Deve lopm en t o f po lyu rethane foam d ressing con tain ing silver an d asiaticoside fo r hea ling o f derm a lw oun d

Nn tporn Nm viriychote,Vim olm s Lipipun,Yd Akkhw ttnngku l,Phingphol Chroonru t,Grnpim ol C.Ritthidej,∗

a Facu lty ofPharm aceutica l Sciences,Chu lalongkorn University,Bangkok 10330,Thailand

b Facu lty of Veterinary Science,MahidolUniversity,Nakhon Pathom 73170,Thailand

Keyw ords:Po lyu rethane foam d ressing Natu ra l po lyo ls Silver nanoparticles Asiaticoside Wound healing

A B S T R A C TPo lyu rethane foam d ressings fo r derm a l w ounds w ere form u lated w ith natu ra l po lyo ls in o rder to im p rove the foam characteristics an d the release o f 2 active agen ts,silver an d asiaticoside(AS)as an an tim ic robia l agen t and an herba lw oun d hea ling agen t,respectively.The foam w as instan tly form ed by in teraction of po lyols and diisocyanate.Hyd roxyp ropyl m ethy lcellu lose,ch itosan an d sod ium a lginate w ere in d ividua llym ixed w ith them ain po lyo ls,po lyp ropy lene glyco l,in the fo rm u lation w h ile the active com ponen tsw ere im p regnated in to the ob tained foam d ressing sheets.A lthough the type an d am oun to f the natu ra lpo lyo ls sligh tly a ffected the po re size,w ater so rp tion-deso rp tion p ro f ile and com p ression strength o f the obtained foam sheets,a p rom inen t effect w as found in the release o f bo th active com ponen ts.Am ong natu ral po lyo ls fo rm u lation s,foam sheets w ith a lginate show ed the h ighest silver an d AS re lease.Non-cy to tox icity o f these foam sheets to hum an f ibrob last ce lls w as con f irm ed.An tim icrobia l testing on fou r bac teria strain s show ed that 1m g/cm 2 silver in fo rm u lation sw ith 6%o f natu ral po lyo ls an d w ithou t natu ra l po lyo ls had su ff icien t con ten t o f the silver re lease w ith com parab le inh ibition zone and sign if ican tly larger zone than o ther fo rm u lation s.In p ig study,the foam d ressing w ith 6%a lginate,1m g/cm 2 silver an d 5%AS cou ld im p rove w oun d hea ling in bo th the percen tage o f the w oun d closu re an d h isto logica l param eters o f the derm a lw ound w ithou t any derm ato logic reaction s.In con clusion,th is innovative foam d ressing had po ten tia l to be a good can d idate fo r w oun d treatm en t.

1. In trodu ction

Biodegradab le natu ra l po lym er-based nanoparticles are h igh ly accep ted as d rug delivery carriers fo r can cer treatm en t[1].The th ird m ost com m on can cer,co lo recta l can cer is sim u ltaneous occu rren ce o f co lon can cer and recta l can cer[2].Chem o therapy is a com m on strategy fo r treatm en t o f can cer;how ever,it has m any undesirab le side effects caused by chem otherapy in teraction w ith healthy cells in the body[3,4].There fo re,a new in terd iscip linary f ie ld ca lled nanom ed icine has been developed to m od ify the fate o f the d rug in the body[5,6].Overcom ing m ononu clear phagocy te system(MPS)can p ro long the p resen ce o f d rugs in system ic circu lation;[7]th is can be ach ieved by the p resen ce o f PEG on NPs su rface[8].The p resen ce o f targeting ligands on the su rface o f nanoparticles in creases the in tracellu lar delivery of d rugs to specif ic targets[9].M any su rface recep to rs su ch as transferrin and fo late recep to rs are exp ressed in severa l can cer ce ll types.

