transdermal drug delivery is a noninvasive route of drug administration into the body through the skin .
however , only a few drug candidates have been successfully developed into suitable transdermal formulations because of the formidable skin barrier .
the highly lipophilic nature of the skin restricts the permeation of hydrophilic , high molecular weight and charged compounds through the stratum corneum into the systemic circulation .
transdermal iontophoresis is defined as application of an electrical potential that maintains a constant electric current across the skin and enhances the delivery of ionized as well as unionized molecules .
it uses an electrode of the same polarity as the charge on the drug to drive ionic drug molecules into the body .
the mechanisms of transdermal iontophoresis include electrorepulsion ( a charged ion is repelled from an electrode with the same charge ) , electro - osmosis ( convective flow of solvent through a charged pore that occurs in response to the preferential passage of counter ions when the electric field is applied ) , and current - induced skin permeability increment .
one of the major advantages of iontophoretic drug delivery is the ability to readily and precisely control the drug - delivery profile through modulating the current output .
methotrexate ( mtx ) is an antineoplastic agent used for the treatment of cancer , psoriasis and rheumatoid arthritis .
it is used for the treatment of cancer and at low doses , it has immunosuppressive and anti - inflammatory properties and is used for the treatment of psoriasis and rheumatoid arthritis .
various topical forms like ointments , creams and gels[1518 ] have been tried as the systemic use of this drug causes many side effects mainly , hepatic toxicity and liver damage but are still not available commercially .
the drug can be delivered either in solution formulation or through loading on to the hydrogel patches .
incorporation of the drug into hydrogels facilitates drug handling and release and in case of iontophoretic delivery , allows the patient to remain ambulant .
it has been reported that the iontophoretic delivery of mtx from hydrogels was more effective than passive delivery from aqueous solution .
it has been demonstrated that iontophoresis remarkably improved the transdermal delivery of mtx over passive diffusion .
more interestingly , a case of palmer psoriasis treated with iontophoresis of mtx has been reported using current density for iontophoresis beyond the clinically acceptable limits .
although , the results with iontophoresis are promising , none of the papers give a detailed comparison between high and low current density used for iontophoresis .
the aim of the present study was to analyze the effects of current density on mtx permeation as well as to assess the skin injury caused by the above - mentioned physical enhancer by histological examination .
mtx was a gift sample from dabur ( india ) , acrylamide was obtained from spectrochem pvt .
( india ) , n , n - methylene bis - acrylamide , potassium chloride and potassium dihydrogen phosphate from sisco research laboratory ( india ) , sodium chloride from e. merck india ltd .
( india ) , dihydrogen - o - phosphate anhydrous from qualikems fine chemicals pvt . ltd .
( india ) , sodium hydroxide and ethyl acetate from excelar qualigens fine chemicals ( india ) , ammonium persulphate from thomas baker chemicals ltd .
fine chemicals ( india ) . deionised water having a resistivity of 18 m or greater was used to prepare all solutions and buffers .
the hydrogel patches were synthesized using acrylamide monomer by solution polymerization method as described by prasad et al . , 2007 .
all experiments were conducted according to the protocol approved by the institutional animal ethics committee ( iaec ) of all india institute of medical sciences , new delhi , india .
white albino mice ( n=5 in each group ) were procured from aiims and sacrificed .
the hair was removed from the abdominal region using an animal hair clipper and the full - thickness skin was excised .
fat adhering to the dermis side was cleaned by using a blunt scalpel and isopropyl alcohol , taking care not to damage the skin .
finally , the skin was washed in tap water and observed physically for any gross damage .
the fresh skin was used , for in vitro , attenuated total reflectance - fourier transform infrared ( atr - ftir ) and histopathological studies . for permeation studies
, the mice skin was clamped between the two half - cells of the modified vertical franz diffusion cell with epidermis facing the donor chamber and the area available for permeation was 5.72 cm .
the skin was equilibrated for 1h in phosphate buffer saline ( ph 7.4 ) in the receptor chamber and was magnetically stirred throughout the experiment .
