Yum eiWu,Yuanyuan Liu ,Xinyue Li,Dereje Kebebe,e,Bing Zhang,Jing Ren,Jun Lu ,Jiaw ei Li,c,Shouying Du ,Zhidong Liu ,??

a Engineering Research Center ofM odern Chinese M edicine Discovery and Preparation Technique,M inistry of Education,Tianjin University ofTraditionalChinese Medicine,Tianjin 300193,China

b Tianjin State Key Laboratory ofM odern Chinese M edicine,Tianjin University ofTraditional Chinese M edicine,Tianjin 300193,China

c Departm ent ofExperim ental Departm ent,Tianjin University of Traditional Chinese M edicine,Tianjin 300193,China

d School ofChinese Materia Medica,Beijing University ofChinese Medicine,Beijing 100029,China

e SchoolofPharm acy,Institu te ofHea lth Sciences,Jimm a University,Jimm a,Ethiopia

f SchoolofPharm aceutica l Science and Technology,Tianjin University,Tianjin 300193,China

Keyw ords:In-situ gel Ocu lar Drug de livery Bioavailability Po lym er Corneal reten tion

A B S T R A C TBlindness and vision im pairm en t are the m ost devastating global health p roblem s resu lting in a substan tia l econom ic and socia l bu rden.De livery o f d rug to particu lar parts o f the an terio r or posterio r segm en t has been a m a jor challenge due to various p rotective barriers an d elim ination m echan ism s associated w ith the un ique anatom ica l an d physio logical natu re o f the ocu lar system.Drug adm in istration to the eye by conven tiona l delivery system s resu lts in poor ocu lar bioavailability(＜5%).The design ing o f a novel app roach fo r a sa fe,sim p le,an d effec tive ocu lar d rug delivery is a m a jo r con cern an d requ ires innovative strategies to com bat the p roblem.Over the past decades,several novel app roaches invo lving d ifferen t strategies have been developed to im p rove the ocu lar delivery system.Am ong these,the oph tha lm ic in-situ gelhas attained a great atten tion over the past few years.Th is review d iscussed an d sum m arized the recen t an d the p rom ising research p rogress o f in-situ gelling in ocu lar d rug delivery system.

1. In trodu ction

The eye is a com p lex and un ique part o f the hum an o rgans that has been con sidered as the w indow to the hum an sou l.Broad ly,the hum an eye is d ivided in to tw o segm en ts that are an terior and posterio r segm en ts(Fig.1)[1].The specif ic d isease cond ition s o f the eye are associated w ith each o f these broad segm en ts.For instan ce,con jun ctivitis,glaucom a,blepharitis,and cataract are som e o f the d iseases that a ffect the an terior segm en t o f the eye,w h ile d iabetic retinopathy and age-related m acu lar degeneration are know n to a ffect the posterio r segm en t[2].

Due to the un ique stru ctu re o f the eye,w h ich inh ibits the en try o f d rug m o lecu les in to the desired site,the oph tha lm ic delivery o f the d rug has been one o f the m ost challenging tasks for a pharm aceu tica l scien tist.Eye d rops accoun ts fo r m ore than 90%o f oph tha lm ic p reparations on the m arkets.How ever,they are w ashed aw ay from the eye and resu lts in low ocu lar bioavailability(＜5%)a fter top ica l adm in istration[3]by d ifferen t elim ination m echan ism s.Th is e lim ina tion p rocess in cludes tear tu rnover,naso lacrim a l d rainage,p rotein bind ing,system ic absorp tion,enzym atic degradation and com p lex penetration barriers(Co rnea l Barrier,Blood Aqueous Ba rrier(BAB),and Blood Retina l Barrier(BRB))[4](Fig.2).

One o f the m ain d raw backs in ocu lar d rug delivery is ach ieving and retain ing of op tim al con cen tration of d rug at the desired site o f action in the eye.Severa loph tha lm ic dosage fo rm s such as oin tm en ts,eye d rop so lu tions,gels,and ocu lar inserts have been investigated in order to p ro long the ocu lar residen ce tim e o f d rugs a fter the top ica lapp lication to the eye.W ith these form u lation s,the co rnea l con tact tim e has been in creased to som e ex ten t.Bu t,due to b lu rred vision and poo r patien t com p lian ce resu lted from oin tm en ts and inserts,respective ly,they have no t been fu lly accep ted[5].Fu rtherm o re,d rugs that are adm in istered system ica lly to exert their action in the oph tha lm ic system a lso have know n to access poorly to the eye tissue[6].In travitrea l and pertiocu lar rou tes are recomm ended in order to deliver d rugs to the posterior part o f the eye.How ever,there are d isadvan tages associated w ith these rou tes like the frequen t in travitrea l in jections cou ld be pain fu l,thus a ffecting a pa tien t com p lian ce.The periocu lar rou te is easy for adm in istration,bu t the static and dynam ic barriers con stitu te a p rob lem[7].

The low bioavailability o f m ed ication s from the conventional delivery system is resu lted from a great exten t of p reco rnea l d rug loss by naso lach rym a l d rainage.The rap id clearan ce o f the top ica lly app lied d rug in to the eye o ften resu lts in a sho rt du ration o f pharm aco logica l activity and,therefore,the need for a frequen t dosing regim en.Moreover,50%-100%o f an instilled dose cou ld undergo system ic abso rp tion th rough d rainage via the naso lach rym a l duct.Th is cou ld lead to a possible increased risk o f unw an ted system ic tox ic effects[8].

In last decades,va rious de livery system s su ch as using chem ica l perm eability enhan cers[6],p rod rugs[9]stim u liresponsive in-situ gel[10],and d rug delivery carriers such as liposom es[11]and nano-orm icroparticles[12],noisom es[13],dend rim ers and m icroneed les[14]have been developed to increase ocu lar residen ce tim e,d rug penetration across the ocular barriers and oph tha lm ic bioavailability.In-situ ge lling system is one o f the p rom ising app roaches to im p rove the reten tion tim e o f d rugs on the ocu lar su rface.A fter instilla tion of the aqueous so lu tion con tain ing stim u li-responsive po lym ers su ch as pH-sensitive po lym ers,therm osen sitive po lym ers,and ion-sensitive po lym ers,the v iscous and m ucoadhesive gels are fo rm ed on the eye su rface[15],subsequen tly,ocu lar reten tion tim e an d ocu lar bioavailability of the ophtha lm ic d rugs are im p roved.There fo re,in th is review,w e summ arized and d iscussed the m ost recen t research innovations in stim u li-responsive in-situ gelling system for ocu lar d rug delivery system.

