rna interference ( rnai ) offers potential for treating a wide variety of disorders through selective silencing of disease - relevant rnas [ 1 , 2 ] . topical use of small interfering rna ( sirna ) has been increasingly studied for its applicability in treating skin disease . however , transdermal naked sirna delivery is limited due to its low permeability of skin barriers such as the stratum corneum and the epidermal layer . of particular interest are cationic liposomes for passive transdermal delivery of sirna , since their opposite charges spontaneously result in complexation due to electrostatic interactions . it has been reported that elastic cationic liposomes ( ultradeformable cationic liposomes ) can penetrate intact skin while carrying plasmid dna ( pdna )   and antisense oligonucleotides   when applied under nonoccluded conditions . elastic cationic liposomes consist of a cationic lipid and an edge activator that is responsible for the elasticity of the membrane . an edge activator is usually a single - chain surfactant that destabilizes the lipid bilayers of the liposomes and increases the deformability of the bilayers . for transdermal gene delivery , sodium cholate ( nachol ) , sodium deoxycholate ( nadchol ) , and tween 80 were used as an edge activator [ 5 , 6 , 8 ] ; however , there is little information about the feasibility and effect of an edge activator on elastic liposomes for transdermal sirna delivery in vivo . deformability of elastic liposome enables therapeutic sirna to penetrate the skin barrier by the stratum corneum . a cationic lipid , 1,2-dioleoyl-3-trimethylammonium propane ( dotap ) , has frequently been used as a cationic lipid for a liposomal delivery system of sirna by several research groups [ 912 ] . moreover , we previously reported that nachol and tween 80 incorporated into liposomal membranes confer deformation and flexibility to ultradeformable liposomes [ 13 , 14 ] . therefore , in the present study , for transdermal sirna delivery , we optimized the formulation of elastic dotap liposomes including nachol or tween 80 , which are positively charged liposomes exhibiting some deformability , and evaluated the permeability of sirna in mouse skin after topical application of elastic liposome / sirna complex ( elastic lipoplex ) . sodium cholate hydrate ( nachol ) was purchased from sigma - aldrich japan ( tokyo , japan ) . lissamine rhodamine b 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( rhodamine - dhpe ) , was purchased from invitrogen ( carlsbad , ca , usa ) . the sirnas targeting nucleotides of firefly luciferase ( luc sirna ) and nonsilencing sirna ( cont sirna ) as a negative control were synthesized by sigma genosys ( tokyo , japan ) . the sirna sequences of the luc sirna were passenger strand : 5-ccgugguguucgugucuaaga-3 and guide strand : 5-uuagacacgaacaccacggua-3 . the sirna sequences of the cont sirna were passenger strand : 5-ccguacuagccauuaugcguc-3 and guide strand : 5-cgcauaauggcuaguacgggu-3. 6-carboxyfluorescein - aminohexyl - amidite- ( fam- ) labeled luciferase ( pgl3 ) sirna was obtained from cosmo bio co. , ltd . the sirna sequences of the fam - labeled luciferase sirna were passenger strand : 5-ucgaaguacucagcguaagdtdt-3 and guide strand : 5-cuuacgcugaguacuucgadtdt-3. nachol and tween 80 were dissolved in phosphate - buffered saline ( pbs , ph 7.4 ) by vortexing . dotap liposomes were prepared by vortexing in pbs with a concentration of 10  mg / ml dotap until a milky suspension was obtained as previously reported . elastic liposomes were prepared by vortexing after adding nachol or tween 80 solution to dotap solution at final concentrations of 5 , 10 , and 15% ( w / w ) ( table 1 ) . for preparation of rhodamine - labeled liposomes , rhodamine - dhpe was incorporated into the liposomal formulation at 0.2  mol% in the total lipid . the liposomes were sonicated in a bath - type sonicator ( branson 25010j - mth , usa ) for 1  h at room temperature and then filtered 31 times through 100  nm polycarbonate membrane filters ( whatman , brentfort , uk ) . lipoplexes were prepared by mixing the liposome with sirna at a weight ratio of dotap / sirna of 14/1 ( charge ratio ( + / ) of 6/1 ) , as previously reported , and then incubated at room temperature for 1015  min . average diameters and -potentials of liposomes and lipoplexes were measured by dynamic light - scattering and electrophoresis light - scattering methods , respectively ( els - z2 ; otsuka electronics , osaka , japan ) . all measurements were performed at 25  1c , after diluting the liposome and lipoplex suspension with milli - q water . the elasticity of the bilayer of liposomes was directly proportional to jflux  ( rv / rp ) : 