mtx - loaded patch was placed over the skin and dc iontophoresis using current density of 0.2 ma / cm was applied for 1 h to the hydrogel patch through silver -silver chloride electrode and having same dimensions as the hydrogel patch to study the effect of low current density iontophoresis .
the experiments were done at thermostatically maintained temperature ( 372c ) . for mtx quantification in receptor solution ,
0.5-ml samples were withdrawn at specified intervals from the receiver compartment and analyzed for the amount of drug by uv - vis spectrophotometer ( cary 100 model ) at 302 nm .
the samples were also analyzed by hplc using waters 1525 binary pump attached to uv detector .
the cumulative amount of mtx permeated per unit skin surface area was plotted against time and flux ( j ) was calculated as : j=(dc / dt ) v / a , where v = volume of solution in the receptor compartment of the diffusion cell , a = area of the patch , and dc / dt = change in concentration of drug in the receptor compartment solution of the diffusion cell with time . the percent enhancement in flux
was calculated as follows : % enhancement in flux = { ( flux with enhancer  passive flux)/ passive flux}*100 all experiments were repeated five times and the values are expressed as mean  s.d .
statistical comparisons were made using student 's t - test and the significance level was set at p<0.05 . for curve fitting , a third order polynomial expression , y = at + bt + ct + d , where y = total amount and t = time , was utilized for a best curve fit to compare the rate of iontophoretic permeation of methotrexate with passive .
the samples treated with iontophoresis mentioned above were subjected to atr - ftir spectroscopic study using bio - rad , fts 135 , ftir spectrophotometer .
the peak height and areas of c - h stretching , c = o stretching and amide peak absorbances were measured for each sample .
the albino mice skin was treated with iontophoresis for 1 h and fixed in em fluid . after fixing the samples for 48 h , they were washed with phosphate buffer saline and dehydrated using a graded series of ethanol solutions and finally dipped in acetone .
the samples were air dried and mounted on the base plate and then coated with silver using vapor deposition technique .
the surface of the skin sample was investigated using cambridge stereoscan model s4 - 10 , scanning electron microscope .
the iontophoresis - treated skin area ( both in vitro and in vivo ) was excised after 1 h to study the effect on skin and after 24 h and 48 h in vivo to see the reversal of injury after enhancer application .
the excised skin was fixed in 10% formalin and then subjected to processing for histological examination by light microscope .
the skin samples were dehydrated by a series of graded ethanol then treated with xylene and finally embedded in paraffin blocks .
skin sections of 5-m thickness were cut and stained with hematoxylin - eosin ( hande ) stain .
the mounting of the stained sections was done in dpx and observed under light microscope using a modified score [ table 1 for in vitro scoring ] .
mtx was a gift sample from dabur ( india ) , acrylamide was obtained from spectrochem pvt .
( india ) , n , n - methylene bis - acrylamide , potassium chloride and potassium dihydrogen phosphate from sisco research laboratory ( india ) , sodium chloride from e. merck india ltd .
( india ) , dihydrogen - o - phosphate anhydrous from qualikems fine chemicals pvt . ltd .
( india ) , sodium hydroxide and ethyl acetate from excelar qualigens fine chemicals ( india ) , ammonium persulphate from thomas baker chemicals ltd .
fine chemicals ( india ) . deionised water having a resistivity of 18 m or greater was used to prepare all solutions and buffers .
the hydrogel patches were synthesized using acrylamide monomer by solution polymerization method as described by prasad et al . , 2007 .
all experiments were conducted according to the protocol approved by the institutional animal ethics committee ( iaec ) of all india institute of medical sciences , new delhi , india .
white albino mice ( n=5 in each group ) were procured from aiims and sacrificed .
the hair was removed from the abdominal region using an animal hair clipper and the full - thickness skin was excised .
fat adhering to the dermis side was cleaned by using a blunt scalpel and isopropyl alcohol , taking care not to damage the skin .
finally , the skin was washed in tap water and observed physically for any gross damage .
the fresh skin was used , for in vitro , attenuated total reflectance - fourier transform infrared ( atr - ftir ) and histopathological studies . for permeation studies ,
the mice skin was clamped between the two half - cells of the modified vertical franz diffusion cell with epidermis facing the donor chamber and the area available for permeation was 5.72 cm .