Fig.1-The anatom y o f ocu lar system:the an terio r segm en t invo lves con jun ctiva,cilia ry body,iris,pup il,an terio r cham ber,cornea and len s;the posterior segm en t consists o f sclera,choroid,retina,m acu la and op tic nerve.

Fig.2-The critica l ba rriers to ocu lar d rug de livery system s:the Co rnea l Ba rrier:invo lves o f ep ithe lia l layers attached together by tigh t jun ction s avoid ing en try o f d rug particle fo llow ed by th ick strom a an d en do thelia l cells.The Blood Retina l Ba rrier(BRB):com p rises o f the inner BRB resu lted from retina l cap illa ries.Blood Aqueous Ba rrier(BAB):m ade by the non p igm en ted ce lls o f the ep ithe lium o f the cilia ry body,and the endo the lium o f the iris b lood vesse ls.

2. Anatom y o f the ocu lar system

Genera lly,the eye is d ivided in to tw o im portan t segm en ts:(1)The an terior segm en tw h ich invo lves the cornea,con junctiva,iris,pup il,cilia ry body,an terio r cham ber,aqueous hum o r,lens and trabecu larm eshw o rk.(2)The posterio r segm en t in cludes vitreous hum or,sclera,retina,choroid,m acu la and op tic nerve(Fig.1)[1].

The co rnea is the transparen t and clear avascu lar part o f the ocu lar system that fo rm s the an terio r m ost coat o f the eye.Anatom ically,the cornea is consist o f f ive m ajor layers.Cornea l ep ithe lium is the f irst layer,w h ich is the m ost ex terior[16].The o ther layers in clude Bowm an’s m em brane,strom a,Descem et’sm em b rane and the en dothe lium layer[1].Corneal perm eability is the m ost essen tial factor that determ ines d rug con cen tration in aqueous hum o r.Fo rm ost o f hyd roph ilic d rugs,the ep ithelium is a rate-lim iting barrier o f transcorneal d iffusion o f d rugs[17].The strom a is ow ing to the hyd roph ilic natu re,it acts as a barrier for the d iffusion o f h igh ly lipoph ilic d rugs[17].The co rnea l strom a ism ain ly consisting o f charged and h igh ly o rgan ized hyd roph ilic co llagen that inh ibit the d iffusion of hyd rophobicm o lecu les[18].

Con jun ctiva is a clear th in m em b rane that covers the sclera and lines the inner su rface o f the eyelid.It is consist o f stratif ied ep ithe lium(non-keratin ized)and gob let ce lls.It p rovides p rotection to the eyes by secreting m ucus that p reven ts entry o fm icroo rgan ism s and lub ricating the eyes[1].In hum ans,the con jun ctiva occup ies a 17-tim es larger su rface area than the cornea.Th is allow s for greater absorp tion o f the d rug to occu r th rough the con jun ctiva.Therefore,d rugs are usua lly m o re perm eab le across the con jun ctiva than the co rnea.However,abso rp tion o f the d rug via the con jun ctiva is still no t sign if ican t due to the existence o f con jun ctival blood cap illaries and lym phatics,w h ich leads to a considerab le loss o f d rug in to the system ic circu lation,thereby reducing the overa ll ocu lar bioavailability[19].

Aqueous hum o r consists o f clear liqu id that f ills bo th the posterio r and an terio r cham bers o f the eye.The aqueous hum o r is non-vascu lar structu re that m ust be transparen t to allow ligh t transm ission,w h ich p rovides nu trition for the co rnea[1].It con tains excessive con cen tration o f asco rbate w h ich is abou t 15-fo ld the con cen tration in the p lasm a,and has a pH o f 7.2[16].Itsm ain fun ction is to p rovide nu trien ts,elim inate w aste from non-vascu lar tissues and con tro l the in traocu lar p ressu re that keeps the convex shape o f the cornea[20].

Fig.3-In-situ fo rm ing ge ls p rocess.The fo rm u lation is liqu id w hen in stilled in to the eye w h ich un dergoes ge l fo rm ation rap id ly in the cu l-de-sac o f the eye in respon se to env ironm en ta l changes su ch as p H,tem peratu re and ion;f ina lly re lease the d rug slow ly un de r physio logica l con d itions.

The sclera is the w h itish portion o f the eye,opaque and elastic in natu re,consisting o f co llagen f ibers[1].The so lu tes especia lly hyd roph ilic com pounds a re genera lly m o re perm eab le across the sclera than the cornea and the con jun ctiva,because d iffusion th rough sc lera ism ain ly am atter o f transport across an aqueousm ed ium o f p roteog lycans o r leaky spaces w ith in the co llagen netw o rk rather than d iffusion across ce llu lar m em branes[19].The sclera o ffers a p rotective ou ter layer,m ain tain ing in traocu lar p ressu re and serving as the attachm en t po rtion for the ex traocu larm usc les[17].

The retina is a m u ltip le layers and com p lex stru ctu re that con sists o fvascu lar,glialan d neu ral,cellsan d nerve f ibers[16].The retina is a m a jo r barrier to ocu lar delivery o f d rug w ith largerm o lecu lar w eigh t[19].

3. In-situ ge lling system

Oph tha lm ic in-situ ge lling is com p rising o f environm en ta lly sensitive po lym ers that w ill be a ltered structu ra lly w ith the sm a ll changes in specif ic cond ition s like pH,tem peratu re and ion ic strength in the environm en t.In-situ form ing gels are liqu ids du ring instillation in to the eye and then undergoes rap id ge lation in the cu l-de-sac o f the eye to fo rm viscoe lastic ge ls in response to environm en tal changes(Fig.3);lastly release the d rug slow ly under physio logica lcond itions[21].Consequen tly,the residen ce tim e o f the gel fo rm ed in-situ w ill be ex tended and the d rug is released in a sustained m annerw h ich leads to enhan ced bioavailability,m in im ized system ic absorp tion and reduced frequen t dosing regim en resu lting to im p roved patien t com p lian ce[22].Fu rtherm o re,som e other po ten tia l advan tages su ch as sim p le m anu factu ring p rocess,ease o f adm in istration,and de liveran ce o f accu rate dose have been exh ibited by in-situ ge lling system s[23].

