Shivani Verm a,Puneet Utreja

a Departm ent ofPharm aceutics,Rayat-Bahra College ofPharm acy,Hoshiarpur,Punjab 146001,India

b I.K.Gujral Punjab Technical University,Jalandhar,Punjab 144601,India

c Facu lty ofPharm aceutica l Sciences,Departm ent ofPharm aceu tics,PCTEGroup of Institu tes,Ludhiana,Punjab 142021,India

Keyw ords:Conven tiona l Nanoparticle Span lastics Tran sfersom es Vesicu lar

A B S T R A C TOccu rrence of skin funga l in fection s is in creasing now adays and their p resen ce is m o re p rom inen t in patien ts su ffering from imm unocom p rom ised diseases like AIDS.Skin fungal in fection s are a m a jo r cau se of visits by patien ts to derm ato logy clin ics.A lthough,a large num ber of an tifunga l agen ts are availab le fo r treatm en t of sk in funga l in fection s,bu t,their tox ic p rof ile and physicochem ical characteristics reduce therapeu tic ou tcom e.W hen these an tifunga l agen ts are delivered top ica lly using conven tiona l fo rm u lation s like cream s and gels,they m ay cause various side effects like redness,bu rn ing,and sw elling at the site of app lication.Therefo re,various vesicu lar fo rm u lation s(phospho lip id based or non phospholip id based)have been exp lo red by pharm aceu tica l scien tists to treat skin funga l in fections top ica lly.Vesicu lar fo rm u lation exp lo red fo r the pu rpose are liposom es,ethosom es,tran sfersom es,tran sethosom es,n iosom es,span lastics,o leic acid vesicles,and nanoparticles.These fo rm u lation s show various advan tages like bioavailability enhan cem en tof bioactives,h igh skin perm eation pow er,no side effects at app lication site,dosing frequen cy reduction,and sustained d rug re lease.There fo re,in the p resen t article,w e have d iscu ssed abou t the u tility of various vesicu lar nanocarrier system s to treat sk in funga l in fec tion s.

1. Introduction

Fungiare parasitic m icroo rgan ism s w h ich can a ffect the sk in and m u cous m em b rane a long w ith generation of system ic in fections of various in terna l organ s[1].Funga l in fections of sk in or m u cous m em b rane,in m a jo rity,p rom o te visits of victim s to derm ato logists[2].It has been reported that 20%-25%of hum an popu lation show p resen ce of sk in funga l in fection s[3].In ciden ces of occu rren ce of sk in funga l in fection are very h igh in im m unocom p rom ised patien ts[4].Skin funga l in fections are catego rized in to superf icia l,cu taneous,an d subcu taneous depen d ing upon the level o f tissue invasion[5].W hen attack o f invad ing fungi is lim ited to ou term ost sk in layers on ly then generated in fection is ca lled superf icia l funga l in fection.Tinea versico lo r,w h ite p ied ra,and tinea n igra are exam p les o f superf icial fungal in fections.Superf icia l funga l in fection leads to in crease in the skin pH a long w ith m ild sca ling,redness,and in f lam m ation at the invading site.The barrier natu re o f sk in becom es poor in su ch a state[6].Invasion o f parasitic fungus in to deeper ep iderm a l skin layer develop cu taneous funga l in fection.Th is in fection is a lso know n as derm atom ycoses and it m ay have invo lvem en t o f skin appen dages like nails and hairs[7].Derm atom ycoses can a lso instigate cellu lar im m une respon se develop ing patho logica l variations in patien ts[8].Va rious fungi generating derm a tom ycoses com e under th ree genera,nam e ly Epiderm ophyton,Trichophyton,and M icrosporum.Tinea faciei,tinea barbae,tinea cap itis,and tinea m anuum are the exam p les o f cu taneous funga l in fections[9].Fu rtherm o re,ex tension o f funga l in fection to derm a l o r subcu taneous region resu lts subcu taneous fungal in fection.It is caused by fungi nam ely Sporothrix schenckii and Candida a lbicans[10].Th is funga l in fection is characterized by either u lcerated or in f iltrated nodu lar lesions in the in fected areas[11].M adu ram ycosis an d ch rom om ycosis are other exam p les o f subcu taneous funga l in fections[12].Fig.1 gives a b rie f overview o f skin funga l in fections.cida l o r fungistatic depend ing upon therapeu tic natu re o f in co rporated an tifunga l d rugs[15].Chan ces o f d rug-d rug in teractions are neg ligib le in the case o f top ica l fo rm u lations w h ich are m ore com m on in o ra lly adm in istered an tifunga l d rugm o lecu les[16].How ever,top ica l an tifunga l fo rm u lations like cream s,ge ls,and lotions m ay show redness o f sk in,erythem a,stinging,and bu rn ing sensation as side effects[17].