the skin was equilibrated for 1h in phosphate buffer saline ( ph 7.4 ) in the receptor chamber and was magnetically stirred throughout the experiment .
mtx - loaded patch was placed over the skin and dc iontophoresis using current density of 0.2 ma / cm was applied for 1 h to the hydrogel patch through silver -silver chloride electrode and having same dimensions as the hydrogel patch to study the effect of low current density iontophoresis .
for mtx quantification in receptor solution , 0.5-ml samples were withdrawn at specified intervals from the receiver compartment and analyzed for the amount of drug by uv - vis spectrophotometer ( cary 100 model ) at 302 nm .
the samples were also analyzed by hplc using waters 1525 binary pump attached to uv detector .
the cumulative amount of mtx permeated per unit skin surface area was plotted against time and flux ( j ) was calculated as : j=(dc / dt ) v / a , where v = volume of solution in the receptor compartment of the diffusion cell , a = area of the patch , and dc / dt = change in concentration of drug in the receptor compartment solution of the diffusion cell with time . the percent enhancement in flux
was calculated as follows : % enhancement in flux = { ( flux with enhancer  passive flux)/ passive flux}*100 all experiments were repeated five times and the values are expressed as mean  s.d .
statistical comparisons were made using student 's t - test and the significance level was set at p<0.05 . for curve
fitting , a third order polynomial expression , y = at + bt + ct + d , where y = total amount and t = time , was utilized for a best curve fit to compare the rate of iontophoretic permeation of methotrexate with passive .
the samples treated with iontophoresis mentioned above were subjected to atr - ftir spectroscopic study using bio - rad , fts 135 , ftir spectrophotometer .
the peak height and areas of c - h stretching , c = o stretching and amide peak absorbances were measured for each sample .
the albino mice skin was treated with iontophoresis for 1 h and fixed in em fluid . after fixing the samples for 48 h , they were washed with phosphate buffer saline and dehydrated using a graded series of ethanol solutions and finally dipped in acetone .
the samples were air dried and mounted on the base plate and then coated with silver using vapor deposition technique .
the surface of the skin sample was investigated using cambridge stereoscan model s4 - 10 , scanning electron microscope .
the iontophoresis - treated skin area ( both in vitro and in vivo ) was excised after 1 h to study the effect on skin and after 24 h and 48 h in vivo to see the reversal of injury after enhancer application .
the excised skin was fixed in 10% formalin and then subjected to processing for histological examination by light microscope .
the skin samples were dehydrated by a series of graded ethanol then treated with xylene and finally embedded in paraffin blocks .
skin sections of 5-m thickness were cut and stained with hematoxylin - eosin ( hande ) stain .
the mounting of the stained sections was done in dpx and observed under light microscope using a modified score [ table 1 for in vitro scoring ] .
the flux obtained with dc iontophoresis using 0.2 and 0.5 ma / cm current density was 20.571.02 g / cm / h and 36.82.21 g / cm / h , respectively ( p<0.05 ) .
similar results have been obtained by cesares - delgadillo , 2010 , although they have tried different current densities for a different drug .
a third order polynomial was chosen to model the experimental results as the data could not be presented by a quadratic .
there was no significant improvement in the least square error by using a higher order polynomial , hence was not used for analysis .
the results are presented in figure 1 which shows the effect of dc iontophoresis on the net permeation of methotrexate at a given time calculated by third order polynomial curve fitting . a good correlation between experimental and polynomial simulation
it can be observed from the curve that with the passive experiments ( drug - loaded hydrogel patch ) , initially there is an increase in permeation , then there is decline and steady state is obtained .
the drug diffusion is via the least resistant pathway , which reaches a saturation , so there is fall in permeation .
moreover , the swelling pattern of the hydrogel ( 0.4 mole% ) is fickian in nature ( data not shown ) , which further supports that diffusion is the main mechanism of drug release .
polynomial curve fitting : effect of dc iontophoresis enhancement of permeation is required to achieve the desired drug levels . from the curve
, it can be seen that there is sharp rise in permeation with iontophoresis and then steady level is reached [ figure 1 ] .