3.1. M echanism s ofgelling system

In-situ gel fo rm ation m ay be ach ieved by a num ber o fm echan ism s in clud ing tem peratu re-(Fig.4),pH-and ion-activated system s.Tem peratu re triggered in-situ ge l system w h ich u tilizes the tem peratu re sensitive po lym ers that ex ist as a liqu id fo rm below its low critica l so lu tion tem pera tu re(LCST)and undergoes gelation w hen the environm en ta l tem peratu re reaches or is above the LCST[24].The pH induced in-situ gel con tains po lym ersw h ich possess acid ic or a lka line fun ctiona l groups w ith in the chain m o lecu le and undergoes a so l-gel phase transition on change from a low pH to h igh pH environm en t[23].Ion-activated system s are also know n as osm otica lly triggered in-situ ge lsystem sw herein the po lym er un dergoes a so l-gel transition due to changes o f ion ic con cen tra tion,w h ich is typically triggered bym ono or d ivalen t cations in tear f lu id particu larly Na+,Mg2+an d Ca2+[25].In add ition,so l-gel phase transition has know n to be in duced by enzym atic cross lin king and pho ton po lym erization[25,26].How ever,the pH,tem peratu re,and ion-indu ced in-situ ge l are the m ost ex tensively stud ied app roaches o f in-situ gel,and the concern of th is review.

3.2. Stim u li-responsive in-situ gel system

3.2.1. Temperature-triggered in-situ gelsystems

The tem peratu re sen sitive in-situ gel is the o ldest,the m ost extensively stud ied and com m on type o f stim u li-responsive ge l.It can be easily and p recisely in troduced in to the eye in liqu id fo rm w ithou t p rodu cing irritation o r b lu rred vision.The gel is form ed at the p recorneal tem peratu re(35°C)to endu re the lach rym a l f lu id d ilu tion w ithou t rap id p reco rnea l e lim ination o f in stilled d rug a fter adm in istration[27].It has been recom m ended that a good therm o-responsive ocu lar in-situ gel shou ld possess the gelation tem peratu re above the room tem peratu re and undergo gel-so l transition at a p re-co rnea l tem peratu re in order to avoid storing in a refrigerator before instillation,w h ich m ay som etim es resu lt in eye irritation due to co ld natu re[28].

Fig.4-The ge lation p rocess o f therm osen sitive in-situ ge lling.W hen the tem peratu re is be low the ge la tion tem peratu re(GT),it is clear so lu tion w ith low viscosity,u pon heating it to GT,the so lu tion is converted to the ge lw ith h igh viscosity.

Tab le 1-Som e exam p les o f therm o-sensitive in-situ ge lling system.

Polym ers used in tem perature triggered in-situ gelsystems

Poloxam ers(Pluronic)

Po loxam ers are a trib lock copo lym er po ly(ethy lene oxide)-b-po ly(p ropy lene ox ide)-b-po ly(ethy lene ox ide)(PEO-PPOPEO)exh ibiting am ph iph ilic natu re because o f hyd roph ilic ethy lene oxide dom ains and hyd rophobic p ropy lene ox ide dom ain s[29](Fig.5).The trip le b lock o f copo lym ers PEO-PPOPEO(Plu ron ics or Po loxam ers)undergo gelation at body temperatu re in concen trations above 15%(w/w)[30].The p rincipa l possib le m echan ism s have been p roposed to exp lain the so l-gel phase transition at an in creased tem peratu re are the gradua l deso lvation o f the po lym er,enhan ced m ice llar aggregation,and the increased en tanglem en t of the po lym eric netw o rk[26].The p lu ron ic trib lock copo lym ers are ex isting on the m arket in d ifferen t grades w ith d ifferen t physica l fo rm s and m olecu larw eigh ts.Depend ing upon the physicaldescription fo r the grades are given as L fo r liqu id,P for paste and F fo r f lakes.The com m on ly used po loxam ers are 188(F-68),237(F-87),338(F-108)and 407(F-127)[31].Plu ron ic F-127(F-127)or Po loxam er 407(P407)(copo lym er PEO106-PPO70-PEO106)con sists o f ethy lene oxide(70%)w h ich con tribu tes to its hyd roph ilic p roperty.F-127 is a copo lym erw ith m o lecu larw eigh t o f 12 000Da,a PEO/PPO ratio o f 2:1,non tox ic,w ith low viscosity below 4°C and form ing a sem iso lid gel at body tempera tu re.Fu rtherm ore,F-127 has better so lubility in co ld w ater than in ho tw ater because o f the hyd rogen linkages at low tem peratu res[31,32].

Fig.5-The chem ical structu re o f som e in-situ gel po lym ers.

Xyloglucan

Xy loglucan is a po lysaccharide obtained from tam arind seeds,there fo re it is o ften nam ed tam arind seed po lysaccharide(TSP),w h ich w hen partia lly degraded byβ-ga lactosidase d isp lays therm a lly reversib le ge l form ation in d ilu ted aqueous so lu tion(Fig.5).The sol-gel transition tem peratu re is varying w ith the degree o f ga lactose degradation[33].TSP gels have been repo rted to have a poten tia l for o ra l,ocu lar,in traperiton ia l an d recta l d rug de livery[23,33].TSP is h igh ly w aterso luble and gelation occu rs w hen the galactose elim ination exceeds 35%[34].

Cellu lose derivatives

Cellu lose is a po lysaccharide con tain ing a linear chain m ade up o f severa l hund red to over ten thousandβ(1→4)lin ked D-glucose un its.The cellu lose derivatives used in top ica l oph tha lm ic fo rm u la tions a re m ethy l ce llu lose,hyd roxyethy l ce llu lose,hyd roxyp ropy lm ethy lce llu lose(HPMC),sod ium carboxym ethy l cellu lose(NaCMC)[34].At low con centrations(1-10%),their aqueous so lu tions ex ist as a liqu id bu t form gels upon heating.The h igh phase transition tem peratu re exh ibited by cellu lose derivatives can be low ered by physica l o r chem ica lm od if ication[35].The transition tem peratu re is betw een 40 and 50°C for MC and betw een 75 and 90°C fo r HPMC.Add ition of sod ium ch loride is know n to low ers the ge lation tem pera tu re o f MC to 32-34°C,w h ile the tran sition tem peratu re o f HPMC can be decreased to abou t 40°C by lowering the hyd roxyp ropy lm olar substitu tion[36].