Fo late recep to rs(FRs)are one o f the m ost exp ressed recep tors on can cer cells[10]although they are exp ressed in m any can cer cell types,their exp ression levels are low in no rm a l cells[11].In particu lar,co lo recta l can cer ce ll lines,HT 29 and HCT116 have over exp ressed the fo late recep to r w h ich can easily in teract w ith fo late targeted nanopartic les on their su rface[12]and in creases release and up take o f cargo d rugs at co lon ic area.There fo re,fo late-targeted PEG con jugated nanoparticles cou ld poten tially enhance the effect o f chem o therapeu tic agen ts by p rom o ting in tracellu lar con cen tration o f en trapped chem o therapeu tic agen ts[13].Fu rtherm o re,FA-con jugated nanopa rticles cou ld enhan ce the residen ce tim e of en trapped d rugs in the body by active and passive targeting tum o r sites,w h ich m igh t redu ce tum or cell resistan ce[14].

Ch itosan nanoparticles arew idely used for severalbiom edica l app lica tions as a d rug de livery system[15].The positive ly charged am ino group o f Cs,the po lyan ion ic natu ra l po lym er,fo rm s com p lexes w ith negatively charged po lyelectro ly tes to spon taneously resu lt in nanoparticle form ation[16].Researchers have fabricated nanoparticles,tab lets,pellets,m icrocapsu les,beads,m em branes,ge ls,and f ilm s by using Cs-based po lyelectro ly te com p lexes(PECs)[17,18].Chs,a natu ra l po lysaccharide,is an acid ic m u copo lysaccharide found in cartilage,bone,an d connective tissue.It can form ion ic com p lexes w ith positively charged ch itosan[19].Chs is m echan ically and chem ically stable and is an idea l m atrix fo rm any biom ed ica l app lications in clud ing site-specif ic d rug delivery in chem o therapy and treatm en t o fm any d iseases.

Recen tly,new d rug de livery system w as developed as PECs p repared from natu ra l po lysaccharides,in clud ing Cs and chond roitin su lfate(Chs)com p lexes[20]because in terpo lym er com p lexed nanocarriers fo r chem o therapy easily develop due to the attraction betw een(NH3+)o f Cs and(-OSO3-and-COO-)o f Chs[21].M o reover,th is PECs have bio logica l p roperties such as biocom patibility,biodegradability and p rodu ce better carriers for ion ized d rugs w ith h igh d rug load ing and en trapm en t eff icien cy[22].

Bo rtezom ib(Bo r),used in the treatm en t o f m u ltip le m yelom a,is an inh ibitor o f the 26S p roteasom e[23].It is a lso used as chem otherapeu tic d rug in m any can cers such as hepatocellu lar carcinom a,p rostate can cer,ovarian can cer,and co lon cancer[24];it also inh ibits tran scrip tion factor nuclear facto r-kappa beta(NF-kB).Bor in creases the cellu lar suscep tibility o f can cers to apop tosis by inh ibiting NF-kB[25].

In the p resen t study,w e deve loped a Bo r-loaded na tu ra l polysaccharide nanoparticle system con jugated w ith folatecoup led DSPE-PEG(Fig.1).PEG m oiety in the am ph iph ilic DSPE-PEG cou ld p rom o te and p ro long the b lood circu lation,w hereas the DSPEm oiety cou ld increase the d rug load ing capacity and stability o f the co re.FA w illact as a targeting ligand to in crease the up take o f d rug in co lo recta l can cer ce ll lines.W e stud ied the therapeu tic activity o f Bo r/Cs/Chs-FA nanoparticles in vitro and in tum or bearing xenograftsm ice m odel as in vivo.