Fig.1-Classif ication o f sk in funga l in fection s depen d ing upon the dep th o f penetration o f pa rasitic fungus in to the sk in.

2. Conven tiona l strategies fo r treatm en t o f sk in funga l in fection s

Conven tiona lly,sk in funga l in fections a re treated w ith cream s,gels and lotions con tain ing free an tifungal agen ts[13].Top ica lly de livered an tifunga l agen ts show loca l action,there fo re,they exh ibit less toxic effects compared to o ra l an tifunga l agen ts[14].Top ica l fo rm u lations used for treatm en t o f skin fungal in fections m ay be fungi-

Fig.2-Advan tages o f vesicu la r nanoca rriers over conven tiona l de live ry system s fo r tran sderm a l de livery.

Poor sk in penetration o f hyd roph ilic an tifunga l d rugs and h igh dosing frequen cy o f conven tiona l an tifunga l fo rm u lations reduce their e ffectiveness against sk in funga l pathogens[18].For the top ical delivery,an an tifungal d rug m ust have som e specif ied p roperties and lipoph ilic natu re ism ost im po rtan t am ongst them[19].Lipoph ilic d rugs show exce llen t skin penetration up to un derlying sk in layer,however,their release rates shou ld be con tro lled to obtain su ff icien t loca l con cen trations and p ro longed pharm aco logica l effects[20].M o lecu lar w eigh t o f an tifunga l d rug a ffects its top ica ldelivery and it becom esm o re p rom inen t fo r the de livery o f an tifungald rugs like am photericin Bw hosem o lecu larw eigh t exceeds 500 da ltons(Da)[21].There fo re,severa l nanocarrier system s have been investigated by pharm aceu tica l scien tists to fu lf il these criteria and consideration s fo r top ica ldelivery o f an tifunga l d rugs[22].Nanocarriers can m ake their w ay easily to hair fo llicles and theym ay show accum u lation betw een co rneocy tes,m erging w ith lip id ic layer,and h igh in term ing ling w ith lip ids p resen t in the sk in[23].Advan tages o f vesicu lar nanocarriers over conven tiona l de livery system s fo r transderm a ldelivery are exp lained in Fig.2.Nanocarriers a lso have the capability to sustain the d rug release,w h ich redu ce the side effects and dosing frequency o f an tifungal d rugs[24].

So,in the p resen t review ou rm a jo r aim w as to exp lo re the u tility o f various vesicu lar nanocarrier system s for effective treatm en t o f sk in funga l in fection s.

3. Novel vesicu lar nanocarrier system s in the treatm en t o f sk in funga l in fection s

3.1. Phospholipid-based vesicu lar nanocarriers

3.1.1. Liposom es

Liposom es rep resen t f irstly used phospho lip id based nanoca rrier system s for d rug delivery,w h ich w ere described du ring 1980s[25].Structu ra lly,liposom es are bilayered vesicu lar system s having an aqueous core a long w ith one or several con-cen tric phospho lip id m em b ranes[24].Fig.3 describes various phospho lip id based nanocarrier system s used for top ica l delivery o f an tifungal d rugs[26].Due to their un ique structu ral characteristics,liposom es have the capability to deliver both hyd roph ilic and lipoph ilic bioactive m o lecu les[27].

Other advan tages o f liposom es are h igh d rug load ing,toxicity redu ction,im p roved stability and bioavailability along w ith h igher biocom patibility[28].Cho lestero l is a lso added in to the liposom a l system to enhan ce rigid ity o f bilayer,imp rove vesicles stability,and to sustain the release o f en capsu lated m aterial[29].The top ical d rug delivery rou te is qu ite e ffective as it generates h igh d rug con cen tration in loca l area redu cing dosing frequen cy a long w ith e lim ination o f side e ffects o fd rugs.Top ica ldelivery a lso redu ces costo f therapy and im p rove patien t com p lian ce because o f ease o f app lication an d rem ova l o f fo rm u lation[30].Liposom es show effective penetration up to stratum co rneum,w h ich is a site o f invasion o f parasitic fungus[22].Mechan ism o f tran sderm al delivery th rough va rious vesicu lar nanoca rrier system s is exp lained in Fig.4[31].Sudhakar et a l.eva luated terbina f ine HCl loaded liposom es d ispersed in gum karaya ge l fo r ex-vivo d rug retention in rat skin.Developed liposom es show ed app roxim ately 70%en trapm en t o f terbina f ine HCland p ro longed reten tion o f d rug in rat skin com pared to p lain gum karaya gel con tain ing free terbina f ine HCl up to 24 h[32].