Chitosan

Ch itosan is an am inopo lysaccharide(Fig.5)m ade from the partial deacety lation and depo lym erization o f ch itin,w h ich is found in the exoskeletons o f arth ropods,su ch as crustaceans[37].Com m ercia lly,ch itin is m ain ly derived from the she ll w astes o f sh rim p,crab,k rill,lobster,and squ id[26].Ch itosan has been p roven to possess m any advan tages in biom ed ica l app lications due to its biocom patibility,biodegradability,m ucoadhesiveness w ith low im m unogen icity and low cy to tox icity[38].Recen tly,ch itosan-based therm osensitive gels w ith d ifferen t po lyo ls such as ethy lene glyco l,glycero l,an d so rbito l have attained m u ch popu larity[39].

The derivatization o f p rim a ry am ino groups o f ch itosan(CS)by th io l groups resu lts in the form ation o f Th io lated Ch itosan(TCS).TCS based d rug de livery system is gain ing atten tion because it exh ibits h igh m u coadhesive strength and ex tended d rug release p roperties.TCS show s in-situ gelling p roperties because o f the fo rm ation o f in tra and in term o lecu la r d isu lf ide bonds as a resu lt o f ox idation o f th io l groups at physio logica l pH-va lues[40].

Research progress in tem perature triggered in-situ gel system s

Over the last decades,a large num ber o f stud ies on tempera tu re triggered in-situ form ing system have been repo rted(Tab le 1).Tom en tion som e o f them,Lieta l.form u lated Brinzolam ide d rug-resin in-situ therm osensitive ge lling system,using Po loxam er F127 in com bination w ith Carbopo l 934P.The op tim a l fo rm u lation d isp layed a ge l fo rm ation at 33.2±1.1°C and the diffusion-con tro lled release of the m odel d rug over a period o f 8 h.The in vivo study suggested that the in-situ gel dem onstrated a better ability in retain ing the d rug than comm ercia l fo rm u lations[41].

A l-Khateb et a l.a lso investigated the in-situ gelling system con tain ing o f loxacin using a com bination o f Plu ron ic(PF-127 and PF-68)and sod ium a lginate.The in co rpo ration o f Plu ron ic F68 to Plu ron ic F127 so lu tions w as found to rises the so l-gel tem peratu re o f binary fo rm u lation to above the physio logical range o f tem peratu res.The superior in vitro d rug reten tion perfo rm an ce on glass su rfaces and fresh ly excised bovine co rn w ere exh ibited by 20%(w/w)Plu ron ic F127 in comparison w ith other fo rm u lations.Add itiona lly,in vivo eva luation in rabbits dem onstrated that a reten tion perfo rm an ce o f 20%(w/w)Plu ron ic F127 w as h igher than that o f Plu ron ic F68.Fu rtherm o re,the slugm u cosa irritation assay and bovine cornea lerosion stud ies dem onstrated no sign if ican t irritation w as observed that resu lted from these po lym ers and their com binations[42].

Ossw a ld et a l.p repared an in jectab le m icrospherehyd rogel by load ing the an tivascu lar endothelial grow th facto r,an ti-VEGF(ran ibizum ab o r a f libercep t)in to po ly(lactic-co-g lyco lic acid)m icrospheres that w ere then suspended w ith in an in jectable poly(N-isop ropy lacry lam ide)-based therm o-respon sive hyd rogel DDS.The eff icacy w as eva luated in vivo in a laser-in duced rat m ode l o f cho roida l neovascu larization(CNV).CNV lesion area w as m easu red and quan tif ied by f luorescein angiogram s and a m u lti-Otsu th resho ld ing techn ique,respective ly.In traocu lar p ressu re(IOP)and dark-adap ted electroretinogram(ERG)w ere a lso m easu red p re-and post-treatm en t(1,2,4,8,and 12 w eeks).The resu lt has show n tha t the an ti-VEGF-loaded DDS group had exh ibited sign if ican tly sm a ller CNV lesion areas than a non-treatm en t group o f an im a ls th roughou t the study,w h ich suggests that the DDS o ffer a sign if ican t benef it in the m anagem en t o f posterior segm en t eye d iseases[43].

The add ition o f cellu lose derivates to Plu ron ic F12 hyd rogels assist in in creasing the bioavailability o f the in-situ ge l[44],M orsi et a l.,p repared Keto ro lac trom etham ine nanod ispersions form u lated in to therm o-sensitive in-situ ge l using Eud ragit RL100.The study dem onstrated that reducing the con cen tration o f Plu ron ic F-127 w as found to in crease the gelation tim e and gelling tem peratu re o f the in-situ ge ls.The in corpo ration o f HPMC to p lu ron ic F12 hyd rogels has sign if ican tly im p roved the m u coadhesive strength o f the gel[45].

Add ition o f salts(NaCl and KCl)to in-situ gel system has know n to decrease the ge lation tem peratu re.Bhowm ik et a l.exam ined the in f luen ce o f d ifferen t sa lts on the ge lation p roperties,rheo logy and d rug release o f in-situ gelbased on m ethy lcellu lose(MC).Itw as found that 5-7%(w/v)sod ium ch lo ride,8-9%(w/v)po tassium ch lo ride,o r 5%(w/v)sod ium bicarbonate w as capab le o f reducing the GT be low physio logical tem peratu re,i.e.37°C.The du ration o f d rug release increased from 1.5 h to 3-5 h from sa lt con tain ing MC so lu tions depend ing on the con cen tration an d the type o f sa lt[46].Similarly,Bhowm ick et a l.con f irm ed that the use o f i-carrageenan w ith po tassium ch loride cou ld effectively decrease the GT o f the v irgin MC so lu tion from 60°C to 33.5°C w h ich is be low physio logica l tem peratu re[47].

Them ixtu re of po loxam erw ith am ucoadhesive agen t(ch itosan)is know n to ex tend the reten tion tim e o f d rugs fo r the treatm en t o f oph tha lm ic d iseases.Gratieri et a l.fo rm u lated in-situ form ing ge l consisting the com bination o f po loxam er and ch itosan.The resu lts dem onstrated that the addition o f ch itosan cou ld im p rove the m echan ica l strength as w ell as tex tu re p roperties o f po loxam er fo rm u lations and the in-situ gel in creased a fou r-fo ld reten tion tim e in com parison w ith a conven tiona l so lu tion[48].