 ( 1)elasticity  ( gsec1cm2)=jflux(rvrp)2 , 
 where jflux is the rate of penetration through a permeability barrier , rv is the size of liposomes after extrusion , and rp is the pore size of the barrier : 
 ( 2)jflux(gsec1cm2 )  = j(dotap(g))(extrusion  time(sec)membrane  area(cm2 ) ) . to measure j , the liposomes were extruded through a polycarbonate membrane ( 4.9  cm)with a pore diameter of 50  nm ( rp ) , at a pressure of 0.5  mpa . after 10  min of extrusion , the extrudate was weighed , and then concentration of dotap in the extrudate was calculated by measurement of absorbance at 228  nm after the addition of equal volume of methanol . the average liposome diameter after extrusion ( rv ) was measured by els - z2 as described in the above section . human cervical carcinoma siha cells stably expressing firefly luciferase ( fl - siha ) were donated by dr . fl - siha cells were grown in eagle 's mem , supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) , 100  g / ml kanamycin , and 1  mg / ml g418 at 37c in a 5% co2 humidified atmosphere . fl - siha cells were seeded at a density of 4  10 cells per well in 24-well plates and maintained for 24  h before transfection . for transfection , each lipoplex of sirna was diluted with serum - free medium to a final concentration of 50  nm luc or cont sirna and then gently added to the cells . after incubation for 3  h at 37c , the cells were added with fbs to a final concentration of 10% and then incubated for another 45  h. lipofectamine rnaimax lipoplex ( invitrogen corp . ) forty - eight hours after the transfection , luciferase activity was measured as counts per sec ( cps)/g protein using the luciferase assay system ( promega , madison , usa ) and bca reagent ( pierce , rockford , il , usa ) , as previously reported . fl - siha cells were seeded at a density of 6  10cells per well in 96-well plates and maintained for 24  h before transfection . for transfection , each lipoplex of sirna was diluted with serum - free medium to a final concentration of 50  nm cont sirna and then gently added to the cells . after incubation for 3  h at 37c , the cells were supplemented with fbs to a final concentration of 10% . after 45  h of incubation , the medium was removed , and the cells were treated with wst-8 ( 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2h - tetrazolium , monosodium salt ) solution ( 10  l ) in medium containing serum ( 100  l ) for 30  min . cell viability is expressed relative to the absorbance at 450  nm of untransfected cells . the excised skin of hairless mice ( laboskin , hos : hr-1 male , 7 weeks , hosino laboratory animals , inc . , ibaraki , japan ) was used for the investigation of skin permeability by elastic liposomes . for this purpose , rhodamine - labeled liposomes were nonocclusively applied on the laboskin ( 3.14  cm ) for 6  h. female hr-1/hos hairless mice ( 6 weeks of age ) were purchased from hosino laboratory animals , inc . all in vivo experiments were approved by the institutional animal care and use committee of hoshi university . for observation of the skin penetration of the elastic lipoplexes , the lipoplexes of rhodamine - labeled liposomes and fam - labeled luciferase sirna ( 50  g ) were applied nonocclusively on the dorsal skin ( 3.14  cm ) under anesthesia for 6  h. the mice were kept on a hot plate during the sedation period . after application with elastic liposomes or lipoplexes , the skins were embedded in oct compound ( tissue - tek , sakura finetechnical co. , ltd . , each frozen section was mounted on a silane - coating slide ( muto pure chemicals co. , ltd . , localizations of fam - labeled sirna and rhodamine - labeled liposomes were examined using an lsm5 exciter confocal laser scanning microscope ( carl zeiss , thornwood , ny , usa ) . fam - labeled sirna was imaged using an argon laser at 488  nm excitation , and fluorescence emission was observed with a filter , bp505 - 530 . for rhodamine - labeled liposome , maximal excitation was performed with a 543  nm internal he - ne