2. M ateria ls an d m ethods

2.1. Materia ls

Borw as obtained from LC Labo rato ries(W obu rn,MA,USA).Cs,Ch s,FA,and MTT[2,5-d ipheny ltetrazo lium b rom ide]w ere pu rchased from Sigm a-A ld rich(St Lou is,MO,USA).HCT-116,HT-29,and A549 cells w ere obtained from the Korean Cell Line Bank(Seou l,Sou th Ko rea).DSPE-PEG2000w as pu rchased from Avan ti Po lar Lip id(A labaster,AL,USA).Coum arin-6 and Lyso Tracker Red w ere pu rchased from Therm o Fisher Scien tif ic Inc.(Waltham,MA,USA).Prim ary an tibod ies,ubiqu itin,and P53 w ere pu rchased from Ce ll Signa ling Techno logy(Danvers,MA,USA).A ll other chem ica ls w ere o f reagen t grade and used w ithou t fu rther pu rif ication.

2.2. Preparation of cs/chs NPs and Bor/Cs/Chs-FA

Cs/Chs,Bo r/Cs/Chs,and Bo r/Cs/Chs-FA NPs w ere p repared by po lyelectro ly te com p lexation betw een carboxy late and su lfate groups from chond roitin su lfate and am ino groups o f ch itosan[26].Brief ly,stock so lu tions o f Cs(1.5m g/m l,pH=4.5)and Chs(1.75m g/m l,pH=6.5)w ere p repared using pu rif ied w ater.The stock so lu tions w ere f iltered using 0.2μm f ilter paper(W hatm an,Dasse l,Germ any)and d ilu ted to op tim ized con cen trations.Cs/Chs NPs w ere p repared by m ix ing Cs and Chs w ith stirring at 25°C.Differen t con cen trations o f Chs(0.41-1.25m g/m l)w ere added in to 1.5m g/m l Cs so lu tion to get desired Chs/Cs w eigh t ratios(1:6).For p reparation o f Bor/Cs/Chs and Bo r/Cs/Chs-FA,f irstly,Bor and FA-con juga ted DSPE-PEG,w h ich w as p repared and characterized in a p revious study[27],w ere d isso lved in phosphate bu ffer sa line.Bor so lu tion w as then added to the Chs so lu tion d ropw ise w ith stirring at room tem peratu re and kep t fo r 6 h.Fina lly,FA-con jugated DSPE-PEG w as added to the Bo r-loaded Cs/Chs NPs,(Bor/Cs&Chs w eigh t ratios,1:2)w ith stirring and kep t fo r 12 h at 25°C.The resu lting so lu tion w as cen trifuged at 13 000 rpm for 25m in at 20°C an d the NPpe lletw as re-suspen ded in pu rif ied d istilled w ater a fter d iscard ing the supernatan t to obtain Bor/Cs/Chs-FA(Bor/Cs&Chs/FA w eigh t ratios,1:2:1)for fu rther study o f in vitro and in vivo experim en ts.

Fig.1-Schem atic illustration o f p reparing fo late ta rgeted po lym eric nanopa rticles to de liver bo rtezom ib to fo late recep to r exp ressing co lo recta l can cers.

2.3. M orphologicalanalysis of cs/chs NPs and Bor/Cs/Chs-FA

The m o rpho logy o f Cs/Chs NPs and Bo r/Cs/Chs-FA w as studied using Transm ission Electron M icroscope(CM 200 UT,Ph ilips,MA,USA).Brief ly,sam p les stained w ith phosphotungstic acid so lu tion w ere pu t on copper grid,w h ich w as coated w ith carbon and d ried at room tem peratu re.Tran sm ission electron m icroscope w as operated at 120 kV to take images o f NPs[28].

The physicochem ica l p roperties o f nanoparticles su ch as zeta-poten tial,po lyd ispersity index(PDI),and Z-average hyd rodynam ic pa rticle size w ere cha racterized using the DLS(dynam ic ligh t scattering)m ethod w ith a Zetasizer Nano ZS(M a lvern Instrum en ts,M a lvern,UK)[29,30]on Nano DTS so ftw are(version 6.34).Th ree ind ividua l m easu rem en ts w ere taken to obtain m ean va lue.