In add ition to Po loxam er,Po ly(N-isop ropy lacry lam ide)(PN)has been w idely used as therm o-responsive po lym ers.For instan ce,Hsiue et a l.deve loped oph tha lm ic fo rm u lation using PN as the therm o-sensitive po lym er.The clear so lu tion o f PN w as know n to fo rm a ge lupon the raising o f tem peratu re from the room tem peratu re to abou t 32°C.Ep ineph rine-loaded linear PN and crosslinked PN nanopa rticles w ere deve loped and eva luated.The f ind ing o f the study show ed that the p ressu re decreasing the activity o f the form u lation w ith linear PN and com bination o f linear PN and crosslin ked nanoparticles lasted six-fo ld and eigh t-fo ld longer than tha t o f the conven tiona l eye d rop,respective ly[49].

Recen tly,the stud ies have show n that copo lym erization o f PN w ith hya lu ron ic acid(HA)has in creased the LCST o f PN from 32°C to above body tem peratu re,w h ich is m o re app rop riate fo r the oph tha lm ic app lication.W ith th is aim,Zhu et a l.developed therm o-sensitive in-situ form ing gelling fo rm u lation o f Ketoconazo le(KCL)based on PN/HA.The in vitro gelation,d rug re lease,and an tifunga l activity w ere eva luated fo r the developed form u lations.The gelation tem peratu re o f the PN-HA therm o-gelling so lu tion w as found 33°C.The m oderate release o f KCL from in-situ gels w ithou t bu rst effects w as exh ibited.Nom acroscop ic signs o f irritation,redness,or other toxic effects w ere observed.The in vivo an tim icrobial study ind icated that KCL PN-HA in-situ ge ls d isp layed an im p roved cu re percen t as com pared w ith com m ercia l eye d rops[3].

Very recen tly,Iohara et al.developed a hyd rophobica lly m od if ied hyd roxyp ropy l m ethy lcellu lose(HM-HPMC)gel fo rm ed therm o-responsive hyd rogels by in co rpo ration o f sm a ll am oun t o fα-Cyc lodex trin(α-CD)in to the so lu tion.The form ed HM-HPMC/α-CD gel exh ibited a reversible so lgel transition w ith in the range o f physio logica l tem peratu re w h ich w as to ta lly opposite to the tem peratu re dependen cy has show n by the o rigina lHM-HPMC(w ithou tα-CD).The HMHPMC/α-CD exh ibited the rap id ge lation on the ocu lar su rface and a sign if ican tly im p roved ocu lar d rug(d ic lo fenac sod ium)absorp tion[50].

3.2.2. pH triggered in-situ gelling system s

This in-situ gelling system consists o f pH-sen sitive polym ers w h ich are po lyelectro ly tes con tain an acid ic(carboxy lic o r su lfon ic)o r a basic group(am m on ium sa lts)that either accep t or release p rotons in response to alteration in pH in the su rround ing environm en t.A t low er pH(pH 4.4),the fo rm u lation ex ists as a regu lar so lu tion,how ever,it undergoes gel fo rm ation at pH 7.4,that is the pH o f tear f lu id.

Polym ers used in pH triggered in-situ gel systems Them ost com m on ly used pH-responsive po lym ers in ophtha lm ic p reparation are Po lyacry lic acid(PAA,Carbopo l 940),polycarboph il,and cellu lose acetate ph thalate(CAP)[17].

Carbopol(Polyacrylic acid) Carbopo l is a po lyacry lic acid(PAA)po lym er(Fig.5),that d isp lays a so l-gel phase transition in aqueous so lu tion as a resu lt o f raising the pH above its p K o f abou t 5.5[57].The carboxy lic groups o f PAA accep t and re lease p ro ton s at low pH va lues and h igh pH va lues,respective ly.There fo re,at h igh pH,the PAA sw ells due to the electrostatic repu lsion of the negative ly charged groups,releasing the d rug m o lecu les to the environm en t[17].It is ex tensive ly exp loited in ocu lar fo rm ulation w ith the aim o f im p roving p re-co rnea l reten tion tim e o f d rugs.Carbopo l p rovides the benef it o f exh ibiting superio rm ucoadhesive p roperties as com pared to o ther po lym ers.Mu coadhesive p roperties o f carbopo l is attribu ted to the interaction of po ly(acry lic acid)w ith m u cin that occu rs by fou r m echan ism s viz.e lectrostatic in teraction,hyd rogen bond ing,hyd rophobic in teraction and in ter d iffusion[35].Desp ite carbopo l d isp lays exce llen tm ucoadhesive p roperties,the acid ic natu re of the gel is am ajor d raw back w h ich leads to irritation and dam age to the eye tissues.There fo re,com binations o f carbopo lw ith o ther po lym ers in clud ing ch itosan and HPMCw ere subsequen tly developed to overw helm ed th is p rob lem[25].

Tab le 2-Som e exam p les o f pH-triggered in-situ ge lling system.

Research progress in pH triggered in-situ gelsystems

The in-situ pH-triggered gelling system has a grea t po tentia l to keep d rug p roductm o re stab le and retain d rug re lease(Tab le 2).W ith th is aim,ou r research group(W u et a l.)designed and eva luated pH-triggered ge l con tain ing Baica lin fo r sustained oph tha lm ic d rug delivery using Carbopo l 974P as the gelling agen t along w ith HPMC E4M(0.6%,w/v)that w as a viscosity enhan cing agen t.The in vitro and in vivo eva luations w ere con du cted using con foca l scann ing ligh t m icroscopy,rheom etry,Gam m a scin tigraph ic techn ique an d m icrod ialysism ethod.The resu lt of rheo logical study d isp layed that the gel strength w as sign if ican tly enhan ced under physio logica l cond ition.The gel cou ld p rovide sustained re lease o f the d rug over an 8 h period.Fu rtherm ore,the AUC and Cmaxva lues w ere found 6.1-tim es and 3.6-tim es h igher than those of the con tro l so lu tion,respectively[58].

In add ition,ou r research group(Pang et a l.)have con f irm ed that the ocu lar in-situ gel can redu ce the system ic abso rption o f the d rug and thus reduce the po ten tia l system ic tox icity.Brim on id ine Tartrate in-situ ge lw as p repared using Carbopo l 974P an d HPMC E4M,and its therapeu tic eff icacy and system ic abso rp tion w ere com pared w ith that o f eye d rop.The pharm acodynam ics study on the eye o f rabbit show ed that the gel fo rm u lation cou ld sign if ican tly decrease in traocu la r p ressu re(IOP)as com pared to the eye d rop.M ore im po rtan tly,the in vivo pharm acokinetic stud ies exh ibited that the p lasm a AUC(0→∞)w as found low er for the in-situ gel than the eye d rop,w h ich suggests the decreased system ic absorp tion[59].