atr - ftir spectra of skin sample treated with current of 0.2 ma / cm current density showed all the major peaks of the lipid and protein but with reduced intensity as compared to the control . a decrease of 28.12% and 31.25% in the peak height of asymmetric and symmetric c - h stretching vibration , respectively , as compared to the control , and 37.5% reduction in the ester peak was noticed [ table 2 ] .
furthermore , a decrease of 31.4% and 47.05% was noticed with amide i and amide ii , respectively . with 0.5ma / cm current density iontophoretic samples , a greater reduction of 96.8% , 93.75% and 93.7% [ table 2 ] in asymmetric and symmetric c - h stretching and c = o stretching vibration , respectively ,
was obtained which was attributed to substantial amount of lipid extraction in the lipid protein domains . moreover ,
an increase in the ratio of amide i and amide ii bands from 2.6 to 11 with increase in current density from 0.2 to 0.5 ma / cm was noticed , thus indicating that the hydration levels are associated with iontophoresis and play an important role in increasing the drug permeation .
percentage decrease in peak height for lipids and protein absorption bands after current treatment for 1h the spectra also demonstrated a split in amide ii band into 1553 cm and 1541 cm .
the split could be due to the disruption in hydrogen bonding associated with the head of ceramides , breaking interlamellar hydrogen bonding of lipid bilayer and disrupting barrier property of sc , resulting in loosening of lipid - protein domains thus allowing higher flux as compared to the passive treatment .
the scanning electron micrographs of dc iontophoresis clearly showed increase in the pore size of the hair follicles [ figure 2 ] .
kajimoto et al . , 2011 have also reported the use of follicular pathways during iontophoresis .
scanning electron micrograph showing increase in hair follicles of the mice skin treated with dc iontophoresis figure 3 depicts a comparison between control , dc ( 0.2 ma / cm ) and dc ( 0.5 ma / cm ) . from the histopathological studies it is clear that at higher current density , 0.5 ma / cm , disruption of epidermis in almost half of the sectioned area was noticed [ figure 3b ]
there was severe dermal edema with marked dilatation and loss of appendages and fractured collagen with a dermal score of 18 .
the dermal changes were higher as compared to iontophoresis with 0.2 ma / cm current density .
photomicrographs of treated mice skin ( in vitro ) ( a ) control , ( b ) 0.5ma / cm current density ( c ) 0.2ma / c m current density , ( where , a  appendageal dilatation , b  bullae formation , d  destruction of epidermis ) ( h and e , 200 ) .
iontophoresis using lower current density of 0.2 ma / cm showed focal disruptions of epidermis ( less than  of sectioned area ) and bullae formation [ figure 3c ] and an epidermal score of 24 was obtained .
the dermis showed fractured collagen with moderated edema and mild appendageal damage , thus a dermal score of 12 was obtained .
dc ( 0.5 ma / cm ) resulted in ths of 47 whereas at 0.2 ma / cm dc , ths of 36 was observed indicating lesser skin injury .
the severe edema formation is evident as the hydration effects of iontophoresis are responsible for enhanced permeation of the drug .
this has been depicted by the ratio of amide i / ii band in atr - ftir spectroscopy described previously .
application of current reduces the resistance of skin and the decreased resistance is reflected in the increased permeability of the skin and therefore increased flux .
current causes epidermal destruction as well as appendageal damage , as we could see in all the histological slides as well as from scanning electron microscopy , therefore shunt pathway for drug permeation become more operative on current application.[3032 ] to summarize , with 0.5 ma / cm current density , damage was higher as compared to 0.2 ma / cm , which was in correlation with the flux observed .
the higher current density provides more electromotive force that increases the flux . in vivo results in mice
show that the damages associated with 0.5 ma / cm current density were not reversible in 48 hours [ figure 4 ] .
photomicrograph of mice skin in vivo showing recovery after 48 h of application of iontophoresis ( 0.5 ma / cm current density ) , where f  focal disruption of epidermis .