Fu rtherm ore,a com parative assessm en t betw een p ropylene g lyco l liposom es and conven tiona l liposom es loaded w ith m iconazo le n itrate w as ca rried ou t by Elm oslem any et a l.for transderm a l delivery.Propy lene g lyco l liposom es loaded w ith m iconazo le n itrate show ed m in im um inh ibito ry concen tration(M IC)va lue o f 1.46μg/m l against Candida a lbicans w h ich w as low com pared to the M IC value o f conven tional liposom es(2.93μg/m l).Propy lene g lyco l(PG)liposom es a lso show ed h igh sk in reten tion and skin perm eation o fm iconazo le n itrate com pared to conven tiona l liposom es and m iconazo le n itrate(MN)suspension in hum an skin(Fig.5)[33].

Agarw a l and Katare perfo rm ed an eva luation o f liposom es p repared from tw o d ifferen t phospho lip ids nam ely phosphatidy l cho line satu rated 97.3%con ten t(PCS)and phosphatidy lcho line unsatu rated 98.0%con ten t(PCU)for top ical de livery o f m iconazo le n itra te.Liposom es deve loped from bo th phospho lip ids show ed h igh stability and good co lloida l characteristics.How ever,PCS based liposom es loaded w ith m iconazo le n itrate show ed h igher skin reten tion o fm iconazo le n itra te com pared PCU based liposom es in-vitro in m ouse sk in[34].Tab le 1 gives an overview o f liposom es as effective nanocarriers for treatm en t of skin fungal in fections.

3.1.2. Ethosom es

Ethosom es are nanocarrier system s w h ich are stru ctu ra lly so ft and having h igh ethano l con ten t,phospho lip ids,and w ater in them[38].Ethosom es m ay con tain 2%-5%con ten t of phospho lip ids and 20%-40%con cen tration o f ethano l[39].Sk in penetration capacity o f ethosom es is h igher com pared to liposom es due to capability o f ethano l to f lu id ize various interce llu lar lip ids p resen t in the stratum co rneum o fskin[40].It hasbeen repo rted thatas the am oun to f ethano l in creases size o f ethosom es decreases by keep ing con cen tration o f phospho-

Fig.3-Diffe ren t phospho lip id-based vesicles used in d rug de livery(Rep rodu ced w ith perm ission from re feren ce[26])Copy righ t 2017,Elsev ier.

Fig.4-Schem atic rep resen tation of the m ain perm eation m echan ism s of lip id-based vesicles(Rep roduced w ith perm ission from re feren ce[31])Copy righ t 2018,Elsev ier.

Tab le 1-Ro le of liposom es in e ffective e lim ination of sk in funga l in fections.

Fig.5-MN retained in and perm ea ted th rough sk in a fter 24 h,determ ined at 32°C,using hum an sk in in Fran z d iffusion ce lls un der non-occlusive cond itions(Rep rodu ced w ith perm ission from re feren ce[33])Copy righ t 2012,springer Nature.