laser , and fluorescence emission was observed with an lp560 . the statistical significance of differences between mean values was determined by using student 's t - test . transdermal delivery of naked sirna is limited due to its low stability in skin and low permeability by various skin barriers such as the stratum corneum and epidermal layer . in this study , we investigated whether cationic elastic liposomes could effectively deliver sirna transdermally into the skin . here , we used six formulae for elastic liposomes consisting of dotap as a cationic lipid and nachol or tween 80 as an edge activator ( table 1 ) : dotap liposomes containing tween 80 at 5 , 10 , and 15% ( w / w ) ( t5- , t10- , and t15-liposomes ) and dotap liposomes containing nachol at 5 , 10 , and 15% ( w / w ) ( c5- , c10- , and c15-liposomes ) . average diameter and -potential of dotap liposome were about 110  nm and 40  mv , respectively ( table 1 ) . the addition of nachol to the formulation of dotap liposome decreased the size after preparation of elastic liposome and increased the -potential . c5- , c10- , and c15-liposomes were 8090  nm in size and had -potential of 4252  mv . on the other hand , the addition of tween 80 decreased both the size and the -potential , and t5- , t10- , and t15-liposomes were about 8090  nm in size and had -potential of about 2636  mv . as shown in figure 1 , the elasticity of liposomes containing tween 80 peaked at 10% ( w / w ) tween 80 content and was reduced at 15% ( w / w ) . in contrast , the elasticity of liposomes containing nachol was increased with an increase of nachol content , and c15-liposome showed 2.4-fold - higher elasticity than dotap liposome . these findings indicate that the optimal amount of edge activator for elasticity of dotap liposome differed between the liposomes containing nachol and tween 80 . in the following step the sizes of dotap , c5- , c10- , and c15-lipoplexes were about 1,7001,860  nm , and those of t5- , t10- , and t15-lipoplexes were about 550780  nm . notably , the addition of tween 80 was observed to reduce the size of the lipoplexes . this might be due to effect of polyoxyethylene on the surface of elastic lipoplex containing tween 80 . next , we investigated the silencing effects of luciferase mrna and cytotoxicity in siha - luc cells after transfection of the lipoplexes with luc sirna ( figures 2(a ) and 2(b ) ) . although all formulations showed the suppression of luciferase activity without cytotoxicity , an increase of tween 80 content ( t15-liposome ) decreased the silencing effect by elastic lipoplex . as a control , lipofectamine rnaimax , a commercially available transfection reagent , induced strong suppression by sirna transfection . to examine the skin permeability of elastic liposomes , we applied rhodamine - labeled elastic liposomes on excised mouse skin for 6  h and observed their localization by confocal microscopy . figure 3(a ) shows horizontal sections of mouse skin on which the liposomes were applied . the photographs represent sliced images of mouse skin from the surface and up to a thickness of 50  m . when rhodamine - labeled dotap liposome was applied on the skin , the fluorescence was observed until a thickness of 15  m ( figure 3(a ) ) . however , the liposomes containing nachol improved skin penetration with an increase in nachol content , and fluorescence in the skin after application with c15-liposome was strongly observed up to a depth of 50  m . furthermore , the liposomes containing tween 80 also showed increased skin penetration compared with dotap liposomes , but an increase of tween 80 content decreased skin penetration . fluorescence in skin after the application of t5- , t10- , and t15-liposomes was observed at thicknesses of 45 , 30 , and 35  m , respectively . finally , we applied the elastic lipoplexes on mouse skin ( figure 3(b ) ) . the photographs represent sliced images of mouse skin from the surface and up to a thickness of 70  m . in mouse skin on which naked fam - labeled sirna solution was applied , the fluorescence in contrast , elastic lipoplexes containing nachol showed high skin penetration of both sirna and liposomes with an increase of nachol content , and the fluorescence of fam - labeled sirna and rhodamine - labeled liposomes after application with c15-lipoplex was observed at a thickness of around 70  m although