al., 1995). The TPE which exhibited

lower positive charge than the PE showed higher cumulative

amounts and fluxes of the P in both VED and receiving

solution. This result indicating that the T peptide played an

important role over the charge interaction between the TPE

and the skin. This result was correlated to previous studies

which showed that the interaction of the CPPs including

Tat peptide with skin lipids may be the main transport

across the SC, since this interaction may destabilize the SC

resulting in an increase in the membrane permeability

(Rothbard et al., 2000). Another suggested mechanism of

transport was via the tight junctions which allowed penetra-

tion into the viable skin layers (Lopes et al., 2008). Kang et al.

(2010) studied the in vitro and in vivo skin penetration

effect of Tat-coated elastic liposomes, they found that the

Tat peptide could increase the flux of the liposomes by

about 20%.

For the gel formulations, the cumulative amounts and fluxes

of the plasmid in VED are shown in Figure 7(A) and (B).

The highest cumulative amount and flux of the plasmid in VED

was observed from the TPE gel at 6 h of 4.29� 0.40 mg/cm2

and 25.73� 2.40 mg/cm2/h, respectively, which was 5.39 folds

of P. For TP and PE gel formulations, the cumulative amount

and flux of these formulations in VED were 0.88 and 2.12 folds

of P, respectively. Only plasmid from PE and TPE gel was

found in the receiving solution with the highest cumulative

amount and flux from the TPE gel at 6 h of 1.02� 0.05 mg/cm2

and 6.13� 0.28 mg/cm2/h, respectively (Figure 8A and B).

Higher cumulative amounts and fluxes of the plasmid in

various parts of skin of the gel formulations over the solution

formulations indicated that the gel structure can promote the

penetration of the plasmid DNA owing to the occlusion effects

from the gel formulation, which can enhance skin hydration

and consequently increase the absorption and penetration of

the plasmid DNA across the rat skin (Manosroi et al., 2010b).

The results of this study indicated the potent enhancement of

transdermal absorption through rat skin and the thermal

stability of the human tyrosinase plasmid (pAH7/Tyr) using

the Tat peptide incorporated elastic cationic niosomes both in

solution and gel formulations.

Conclusion

The transdermal absorption through rat skin and thermal

stability of the human tyrosinase plasmid (pAH7/Tyr) could

be potently enhanced using Tat peptide and an entrapment in

elastic cationic niosomes. The TPE solution and gel formu-

lations demonstrated a higher cumulative amount and flux of

the P in the VED and receiving solution and also percentage

remaining of the plasmid than the P, TP and PE solution

and gel formulations kept at 4� 2, 25� 2 and 45� 2 �C for

three months. From the results of this study, the application

of the TPE as a transdermal gene delivery system was

anticipated.

Acknowledgements

This work was supported by the Thailand Research Fund

(TRF) under the RGJ-PhD program, Natural Products

Research and Development Center (NPRDC), Science

and Technology Research Institute (STRI), Chiang Mai

University, Chiang Mai 50200, Thailand. The authors grate-

fully acknowledge the supporting of Boehringer Ingelheim

and University of Tubingen, Germany, for providing tyrosi-

nase (pAH7/Tyr) plasmid used in this study.

Declaration of interest

The authors report no declarations of interest.

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