3.2.3. Ion-activated in-situ gel system

Ion-activated in-situ ge lling system s fo rm a crosslink w ith cations exists in the tear f lu id(Na+,Ca2+and Mg2+),thus fo rm ing a ge l on the ocu la r su rface,w h ich give rise to an extended co rnea l con tact tim e(Tab le 3)[17,64].

Polym ers used in Ion-activated in-situ gelsystem

The m ost com m on ly used ion-activated po lym ers in ocular fo rm u lations are ge llan gum(Gelrite?),hya lu ron ic acid and sod ium a lginates[65].

Gellan gum

Ge llan gum are po lysaccharides that can be used to in duce ion-sen sitive hyd rogels.It is a linea r an ion ic heteropo lysaccharide(Fig.5)m ade up o f a tetrasaccharide repeating un it o f glucose,glucu ron ic acid and rham nose in the ratio o f 2:1:1[66].Gellan com p rises hyd roxy land carboxy lic fun ctiona l groups,w h ich m ay in teract w ith other po lym ers via hyd rogen bond ing an d/or electrostatic attraction s[67].A low-acety l ge llan gum is com m on ly availab le in the m arket as Gelrite?,w h ich undergoes gelation in the p resen ce o f m ono-o r d ivalen t cation s.The e lectro ly tes o f the tear f lu id especia lly Na+,Mg2+an d Ca2+cations are pa rticu larly know n to in du ce gel fo rm ation o f the po lym er upon instillation as a liqu id so lu tion in to the cu l-de-sac[68].The in corpo ration o f op tim a l quan tities o f ca lcium g lu conate to gellan fo rm u lations lead to the form ation o f gellan calcium gluconate-sim u lated tear f luid(STF)ge ls w ith a sign if ican tly h igher strength than w hen ge llan a lone w as m ixed w ith STF[67].It undergoes ge lation by bo th tem peratu re sensitive o r cations induced m echan ism.The possib le m echan ism o f gelation in cludes the fo rm ation o f doub le helica l jun ction zones fo llow ed by aggregation o f the doub le he lica l segm en ts to form a th ree-d im ensiona l netw ork by hyd rogen bon d ingw ith w ater and com p lexation w ith cations[26].

A lginate/A lginic acid

A lginate is a linear co-po lysaccharide derived from b row n seaw eeds and som e bacteria.Chem ica lly it is a(1-4)-linked b lock copo lym er o fa-D-m annu ronate(M)an d its C-5 ep im er R-L-gu lu ronate(G),w ith residues arranged in hom opo lym eric sequen ces o f bo th k inds and in region w h ich app rox im ate to the d isaccharide repeating structu re(MG)[69].Sod ium alginate undergoes ge l fo rm ation as a resu lt o f ca lcium a lgina te fo rm ation by virtue o f its in teraction w ith a d iva len t cation(Ca2+)p resen t in lach rym a l f lu id(pH 7.4)[5].Various p roperties o f the po lym er su ch asm echan ica l strength,po rosity,etc.are h igh ly depend on the ratio o fβ-D-m annu ron ic acid and α-L-g lucu ron ic acid.A lginate w ith a h igh gu lu ron ic acid conten t exh ibit a better gelling p roperties and m in im ize the concen tration o f po lym er requ ired to fo rm stiff ge l[26].

Tab le 3-Som e exam p les o f ion-activated in-situ ge lling system.

Pectin

Pectins are a po lysaccharides fam ily,w here the po lym er backbone m ostly consists o fα-(1,4)-D-ga lactu ron ic acid residues.Low m ethoxy pectins w h ich are w ith a degree o f esterif ication＜50%can fo rm ge l in aqueous so lu tion in the existen ce o f free calcium ions,that cross lin k the galactu ron ic acid chain s.Its w a ter so lubility is one o f the im po rtan t advan tages o f pectin,therefo re o rgan ic so lven ts can be avoided in the form u lation s[70].The in-situ ge lling o f pectin indu ced by ca lcium ions exists in lacrim a l f lu id has been reported in a US paten t.In add ition,pectin based in-situ ge l has been used to p ro long d rug re lease from the form u lation s such as theophy lline,acetam inophen,and cim etid ine[71].

Research progress in ion-activated in-situ gelsystems

Va rious ion-activated in-situ gelling system s have p reviously been reported.Rupen thaletal.form u lated ion-activated in-situ based on ge llan gum,xan than gum and ca rrageenan,and in vivo release,p recornea l reten tion tim e and the ocular irritan cy w ere characterized for the form u lations.The resu lts show ed that the in-situ system w as non-irritan tw ith increased AUC and them iotic response o f p ilocarp ine by 2.5-fo ld com pared to an aqueous so lu tion[64].

Zhu et al.also developed an ion-activated ketotifen ocu lar fo rm u lations using a natu ra lpo lysaccharidew h ich is deacetylase ge llan gum.The study dem onstrated tha t deacety lase gellan gum had a po ten tia l to p ro long the residen ce tim e o f the fo rm u lation.The in-situ gels exh ibited a characteristic sustained and ex tended d rug effects behavior com pared w ith the conven tiona l eye d rops at the sam e dose[72].

Kesarla et al.form u lated nanoparticles-loaded oph thalm ic in-situ gel using the ion-sensitive po lym er ge llan gum used as a ge lling agen t w h ich cou ld fo rm ge l im m ed iately and rem ained fo r the ex tended tim e o f period.The deve loped fo rm u la tion w as found stab le and d isp layed im p roved co rnea l con tact tim e and m in im izing the frequency o fadm in istration.The con foca l m icroscop ic study show ed a c lear cornea perm eation o f d rug-loaded nanopartic les[73].Tayel et a l.deve loped a novel ion-sensitive in-situ oph tha lm ic nanoem u lsion(NE)gels con tain ing terbinaf ine hyd roch loride.The op tim ized in-situ NE gel exh ibited a sign if ican tly h igher Cmax,de layed tmax,p ro longed m ean residen ce tim e and enhan ced ocu lar bioavailability[74].