FTIR spectra o f d ried Bo r/Cs/Chs and Bor/Cs/Chs-FA w ere obtained using an FTIR spectrophotom eter(Therm o Scien tif ic Nico let Nexus 670)and com pa red w ith Bo r,Cs,and Chs spectra[31].

2.4. Drug loading and encapsu lation efficiency

To estim ate the en trapm en t e ff icien cy(EE)and load ing capacity(LC)o f Bo r in Bo r/Cs/Chs-FA,w e used an Am icon®centrifuga l f ilter device(m o lecu lar w eigh t cu t-o ff,10 000Da,M illipo re)[32].Unbound d rug in Bo r/Cs/Chs-FA w as separated by cen trifugation for 10m in at5000 rpm;f iltratew as analyzed on an HPLC system(Hitach i,Japan),w h ich com p rised:an L-2130 pum p,L-2200 au tosam p ler,L-2420UV-Vis detecto r,L-2350 co lum n oven,and Ezch rom elite so ftw a re(318a,Japan)[33].PBS(pH 7.4):m ethano l(50:50,v/v)w as used as a m obile phase fo r isocratic e lu tion and an InertsilC18co lum n(150m m×4.6m m,5μm partic le size,Cosm osil,Naca lai Tesque In c.,USA)w as used as the stationary phase.A 20μl sam p le w as in jected;the f low rate o f them obile phasew as 1.0m l/m in and co lum n tempera tu re w as 25°C.UV abso rban ce w as detected at a w avelength o f 278 nm.The percen tages o f EE and LC w ere ca lculated accord ing to the fo llow ing equations:

w here WDis the w eigh t o f Bo r en capsu lated in Bo r/Cs/Chs-FA and WTis the tota l w eigh t o f Bor w hen p reparing the fo rm u la tion.

w here WDis the w eigh t o f Bo r en capsu lated in Bo r/Cs/Chs-FA and WTNis the tota lw eigh t o f nanoparticles in clud ing the am oun t o f add ing Bor,and to ta lw eigh t o f Cs/Chs.

2.5. In v itro drug release study

In vitro release rate o f Bor from Bo r/Cs/Chs and Bor/Cs/Chs-FA w as stud ied in phosphate-bu ffered sa line(PBS;physio logica l pH 7.4)and acetate-bu ffered sa line(ABS;acid ic pH 5.0)using a d ia lysis m em b rane(Spectra/Po r 3500Da-MW CO)[34].Predeterm ined vo lum e o f fo rm u lation w as added to the d ia lysis m em brane and itw as imm ersed in 30m lo f either ABS(0.01M,pH 5.0)o r PBS(0.01M,pH 7.4).Release study w as carried ou t at 37°C w ith shaking at 100 rpm.At specif ied tim e poin ts,samp lesw ere co llected and a liquots o f fresh m ed ia w ere rep laced.The am oun t o f Bor release w as calcu lated by HPLCm ethod as described in Section 2.3.

2.6. Stability of Bor/Cs/Chs and Bor/Cs/Chs-FA

The m ost im po rtan t param eter to characterize the op tim ization o f fo rm u lated nanoparticles is the stability o f created d rug loaded nanopartic les[35].For physical stability,Bor/Cs/Chs and Bo r/Cs/Chs-FA w ere kep t at 4°C an d room tem peratu re,40°C an d the con ten t o f d rug and partic le size w ere determ ined at specif ied tim e poin t.The stability o f Bor/Cs/Chs and Bor/Cs/Chs-FA in physio logica l cond ition w as carried ou t in p lasm a ob tained from the b lood o fm ice by loading Bor/Cs/Chs and Bor/Cs/Chs-FA in p lasm a and them ix tu re o f form u lations and p lasm a w as kep t in shaking incubator at 40°C.The am oun t o f d rug con ten t in nanoparticles an d particle sizes w erem easu red at p redeterm ined in terva l[36].