Ethosom es are nanocarrier system s w h ich are stru ctu ra lly soft and having h igh ethano l content,phospho lip ids,and w ater in them[38].Ethosom es m ay con tain 2%-5%content of phospho lip ids and 20%-40%con cen tration of ethano l[39].Sk in penetration capacity of ethosom es is h igher com pared to liposom es due to capability of ethano l tof lu id ize various interce llu lar lip ids p resen t in the stratum co rneum ofskin[40].It hasbeen repo rted thatas the am oun tof ethano l in creases size of ethosom es decreases by keep ing con cen tration of phospholip id constan t[41].Presen ce of ethano l in ethosom e a lso p rovides a negative cha rge to its su rface enhan cing its co lloida l stability[42].How ever,ethosom es show h igh leakage of hyd roph ilic/ion ized d rugs com pared to liposom es due to d isruption of close pack ing of phospho lip id bilayer by the p resen ce of h igh ethano l am oun t[43].Bha laria et a l.investigated f lu conazo le loaded ethosom es for treatm en t of cu taneous cand id iasis in eigh t patien ts fo r a period of onem on th.Ethosom a lge lcontain ing f lu conazo le show ed 50%-70%reduction in skin lesions in patien ts,w h ich w as very h igh com pa red to liposom es(30%-60%)and com m ercia l f luconazo le cream(25%-30%)[44].Later on,econazo le n itrate loaded ethosom es w ere com pared w ith liposom es loaded w ith the sam e for the treatm en t of deep funga l in fection by Verm a and Pa thak in gel form.Ethosom a l gel show ed 2 fo ld h igher d iffusion of the d rug in the a lbino rat skin com pared to liposom a l ge l a fter 12 h of app lication.Resu lts of CLSM(con focal laser scann ing m icroscopy)studies revea led accum u lation of econazo le n itrate loaded ethosom es in the stratum basa le layer of an im a l sk in(Fig.6)[45].M aheshw ari et a l.carried ou t a com parative assessm en t betw een ethosom es and u ltradeform able liposom es loaded with clotrim azo le for the treatm en t of cu taneous cand id iasis.D rug loaded ethosom es show ed h igher in vitro an tifunga l activity against Candida albicans by show ing 34.6m m zone of inh ibition com pared to u ltrade fo rm ab le liposom es w h ich show ed 29.6mm inh ibition zone.Resu lts of Fou rier-transform in frared spectroscopy revea led h igher in vitro sk in penetration of ethosom es com pared to u ltrade fo rm ab le liposom es[46].

Fig.6-Con foca l laser scann ing m icroscopy.(A)CLSM im age of con tro l ge l;(B)CLSM im age of liposom a l ge l show ing less penetration of d rug;(C)CLSM im age of ethosom a l ge l show ing penetration of d rug as fa r as the last layer(stratum basa le)of ep ide rm is(Rep rodu ced w ith perm ission from re feren ce[45])Copy righ t 2012,Elsev ier.

Fu rtherm o re,vo riconazo le loaded ethosom es w ere investigated by Faisal et al.for effective skin deposition.Developed ethosom a l fo rm u lation show ed six fo ld m o re ex vivo d rug perm eation in the rat abdom ina l sk in com pared to hyd roethano lic so lu tion of vo riconazo le[47].Ethosom es having con cen tration of ethano l m ore than 30%cause excessive re lease of en trapped m ateria l and irritation of sk in[48].Therefore,Akh tar and Pathak developed Cavam ax W 7 composite ethosom es to m in im ize these harm fu l e ffects of h igh ethano l con cen tration in vesicles by low ering its am oun t in vesicles.Cavam ax W 7 is a perm eation enhan cer and it show s synergistic e ffect on ethano l's sk in penetra tion pow er.Developed Cavam ax W 7 com posite ethosom es show ed h igh stability and ex vivo skin perm eation,an tifunga l activity against Candida a lbicans and Aspergillus niger com pared to conven tiona l ethosom es[49].

3.1.3. Transfersom es

Conven tiona l liposom es have poo r penetration th rough the sk in,w h ich can be im p roved by m od ifying their bilayer com position[50].Liposom es w ere f irstly m od if ied by Cevc and Blum e by the add ition of edge activato rs to liposom a l com position and resu lted m od if ied liposom es w ere called‘deform able liposom es',‘elastic liposom es',or‘transfersom es'[51].Various exam p les of edge activato rs used in transfersom es are sod ium deoxycho late,sod ium cho late,d ipotassium glycy rrh izinate,Tw een 80,Tw een 60,Tw een 20,Span 80,Span 65,and Span 60[52].Transfersom es show enhanced de fo rm ability due to the w eaken ing of their lip id bilayers because of edge activa to rs[53].Transfersom es have h igher sk in penetration com pared to conven tiona l liposom es due to their h igher deform ability and they can easily cross th rough the pores having d iam eter 5-10 tim es less com pared to their ow n d iam eter[54].Pand it et a l.investigated top ica l an tifunga l e ff icacy of transfersom es loaded w ith m iconazo le n itrate in Sp rague-Daw ley rats.Developed m od if ied liposom es show ed h igh in vivo an tifunga lactivity and reduced toxicity com pared to conven tiona l liposom es and free d rug so lu tion[55].Aggarw a land Goind icarried ou t eva luation of transfersom es loaded w ith griseofu lvin in gu inea p igs fo r erad ication of M icrosporum canis induced derm atophytosis.Op tim ized tran sfersom al form u lation show ed better sk in reten tion and perm eation compared to conven tiona l liposom es.Histopatho logica l ana lysis revea led com p lete erad ication of funga l spores from gu inea p ig skin w ith in 10 d treatm en t by using griseofu lvin loaded transfersom es(Fig.7)[56].