( h and e , 200 ) reversibility studies were conducted in vivo after 24 and 48 h of the application of iontophoresis .
it was observed that recovery process had started in 24 h and almost total recovery of epidermal as well as dermal changes was found in 48 h with low current density dc iontophoresis , however with iontophoresis using 0.5 ma / cm current density , edema along with focal disruption of the epidermis persisted [ figure 4 ] .
the partial denudation of the epidermis with left over basal layer showed a rapid recovery as compared to areas with loss of basal layers as seen with 0.5 ma / cm current density iontophoresis .
the flux obtained with dc iontophoresis using 0.2 and 0.5 ma / cm current density was 20.571.02 g / cm / h and 36.82.21 g / cm / h , respectively ( p<0.05 ) .
similar results have been obtained by cesares - delgadillo , 2010 , although they have tried different current densities for a different drug .
a third order polynomial was chosen to model the experimental results as the data could not be presented by a quadratic .
there was no significant improvement in the least square error by using a higher order polynomial , hence was not used for analysis .
the results are presented in figure 1 which shows the effect of dc iontophoresis on the net permeation of methotrexate at a given time calculated by third order polynomial curve fitting . a good correlation between experimental and polynomial simulation
it can be observed from the curve that with the passive experiments ( drug - loaded hydrogel patch ) , initially there is an increase in permeation , then there is decline and steady state is obtained .
the drug diffusion is via the least resistant pathway , which reaches a saturation , so there is fall in permeation .
moreover , the swelling pattern of the hydrogel ( 0.4 mole% ) is fickian in nature ( data not shown ) , which further supports that diffusion is the main mechanism of drug release .
polynomial curve fitting : effect of dc iontophoresis enhancement of permeation is required to achieve the desired drug levels . from the curve
, it can be seen that there is sharp rise in permeation with iontophoresis and then steady level is reached [ figure 1 ] .
atr - ftir spectra of skin sample treated with current of 0.2 ma / cm current density showed all the major peaks of the lipid and protein but with reduced intensity as compared to the control . a decrease of 28.12% and 31.25% in the peak height of asymmetric and symmetric c - h stretching vibration , respectively , as compared to the control , and 37.5% reduction in the ester peak was noticed [ table 2 ] .
furthermore , a decrease of 31.4% and 47.05% was noticed with amide i and amide ii , respectively . with 0.5ma / cm current density
iontophoretic samples , a greater reduction of 96.8% , 93.75% and 93.7% [ table 2 ] in asymmetric and symmetric c - h stretching and c = o stretching vibration , respectively , was obtained which was attributed to substantial amount of lipid extraction in the lipid protein domains . moreover ,
an increase in the ratio of amide i and amide ii bands from 2.6 to 11 with increase in current density from 0.2 to 0.5 ma / cm was noticed , thus indicating that the hydration levels are associated with iontophoresis and play an important role in increasing the drug permeation .
percentage decrease in peak height for lipids and protein absorption bands after current treatment for 1h the spectra also demonstrated a split in amide ii band into 1553 cm and 1541 cm . the split could be due to the disruption in hydrogen bonding associated with the head of ceramides , breaking interlamellar hydrogen bonding of lipid bilayer and disrupting barrier property of sc , resulting in loosening of lipid - protein domains thus allowing higher flux as compared to the passive treatment .
the scanning electron micrographs of dc iontophoresis clearly showed increase in the pore size of the hair follicles [ figure 2 ] .
kajimoto et al . , 2011 have also reported the use of follicular pathways during iontophoresis .
scanning electron micrograph showing increase in hair follicles of the mice skin treated with dc iontophoresis
figure 3 depicts a comparison between control , dc ( 0.2 ma / cm ) and dc ( 0.5 ma / cm ) . from the histopathological studies it is clear that at higher current density , 0.5 ma / cm , disruption of epidermis in almost half of the sectioned area was noticed [ figure 3b ] .
there was severe dermal edema with marked dilatation and loss of appendages and fractured collagen with a dermal score of 18 .
the dermal changes were higher as compared to iontophoresis with 0.2 ma / cm current density .