the size of c15-lipoplex was large ( about 1.8  m ) . moreover , the elastic lipoplex containing tween 80 also showed improvement of skin penetration . among the lipoplexes containing tween 80 , t5-lipoplex ( 600  nm ) showed improved skin penetration , and the fluorescence of both sirna and liposomes the fluorescence of fam - labeled sirna after application of c15- and t5-lipoplexes largely merged with that of rhodamine - labeled c15- and t5-liposomes , respectively , indicating that sirna penetrated into the skin as a lipoplex . from these findings , the elastic liposomes might squeeze between cells in the stratum corneum and penetrate the intact skin in vivo through a transcutaneous hydration gradient because the elastic liposome was shown to have high stress - dependent adaptability . in the liposomes containing nachol , c15-liposome and lipoplex showed the highest elasticity and skin penetration , respectively ; in contrast , in the liposomes containing tween 80 , t10-liposome showed the highest elasticity , but t5-lipoplex showed the highest skin penetration , indicating that the optimal amount of tween 80 for elasticity and penetration might differ between liposome and lipoplex although we did not measure the elasticity of elastic lipoplex . in pdna delivery , nachol , nadchol , and tween 80 have often been used as an edge activator of elastic liposomes . ultradeformable neutral liposome composed of egg phosphatidylcholine ( epc)/nachol or epc / nadchol displayed the highest level of in vivo transdermal pdna absorption . ultradeformable cationic liposomes composed of dotap / dope / nadchol   and dotap / nachol   have been used for the topical delivery of pdna and antisense oligonucleotide , respectively . however , there is little information about the effect of an edge activator on elastic liposomes for transdermal sirna delivery in vivo . reported that ultradeformable cationic liposomes composed of dotap and nachol at a ratio of 6/1 ( w / w ) ( about 14% ( w / w ) ) showed a high gene knock - down effect in melanoma cells by sirna ; however , in in vivo sirna transfer , they reported that secosome ( surfactant - ethanol - cholesterol ) consisted of dotap , nachol , and cholesterol at a ratio of 6/1/1 ( w / w / w ) in 30% ethanol , penetrated into epidermis of excised human skin , and suggested that the combination of ethanol and sodium cholate was needed for effective penetration of lipoplexes into skin . in this study , we prepared elastic liposomes containing nachol or tween 80 as an edge activator and found that c15- and t5-lipoplexes of sirna showed high in vitro gene suppression and skin penetration . these findings suggested that c15- and t5-liposomes are outstanding tools for transdermal sirna delivery in mouse skin . our results suggest that 15% ( w / w ) nachol or 5% ( w / w ) tween80 is the most effective formulation of dotap - based liposome for transdermal sirna delivery . further study should be performed to examine the effect of gene expression after in vivo sirna application .
we developed elastic cationic liposomal vectors for transdermal sirna delivery . 
 these liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium - propane ( dotap ) as a cationic lipid and sodium cholate ( nachol ) or tween 80 as an edge activator . when nachol or tween 80 was included at 5 , 10 , and 15% ( w / w ) into dotap liposomal formulations ( 
 c5- , c10- , and c15-liposomes and t5- , t10- , and t15-liposomes ) , c15- and t10-liposomes showed 2.4- and 2.7-fold - higher elasticities than dotap liposome , respectively . 
 although the sizes of all elastic liposomes prepared in this study were about 8090  nm , the sizes of c5- , c10- and c15-liposome / sirna complexes ( lipoplexes ) were about 1,7001,800  nm , and those of t5- , t10- , and t15-lipoplexes were about 550780  nm . 
 their elastic lipoplexes showed strong gene suppression by sirna without cytotoxicity when transfected into human cervical carcinoma siha cells . following skin application of the fluorescence - labeled lipoplexes in mice , among the elastic lipoplexes , c15- and t5-lipoplexes showed effective penetration of sirna into skin , compared with dotap lipoplex and free sirna solution . 
 these data suggest that elastic cationic liposomes containing an appropriate amount of nachol or tween 80 as an edge activator could deliver sirna transdermally .