2.7. Cellu lar cytotoxicity ofBor/Cs/Chs-FA

The cy to toxic e ffect o f Bo r/Cs/Chs-FA w as determ ined by using MTT assay(Prom ega,USA)[37].The percen tage cell viability a fter treatm en tw ith Bo r/Cs/Chs-FA w as com pa red w ith those o f free Bo r and Bo r/Cs/Chs in HT-29 and HCT-116 cells(fo late recep tor exp ressing can cer ce ll lines)and A 549,lung can cer cells(cell line that does not exp ress fo late receptors).A ll ce lls(1×104/w ell)seeded in 96-w ells p lates w ere in cubated for 24 h and treated w ith Cs/Chs NPs,Bo r,Bor/Cs/Chs,Bo r/Cs/Chs and Bo r/Cs/Chs-FA.A fter treatm en t w ith MTT,absorban ce w as determ ined at 570 nm by using an au tom atedm icrop late reader.Un trea ted ce llsw ere designated as con tro ls and the percen tage cell viability o f each ce ll line w as ca lcu lated using the fo llow ing fo rm u la.

W here,A is the absorban ce.

2.8. Cellu lar up take efficacy of Bor/Cs/Chs-FA

To study the loca liza tion and up take o f d rug carrier in bo th folate recep tor exp ressing and de f icien t cell lines,con foca l laser scann ing m icroscopy w as used.All cells(1×105/w e ll)w ere seeded in 12-w ells p lates con tain ing cover slips.A fter 24 h,coum a rin-6-loaded fo late targeted nanoparticles w ere added to each w e ll,fo llow ed by stain ing w ith Lyso Tracker®Red(1μl)for 10m in.Cells in one group from each cell line w ere treated w ith FA(20μg)fo r one hou r as p retreatm en t to quan tify the up take o f fo late-targeted nanoparticles via fo late recep tors.All treated cells w ere kep t in the dark and f ixed w ith 4%para form a ldehyde so lu tion.Fo r visua lization under con foca l laser scann ing m icroscope(Leica M icrosystem s,W etzlar,Germ any),the cover slips con tain ing trea ted cells w ere m oun ted on glass slides and sea led w ith glycerin[38].

The up take o f fo late-targeted nanoparticles w as studied using f luo rescen ce-activa ted ce ll so rting(FACS;BD Bioscien ces,San Jose,CA,USA).Fo late recep tor exp ressing cell lines,HCT-116 an d HT-29(1×105/w ell),w ere seeded in 12 w ells p lates and in cubated fo r 24 h.Subsequen tly,d ifferen t con cen trations o f coum arin-6-loaded targeted nanopartic les w ere added to the ce lls at p redeterm ined tim es.The sam e p rocedu re w as perfo rm ed fo r fo late recep tor def icien t A 549 cell line.To con f irm the fo late recep tor-m ed iated up take e ff icacy o f fo rm u lations,one group o f cellsw as p retreated w ith free FA(20μg).Finally,cells w ere co llected,w ashed and d ispersed in 0.5m l PBS,and ana lyzed[32].

2.9. In v itro cellu lar study

To investigate ce ll cyc le status in clud ing ce ll rep lication,p roliferation,and d ivision at fou r criticalstages(G1,S,G2,and M),cell cycle ana lysis w as carried ou t using a f luorescen ce assay kit(Ce llClockTM;Bioco lo r Ltd.,UK).Brie f ly,a fter in cubation fo r 24 h,cells(1×105/w ell in a 12-w ells p late)w ere treated w ith Bor,Bor/Cs/Chs,an d Bor/Cs/Chs-FA,an d then incubated for ano ther 24 h.Ce lls w ere then stained w ith a redox dye(Ce ll-Clock Dye Reagen t)fo r 1 h at 37°C and im aged using a f luorescen ce m icroscope(N ikon Ec lipse Ti).The num ber o f ce lls arrested in each phase from d igitized pho tom icrographs w as determ ined using Im age Jso ftw are[39].