Recen tly,transfersom es loaded w ith am pho tericin B w ere deve loped by Perez et a l.and w ere eva luated fo r in vitro antifungal activity and hum an skin perm eation.They reported m axim um defo rm ability in transfersom es using Tw een 80 as an edge activato r.In vitro sensitivity of c lin ica l iso lates of Candida albicans w as very h igh tow ards transfersom es loaded w ith am photericin B com pared to m am m a l cells.Transfersom es show ed forty tim es better accum u la tion in hum an sk in compared to a m arketed liposom a l form u lation of am pho tericin B(Am Bisom e)[57].

3.1.4. Transethosom es

Transethosom es are h igh ly advan ced vesicu lar nanocarrier system s w h ich en com pass the advan tages of transfersom es and ethosom es.Their com position is exactly sim ilar to ethosom es add itiona lly having the p resen ce of a penetration enhancer or an edge activator[58].Song et al.evaluated vo riconazo le loaded transethosom es for in vivo sk in deposition of d rug in m ice.Tran sethosom es(TEL)show ed in creased in vivo skin deposition of voriconazo le in the derm is and ep iderm is area com pared to other nanocarriers like deform ab le liposom es(DL),conven tiona l liposom es(CL),ethosom es(EL),and po lyethy lene g lyco l d rug so lu tion(PG)(Fig.8)[59].Tab le 2 gives a b rie f sum m ary of research w o rk done on phospho lip id based nanocarriers other than liposom es for tran sderm al delivery ofan tifungald rugs.Overview of various advan tages and d isadvan tages of phospho lip id based nanocarrier system s is exp lained in Tab le 3.

Fig.7-Histopa tho logy of sk in of gu inea p ig in fected w ith M.can is a fter treatm en tw ith(A)test fo rm u lation(g riseofu lv in loaded tran sfersom es)show ing com p lete absen ce of funga l e lem en ts(B)p lacebo,a rrow s show p resen ce of spo red hyphae in hair fo llicles(n=5)(Rep rodu ced w ith perm ission from re feren ce[56].Copy righ t 2012,Elsev ier.)

Fig.8-Am oun t of vo riconazo le retained in sk in a t the of in-vivo sk in deposition stud ies a fter app ly ing lip id vesicles or con tro l PG so lu tion.Each va lue is the m ean±S.D.(n=4)(?P＜0.05 vs ELs)(Rep rodu ced w ith perm ission from re feren ce[59].Copy righ t 2012,Elsevier.)

3.2. Non phospholipid-based vesicu lar nanocarriers

3.2.1. Niosom es

Fig.9-The Am oun t of ITZ de livered in to the stratum co rneum(SC),Strip ped sk in and the recep to r f lu id a fter 6 h of in cubation w ith rat sk in after app lying d ifferen t type of n iosom es(Rep rodu ced w ith perm ission from re feren ce[70])Copy righ t 2015,Elsev ier.

N iosom es are bilayered vesicu lar system s w h ich are m ade up of single alky l chain non-ion ic su rfactan ts[60].Hand jan i-Vila et a l.gave f irst descrip tion of n iosom es in 1979[61].Structu ra lly,they have a hyd roph ilic head of su rfactan t orien ted tow ards the ex terior and in terio r of bilayer w h ile,hyd roph ilic tail en do rsed inside the bilayer[62].There fo re,n iosom es are capab le of en capsu lating both hyd roph ilic o r lipoph ilic d rugs[63].Cho lestero l is a lso added in the p rodu ction of n iosom es to enhan ce the rigid ity of bilayer and reduction of p rem atu re d rug leakage[64].Various characteristics like low p rodu ction cost,h igher chem ica l stability,h igh loading capacity,and regu lar cond itions storage m ake them e ff icien t carries than liposom es[65].Characteristics of n iosom es can be easily m od if ied by varying their com position and p reparation m ethods[66].N iosom es are su itab le to de liver various therapeu tic agen ts th rough various rou tes like top ical,oral,and paren teral[67].Exam p les of su rfactan ts w h ich are used fo r n iosom e p rodu ction are Tw een s,Spans,po lyglycero la lky lethers,po lyoxy-ethy lene a lky lethers,Brij,and esterlinked su rfactan ts[68].Kassem eta l.carried ou ta com parative assessm en t betw een n iosom a l gel loaded w ith griseofu lvin,and liposom al gel loaded w ith sam e for top ical treatm en t of tinea co rporis.These fo rm u lationsw ere investigated clin ica lly in six teen patien ts for abou t th reew eek treatm en t period.N iosom a l ge l show ed app roxim ate ly 80%cu re rate,w h ich w as very h igh com pared to liposom a lgel(50%)[69].Later on,Alomran i et a l.p repared n iosom es loaded w ith itraconazo le(ITZ)using d ifferen t non ion ic su rfactan ts fo r transderm a l de livery.A com parison w as done using Span su rfactan ts[like Span 60(SP60),Span 80(SP80),and Span 85(SP85)],Tw een su rfactan ts[like Tw een 60(TW 60),Tw een(TW 80),and Tw een 85(TW 85)],and Brij35(BR35).N iosom es p repared from Span 85 and Tw een 85 show ed m axim um skin penetration,how ever,skin perm eation pow er of Tw een 85 n iosom es w as h igher com pared to Span 85 n iosom es and Brij 35 n iosom es(Fig.9)[70].