photomicrographs of treated mice skin ( in vitro ) ( a ) control , ( b ) 0.5ma / cm current density ( c ) 0.2ma / c m current density , ( where , a  appendageal dilatation , b  bullae formation , d  destruction of epidermis ) ( h and e , 200 ) .
iontophoresis using lower current density of 0.2 ma / cm showed focal disruptions of epidermis ( less than  of sectioned area ) and bullae formation [ figure 3c ] and an epidermal score of 24 was obtained .
the dermis showed fractured collagen with moderated edema and mild appendageal damage , thus a dermal score of 12 was obtained .
dc ( 0.5 ma / cm ) resulted in ths of 47 whereas at 0.2 ma / cm dc , ths of 36 was observed indicating lesser skin injury .
the severe edema formation is evident as the hydration effects of iontophoresis are responsible for enhanced permeation of the drug .
this has been depicted by the ratio of amide i / ii band in atr - ftir spectroscopy described previously .
application of current reduces the resistance of skin and the decreased resistance is reflected in the increased permeability of the skin and therefore increased flux .
current causes epidermal destruction as well as appendageal damage , as we could see in all the histological slides as well as from scanning electron microscopy , therefore shunt pathway for drug permeation become more operative on current application.[3032 ] to summarize , with 0.5 ma / cm current density , damage was higher as compared to 0.2 ma / cm , which was in correlation with the flux observed .
the higher current density provides more electromotive force that increases the flux . in vivo results in mice
show that the damages associated with 0.5 ma / cm current density were not reversible in 48 hours [ figure 4 ] .
photomicrograph of mice skin in vivo showing recovery after 48 h of application of iontophoresis ( 0.5 ma / cm current density ) , where f  focal disruption of epidermis .
reversibility studies were conducted in vivo after 24 and 48 h of the application of iontophoresis . it was observed that recovery process had started in 24 h and almost total recovery of epidermal as well as dermal changes was found in 48 h with low current density dc iontophoresis , however with iontophoresis using 0.5 ma / cm current density , edema along with focal disruption of the epidermis persisted [ figure 4 ] .
the partial denudation of the epidermis with left over basal layer showed a rapid recovery as compared to areas with loss of basal layers as seen with 0.5 ma / cm current density iontophoresis .
it was observed that the transdermal permeation increased with increase in injury caused to the tissue . the higher current density ( 0.5 ma / cm ) increased the mtx flux tremendously , but the reversibility studies did not confirm its skin tolerance as the histological changes were not reversible in 48 h.
 introduction : the effect of dc iontophoresis using low ( 0.2 ma / cm2 ) and high current density ( 0.5 ma / cm2 ) on transdermal permeation of methotrexate loaded into polyacrylamide hydrogel patch was investigated.results:flux of 20.57  1.02 g / cm2/h and 36.8  2.21 g / cm2/h was achieved with low and high current density dc iontophoresis , respectively . 
 attenuated total reflectance - fourier transform infrared ( atr - ftir ) spectra and microscopic studies of the treated skin samples supported the permeation results . a greater decrease in the peak height of asymmetric , 
 symmetric c - h stretching vibration and ester peak was noticed with 0.5 ma / cm2 current density as compared to 0.2 ma / cm2 current density samples . 
 furthermore , an increase in the ratio of amide i and amide ii bands from 2.6 to 11 with increase in current density was noticed , thus indicating that hydration levels are associated with iontophoresis and play an important role in increasing the drug permeation . 
 scanning electron microscopy revealed increase in pore size of the hair follicles . 
 light microscopy studies of the skin samples treated with low current density dc iontophoresis demonstrated epidermal thinning and focal disruptions , spongiosis and appendageal dilatations . with higher current density , disruption of epidermis in almost half of the sectioned area , loss of appendages and fractured collagen in the dermis was noticed . 
 moreover , the reversibility studies conducted in vivo on mice revealed that the recovery process had started within 24 h and is complete in 48 h for lower current density treated animals . 
 however , the histological changes associated with 0.5 ma / cm2 current density were not reversible in 48 h and edema , appendageal dilatations along with focal disruption of epidermis persisted.conclusion:hence our study suggests that high density current is not well - tolerated by the skin . 