In the developm en t o f bioadhesive ion-sensitive hyd roge ls,the in co rporation o f the poo rly w ater so lub le d rug is very cha llenging.Cyclodex trins(CDs)are benef icialpharm aceu ticalexcip ien ts that assist in the fo rm u lation o f poorly aqueous so lub le d rugs.The in clusion o f hyd roxyp ropy l-β-cyclodex trin(HPBCD)in the in-situ fo rm ed gel has show n to a llow a m ore effective con tro l an d a sign if ican t im p rovem en t in the f luconazo le re lease[66].

3.2.4. Mu lti-stim u liresponsive in-situ gel

One o f the recen t exce llen t stra tegies in ocu lar in-situ gelling system is the use o f a com bination o f po lym ersw ith the d ifferen t ge llingm echan ism,w h ich have show n an im p roved therapeu tic eff icacy an d better patien t com p lian ce[20].Over last cu rren t years,a num ber o f investigation s that invo lved the com bination o f therm o-responsive po lym ers,pH-sensitive po lym ers o r ion-activated po lym ers in the sam e oph tha lm ic form u lation have been reported(Table 4).

Khan et a l.developed and eva luated sparf loxacin-loaded nove l in-situ gelling system for sustained oph tha lm ic d rug delivery using a com bination o f ion and pH activated gelling system,w h ich w ere sod ium a lginate andm ethy lce llu lose,respective ly.The fo rm u lation w as in so l fo rm at pH(4.7)and has undergone qu ick so l-gel transition upon raising pH to 7.4.The insitu gel form u lation dem onstrated in vitro sustained release o f sparf loxacin over a period o f 24 h as com pared to eye d rop.The ex vivo co rnea lperm eation study on goa teye revea led that a d ram atica lly im p roved perm eation as com pared to eye d rop[21].

In add ition,Yu et a l.repo rted nepa fenac in-situ ge l using carboxym ethy l ch itosan(CMC)and po loxam er com posed o f PEO-PPO-PEO block copo lym er w h ich w as found to undergo a reversib le so l-ge l transition upon a change in a tem peratu re and/or pH at a very low con cen tration.The resu lt o f a CCK-8(Ce llCoun ting Kit-8)study show ed that the form u lation w as no t tox ic to hum an co rnea l ep ithelia l ce lls at a low concen tration.The form u lation o f po loxam er-CMC/NP show ed a sustained re lease o f nepa fenac from the hyd rogel.The re lease rate w as found to bem axim um at 35°C an d pH 7.4[79].

Davaran et a l.deve loped a dua l therm o-/pH-responsive nanocarriers in-situ gel fo r cip ro f loxacin.Cip ro f loxacin re leased from the nanoparticles in-situ ge lling system dem onstrated an im p roved an tim icrobia l activity as determ ined bym in im al inh ibitory con cen trations[80].Gup ta et al.,a lso form u lated in-situ ge l using the com bination o f ge llan gum(ion-sensitive)and ch itosan(pH sensitive)so as to im p rove p reco rnea l residen ce tim e o f sparf loxacin.Acco rdingly,the developed sparf loxacin in-situ form ing gel w as found non irritan t and show ed the p ro longed reten tion at the cornea l site w ith the p ro longed d rug re lease[81].

Tab le 4-Som e exam p les o fm u lti-stim u li respon sive in-situ ge lling system.

Tab le 5-Som e exam p les o f nanocarrier in-situ gelling system.

3.3. Nano-in-situ gelling system s

Now adays nanotechno logy is the m ost em erging concep t in pharm aceu tica l scien ces[85].Severa l nano-techno logy based fo rm u la tions have been developed to ex ten d ocu lar residen ce tim e and to im p rove bioavailability o foph thalm ic d rugs.However,nanoparticles have no t m u coadhesive p roperty,so are cleared ou t o f eyes rap id ly[86].The suspend ing o f fab ricated nanoparticles in an in-situ ge lling veh icle w h ich undergoes so l to gel phase tran sition upon exposu re to physio logica l cond ition is know n to so lve th is p rob lem.The nanoparticulate in-situ ge lw as designed to exp lore the doub le benef it o f nanoparticles an d in-situ gelling system,for its oph tha lm ic de livery(Tab le 5)[60,87].Th is resu lts in ex tend ing the p reco rnea l residen ce tim e o f the nanoparticles and im p roving ocu la r bioavailability[26].

The form u lation s of in-situ gel in novel d rug delivery system as co lloida l carriers system s su ch as nanosuspensions,lip id-based nanocarriers,have p roven to be the m ost e ffective strategy,causing an exponen tia l in crease in the bioavailability o f the oph tha lm ic d rugs.For instan ce,Liu et a l.deve loped the cu rcum in(CUR)-loaded ocu la r nanogel by using cation ic nanostru ctu red lip id carriers(CNLC)and therm osensitive gelling agen t.The in vitro release,corneal perm eation,ocu la r irritation and p reocu lar reten tion w ere eva luated.A lso,the pharm acok inetic p ro f ile in the aqueous hum o r w as eva luated by m icrod ia lysis techn ique.The AUC o f the nanogel(CUR-CNLC-GEL)w as found 9.24-tim es h igher than those o f cu rcum in so lu tion(CUR-SOL),ind icating a sign if ican tly imp roved bioavailability[88].

Tab le 6-List ocu la r in-situ ge ls ap p roved fo rm a rket.

Tab le 7-List o f som e paten ts o f in-situ ge lling system fo r ocu la r de livery.

Pandu rangan et al.form u lated so lid lip id nanoparticles(SLNs)-loaded in-situ ge lw ith vo riconazo le w h ich w as found to be a p rom ising veh icle fo r ocu lar delivery w ith good stability and exce llen t zone o f inh ibition in the m icrobia l assay of voriconazo le[89].Paradkar et al.developed a n iosom a l in-situ gel using Po loxam er 407 and HPMC K4M.The p repared Natam ycin n iosom a l in-situ gel form u lation exh ibited an in creased co rnea l reten tion tim e due to bioadhesive p roperty o f gel and d isp layed ex tended d rug release up to 24 h w h ich as com pared to m arketed p roducts.The fo rm u lation w as a lso foun d to exh ibit a better tran sco rnea l perm eation[90].Shuk r et al.also form u lated voriconazole-loaded in-situ gelling noisom e fo r ocu lar inserts using span 40 and span 60 w ith p lu ron ic F127 and p lu ron ic L64.The op tim ized in-situ gelling ocu la r insert show ed a sign if ican tly h igher Cmax,delayed tmaxand in creased bioavailability,and w as foun d nonirritan t[91].