A com parative assessm en t betw een n iosom a l fo rm u lation loaded w ith terbinaf ine hyd roch loride and its m arketed cream w as carried ou t by Satha li and Ra ja lakshm i in-vitro against patho logica l fungus Aspergillus niger.N iosom es contain ing en capsu lated d rug(pu rif ied n iosom es)and n iosom es con tain ing en capsu lated d rug and free d rug both w ere d ispersed in to gel base and their an tifunga l effects w ere compared w ith m arketed fo rm u lation.A zone of inh ibition w as found in the order-n iosom a l gel con tain ing en capsu lated d rug and free d rug both＞n iosom al gel con tain ing encapsulated d rug on ly＞m arketed cream form u lation＞ge l con taining pu re d rug[71].A b rie f overview of n iosom es fo r treatm en t of sk in funga l in fection is given in Tab le 4.

Tab le 2-Phospho lip id based vesicles o ther than liposom es as e ffective nanocarrier fo r treatm en t of sk in funga l in fections.

3.2.2. Spanlastics

Fig.10-Electron m icrographs of freeze-etched o leic acid spheres(×41 650).(A)A cen tra l aqueous region su rroun ded by con cen tricm em b ranes-the f lat p itted a rea w as cu t by the m icro tom e.(B)Sm a ller spheres en closed by a com m on enve lope(Rep rodu ced w ith perm ission from re feren ce[80].Copy righ t 1973,Natu re.)

Tab le 3-Advan tages and d isadvan tages of phospho lip id based nanocarrier system s.

Tab le 4-Niosom es as effective nanocarrier for treatm en t of sk in funga l in fection s.

Span lastics are novelvesicu lar carriers,w h ich are a lso term ed as‘m od if ied n iosom es'as they con tain edge activator in n iosom a l com position.Span lastics usua lly con tain Spans a lonw ith p resen ce of edge activato rs like Tw eens and m any o thers[76].Kakkar and Kau r f irstly deve loped span lastics loaded w ith ketoconazo le using Span 60 as su rfactan t and Tw een 80 as an edge activator for ocu lar d rug de livery[77].Elsherifeta l.developed and eva luated span lastics loaded w ith terbinaf ine hyd roch loride for treatm en t of nail fungal in fection nam ely onychom ycosis.Study w as carried ou t by using Span 60 and Span 65 as su rfactan ts w h ile Tw een 80 and sod ium deoxycho late as edge activato rs.Span lastics fo rm ulated w ith Span 65 as su rfactan t and sod ium deoxycho late as edge activa to r show ed m ax im um d rug en trapm en t,sm a ller size and good co lloida l p roperties there fo re,catego rized as op tim ized form u lation.Con focal laser scann ing m icroscopy(CLSM)revea led e ff icien t ex vivo nail perm eation of op tim ized span lastic fo rm u lation[78].

Tab le 5-Ro le of va rious non-phospho lip id based vesicu lar ca rriers in trea tm en t of sk in funga l in fection s.

3.2.3. Oleic acid vesicles

Fatty acids are am ph iph ilic m o lecu les con sisting of a carbon atom chain behaving as po lar part w h ile term inal carboxy lic group behaving as non po lar part.The p resen ce of doub le bon ds in stru ctu re of fatty acid governs w hether they are satu rated o r unsatu rated[79].Gebick iand Hicks f irstly reported ability of unsatu rated fatty acid like o leic acid toform vesic les[80].Fig.10 gives stru ctu ra l elucidation of spherica l vesic les p repared by using o leic acid.