M icrosphere-loaded ion-activated in-situ gel o f o f loxacin(OFX)w as a lso fo rm u lated.In vivo resu lts in rabbits exh ibited that OFX-loaded m icrospheres in-situ ge l cou ld im p rove the relative bioavailability by 11.7-tim es relative to the comm ercia l OFX eye d rops.Fu rtherm o re,the ex tended du ration o f action o f OFX-loaded m icrospheres in-situ gel p reparation is though t to avoid frequen t adm in istration,w h ich im p roves patien t com p lian ce[92].

No f loxacin-loaded nanocarriers w ere designed u tilizing ch itosan as a m atrix form ing po lym er,crosslin ked by an anion ic cross-linker sod ium tripo lyphosphate.The developed ch itosan nanoparticu late in-situ gel exh ibited superior perfo rm an ce over the m arketed eye d rops[60].Levo f loxacin nanoparticles laden in-situ ge l w as fo rm u lated and show ed the im p roved ocu lar reten tion tim e.Gup ta et a l.designed nanoparticle laden in-situ ge l en capsu lated PLGA nanoparticle,con tain ing levo f loxacin,added in to ch itosan in-situ ge l.Ocu lar reten tion w as eva luated by gam m a scin tigraphy in rabbits.The developed nanoparticle laden in-situ gel form u lation exh ibited slow rate o f clearan ce an d retained at the co rnea l su rface form o re ex tended du ration than com m ercia lly availab le fo rm u lation,in-situ gel and nanosuspension a lone[93].Fu rtherm ore,the sam e group o f research con f irm ed for excellen t sustained release o f the nanoparticle in-situ ge lling system con tain ing sparf loxacin[94].

More recen tly,Ahm ed et al.form u lated ketoconazo le po ly(lactide-co-g lyco lide)nanoparticles w ith subsequen t load ing in to in-situ fo rm ing ge l fo r oph tha lm ic d rug de livery system.The in vitro release o f the d rug from the fo rm u lations loaded w ith nanoparticles d isp layed a sustained and greater d rug release com pared to free d rug fo rm u lations.In add ition,the in-situ gelling w ith nanopartic les show ed im p roved an tifunga l activity in com parison to pu re d rug fo rm u lations.A lginate-ch itosan in-situ gel con tain ing op tim ized ketoconazo le nanoparticles d isp layed h igher d rug perm eation via epithelial cell lines[95].

4. Clin ica l ap p lication o f in-situ gelling

To date,som e o f in-situ gel fo rm u lations have been com m ercially available for ocu lar d rug delivery(Tab le 6).For instan ce,Tim op tic-XE?,con tain ing tim o lo lm a leate(0.25%and 0.5%)in ge llan gum has been availab le on m arket sin ce 1994,w h ich is app lied top ica lly on the eye to treat glaucom a.Fu rtherm o re,som e o f the paten ts on in-situ gel for ocu lar delivery system have been issued in the last decades,and are being sum m arized in Tab le 7.

5. Con clusion s an d fu tu re p rospects

Desp ite the cha llenges in ocu lar d rug de livery,over the past few years,m any innovative app roaches are being developed to overcom e the p roblem s associated w ith conven tional o f ophtha lm ic p reparations.The in-situ gelling system is one the p rom ising and ex tensively stud ied strategies that cou ld p rolong p reco rnea l residen t tim e an d o ffer the sustained re lease d rug delivery,thus im p rove ocu lar bioavailability and therapeu tic eff icacy and redu ce system ic absorp tion and tox icity.Fu rtherm ore,due to its d rug release sustain ing ability and decrease the frequen cy o f adm in istration,in-situ gel cou ld imp rove patien t com p lian ce.In in-situ ge l fo rm u lation w ith d ifferen t stim u li-responsive po lym ers that have h igh sensitivity to change in pH,tem peratu re,and ion con cen tration are used.How ever,the com bination of tw o orm ore stim u li-responsive po lym ers in the sam e form u lation is know n to exh ibit greater com p lian ce and im p roved therapeu tic eff icacy.Mo reover,exp lo ring the com bination o f d ifferen t d rug delivery app roaches(i.e.nanopartic les loaded in-situ gelling)to deve lop in-situ gel has been the attractive strategies to im p rove ocu lar d rug delivery system.

As the eye is the m ost essen tial and sen sitive part o f the body,the sa fety issues o f oph tha lm ic fo rm u lations is critica lly im po rtan t.The m a jo rities o f the cy to toxicity and irritability stud ies in c luded in th is review show ed that no sign if ican t a ltera tions or sign o f tox icity due to the app lication o f in-situ gel.How ever,fu rther stud ies are requ ired to eva luate the possib le tox icity due to repeated and long term app lications and m aterials for the p reparation o f nanopartic les in nano-gelsystems.In add ition,the in creased viscosity o f in-situ ge lm ay cause som e lim itations like b lu rred vision and d iscom fo rt to patien t resu lting in a faster e lim ination due to re f lex tears and b links.Therefore,critical con trolo f the viscosity shou ld be taken in to con sideration du ring design ing and op tim ization o f in-situ gel fo rm u lation in o rder to reduce the lim ita tions to the to lerab le level.

Desp ite the p rom ising po ten tia lo f in-situ ge l in ocu lar d rug de livery,on ly a lim ited num ber o f d rugs in the fo rm o f in-situ ge l are cu rren tly in clin ica l use.Consequen tly,fu rther w o rks shou ld be done to exp lore th is d rug delivery system for the clin ica l app lication o f o ther oph tha lm ic d rugs.

A t p resen t,m ost o f the oph tha lm ic in-situ ge ls w ere designed on ly for the fo rm u lations con tain ing o f single active ingred ien t.In the fu tu re,som em o re su itab le strategies shou ld be developed fo r the fo rm u la consisting o f m u ltip le ingred ien ts such as Trad itiona l Ch inese M ed icine in particu lar,w h ich invo lves a m u lti-target app roach to p roduce their action.Lastly,in the fu tu re,w e expect the innovation o f new and m ore reliab le in-situ form ing po lym ers w h ich m ay be responsive to som e biochem icalm arkers associated w ith the d isease cond itions o f the eye.

Con f lict o f in terest

The au tho rs a ff irm and con f irm that there are no any con f lict o f in terest issues w ith regard to the con ten t o f th is a rticle.