Zakir et a l.eva luated o leic acid vesic les loaded w ith f luconazo le for eff icien t tran sderm al d rug delivery.Developed vesic les show ed m ax im um d rug en trapm en t,accep tab le size,and good co lloida l p roperties at 7:3 o leic acid to d rug ratio.Resu lts of ex-vivo skin perm eation and con foca lm icroscop ic stud ies revea led accum u lation of d rug loaded o leic acid vesic les in the low er ep iderm is area of skin a fter top ica l app lication ind icating their effectiveness in the loca lized d rug delivery[81].Later on,clotrim azole loaded oleic acid w ere investigated by Verm a et a l.for cu taneous cand id iasis treatm en t in gu inea p igs.Developed o leic acid vesicles show ed h igh skin perm eation a long w ith good skin reten tion in an im alskin.In-vivo study revealed capability of d rug loaded o leic acid vesicles to re lease clo trim azo le up to 5 d a fter the tim e of app lication[82].A b rief sum m ary of va rious non phospho lip id based vesicu lar nanocarriers fo r skin delivery of various an tifunga l d rugs is given in Table 5.Table 6 describes advan tages and d isadvan tages of various non phospho lip id based nanocarrier system s.

3.3. Nanoparticles

Various types of nanoparticles exp lored for transderm a l de livery of an tifunga l d rugs are po lym eric nanoparticles(NPs),so lid lip id nanoparticles(SLNs),and nanostru ctu red lip id carriers(NLCs)[83].Po lym eric nanoparticles m ay be p resen t either in particu late d ispersion fo rm or so lid pow der form having size range 10-1000 nm.Nanopartic le m atrix m ay have d rug in en trapp ing,encapsu lated,or d isso lved form[84].Various exam p les of natu ra l po lym ers used fo r NPs p reparation are gelatin,a lbum in,ch itosan,and a lginate.Syn thetic po lym ers used fo r NPs p reparation m ay be biodegradab le[poly(lactide-coglyco lide)(PLGA),po ly(ε-cap rolactone)]and non-biodegradab le[po ly(m ethy l m ethacry late),po lysty rene and po lyacry la tes][85].Kum ar et a l.eva luated clotrim azo le loaded PLGAm icroparticles in gu inea p igs fo r su ccessfu leradication of cu taneous cand id iasis.Gel con tain ing d rug loaded PLGA m icrosphere show ed penetration up to 50μm in the derm is of an im a l skin a long w ith better an tifunga l e ff icacy a fter 4 d of app lication[86].So lid lip id nanopartic les(SLNs)are spherical structu res having so lid lip id core w h ich m ay so lubilize non po lar d rug m o lecu les[87].Exam p les of various so lid lip ids im p lem en ted fo r the p reparation of SLNs are g lycero l bahenate,tristearin,glycero l m onostearate,stearic acid,and cety l pa lm itate[88].SLNs m anu factu ring invo lves the use of physio logica l lip ids and less use of o rgan ic so lven t w h ich m ake them eff icien t carrier fo r transderm a l d rug de livery[89].Bha lekar et a l.perform ed ex-vivo skin perm eation stud ies of m iconazo le n itrate loaded SLNs p repared using so lid lip id Com p rito l 888 ATO(g lycero l bahenate).Developed SLNs form u lation show ed better ex-vivo accum ulation of d rug in the skin along w ith better skin targeting e ffect com pared to the m arketed gel[90].Fu rtherm o re,g lycero l pa lm itostearate(Preciro l ATO 5)based SLNs loaded w ith econazo le n itrate w ere eva luated by Sanna et a l.in vivo by using f ive healthy living sub jects(fem ales).SLNs revealed better d iffusion of d rug in low er ep iderm al skin layers a fter 180 m in of app lication com pared to m arketed gel in living sub jects[91].Nanostructu re lip id carriers(NLCs)are nanocarriers consisting of the lip id m atrix em bedded w ith special nanostru ctu res[92].NLCs p rodu ced using h igh p ressu re hom ogen ization techn ique m ay be in lip id particle d ispersion fo rm having 60%-80%so lid content[93].Gup ta and Vyas eva luated f luconazo le loaded NLCs in vivofo r effective erad ication of cu taneous cand id iasis in albino rats.Drug loaded NLCs show ed 3.3 fo ld h igher sk in reten tion,better skin targeting effect,and h igh therapeu tic e ff icacy com pared to d rug loaded SLNs[94].Fu rtherm o re,econazo le n itrate loaded therm odynam ically stable NLCs w ere developed by Kesh ri and Pathak by the use of cen tra l com posite design for transderm a l d rug delivery.Resu lts of con foca l m icroscopy revea led penetration of d rug loaded NLCs up to stratum basa le layer of an im a l skin.The developed form u lation show ed a m ino r change in particles size and zeta poten tia l du ring 90 d stability ana lysis[95].So,a fter review ing literatu re,it can be considered that nanopartic les m ay be a good alternative to im p rove transderm a l an tifunga l therapy.

Tab le 6-Advan tages and d isadvan tages of non phospho lip id based nanocarrier system s.

Tab le 7-List of paten ts regard ing the use of vesicu lar nanoca rriers fo r treatm en t of sk in funga l in fections.

4. In te llectua l p roperty righ ts(IPR)re lated to use of va riou s vesicu lar nanocarriers fo r treatm en t of sk in funga l in fection s

Vesicu lar nanocarriers show h igh therapeu tic poten tial in the treatm en t of skin funga l in fections.These novel carriers can be considered as an effective a lternative to cu rren tly availab le m arketed p rodu cts to treat sk in funga l in fections.There fo re,pharm aceu tica lscien tists have f iled various paten ts regard ing the use of various vesicu lar nanocarriers for treatm en t of sk in funga l in fections.Tab le 7 gives an overview of various paten ts gran ted regard ing the use of vesicu lar nanocarriers in treatm en t of sk in funga l in fection s.

5. Lim itation s and ch a llenges in the use of vesicu la r nanoca rriers fo r treatm en t of sk in funga l in fection s

Vesicu lar nanocarrier system s are very e ffective to treat sk in funga l in fection s due to their capability to m od ify the delivery of bioactive m o lecu les to d ifferen t sk in layers and target d iseased portion of the skin.Vesicu lar nanocarriers show h igh penetration of d rug m o lecu les in the skin th rough various m echan ism s like fusion,abso rp tion,and lip id exchange in sk in layers.Bu t,the excessive skin penetration enhan cem en tm ay becom e a doub le edged sw o rd because a fter heavy penetration,d rug m olecu les m ay reach in blood circu lation w h ich can be harm fu l fo r loca lized treatm en t of sk in funga l in fections.There fo re,serious con cern s regard ing th ism ust be taken by pharm aceu tica l scien tists.M any other facto rs like safety p rof ile,c lin ical eff icacy,scale up techn iques,and the fate of vesicu lar nanocarriers in transderm a l therapeu tics are still cha llenging for the scien tists.Successfu l fu tu re research w ill he lp to answ er these cha llenges and develop an idea l m ode l of vesicu lar nanocarrier for effective transderm a l antifunga l therapeu tics.

6. Con clu sion s

Sign if ican t in crease in m orta lity rate has been observed in patien ts from last one decade due tofunga l in fection s w hether top ical or system ic.There are several effective an tifungal d rugs availab le,bu t their therapeu tic e ff icacy is lim ited due to un favo rab le physicochem ica l characteristics and h igh toxicity p rof iles.Vesicu lar nanocarriers have capability tom in im ize these d raw backs of an tifunga ld rugs due to their un ique p roperties like h igh biocom patibility,ease of su rface m od if ication,and sm a ller size.Vesicu lar nanocarriersm ay be very effective to treat invasive skin fungalassociated w ith imm unosupp ressive d isease like AIDS as they show con tro lled d rug re lease w h ich do no t activate the im m une system of the patien t.Beside a ll th is,vesicu lar nanocarriersm ay im p rove stability and targeting effect of an tifungal d rugs to in fected tissues along w ith the enhan cem en t of their so lubility and an tifunga l e ff icacy.Clin ica leva luation of vesicu lar carriers is stilla cha llenge because am photericin B is the on ly an tifunga ld rug w h ich has been clin ically evaluated in liposom al form and available in the m arket therefo re,their p resen ce in pharm aceu tica lm arket w ill be governed by successfu l clin ica l eva luation.

Con f licts of in terest

The au thors declare that there are no con f licts of in terest.

Acknow ledgm en ts

Au thors are gratefu l to the Departm en t of Research,Innovation&Consu ltan cy(RIC),I.K.Gu jra l Pun jab Techn ica l Un iversity,Ja landhar,Pun jab fo r p rovid ing access to repu ted scientif ic jou rna ls via Open A thens accoun t to carry ou t th is w ork.