To determ ine the tox icity o f Bo r/Chs/Cs-FA against bo th folate recep tor exp ressing an d def icien t cell lines,live/dead assay w as perfo rm ed[40].Brief ly,ce lls(1×105/w ell)w ere seeded in a 12-w ell p la te,and treated w ith Bo r,Bo r/Cs/Chs,and Bor/Cs/Chs-FA fo llow ed by in cubation fo r 24 h.Subsequen tly,cells w ere w ashed w ith PBS and then stained w ith p rop id ium iod ide(red f luo rescen ce)and acrid ine orange(green f luo rescen ce).Fina lly,the cells w ere observed under a f luo rescen ce m icroscope(Nikon Ec lipse Ti,Nikon Instrum en ts In c.).

A cellm igration study w as perform ed to estim ate the an tim etastatic effect o f Bor/Cs/Chs-FA.Cells(1×105/w e ll in 12-w ell p lates)w ere seeded an d in cubated fo r 24 h.To get a straigh t cell-free‘scratch’,them ono layer o f cells w as scraped off using a p ipette tip.The cells from the scraped area w ere rem oved by w ash ing w ith PBS an d im ages w ere taken(designated as 0 h).Then,cells w ere in cuba ted fo r 24 h w ith Bo r,Bor/Cs/Chs,and Bo r/Cs/Ch s-FA.Subsequen tly,the ce lls w ere pho tographed and the im ages w ere com pared w ith those taken at 0 h[41].

2.10. Western blot analysis

A ll cells(2×105/w ell)w ere seeded in 6-w e lls p lates.A fter 24 h,a ll seeded cells w ere in cubated w ith Bo r,Bor/Cs/Chs,and Bo r/Cs/Chs-FA for ano ther 24 h.Then,the cells w ere harvested,lysed,and in cubated fo r 40m in on ice w ith p ro teinase inh ibitors.The reaction m ixtu re w as then cen trifuged at 13 000 rpm fo r 20m in at 4°C,and the supernatan t ob tained w as used to quan tify the con cen tration o f p ro teins using a BSA Protein Assay Kit(Therm o Scien tif ic,IL,USA).Bis-Tris po lyacry lam ide gel(10%)w as used to separate p roteins,w h ich w ere then transferred to po lyviny lidene f luoride m em brane.A fter b lock ing w ith 5%non fat m ilk pow der suspension,the m em branes w ere in cubated overn igh t w ith p rim ary an tibodies,GAPDH,p53,an d Ubiqu itin.The exp ression leve ls o f p roteins w ere determ ined a fter in cubation w ith su itab le secondary an tibod ies fo r 1 h using enhan ced chem ilum inescen ce[33].

2.11. In vivo an titum or effects

A tum o r xenogra ftm odelw as deve loped by in jecting HCT-116 cells(1×107)in to the righ t f lanks o f fem a le ba lb/c nudem ice to exam ine in vivo an titum or eff icacy o f Bo r,and its fo rm ulations w ith an d w ithou t fo late targeting.The experim en ta l an im a ls w ere random ly grouped in to con tro l and th ree o ther experim en tal groups(Group 1:con tro l,Group 2:Bor,Group 3:Bo r/Cs/Chs and Group 4:Bo r/Cs/Chs-FA).W hen the tum o r vo lum es reached app rox im ate ly 100m m3,free d rug an d d ifferen t fo rm u la tionsw ere in jected th ree tim es every fou r days via the tail vein.The length an d w id th o f tum ors w ere m easu red by using Vern ier ca lipers and vo lum e w as ca lcu lated as:

Moreover,loss o f body w eigh t,w h ich is one o f the param eters used to characterize the tox icity o f Bo r/Cs/Chs-FA,w as a lso determ ined.A t the end o f the study,a llm ice w ere sacrif iced by CO2inha lation to harvest tum o rs and p rin cipa l organs fo llow ing the p rotoco ls of the Institu tional An im al Eth ica l Com m ittee o f Yeungnam Un iversity,Sou th Korea.A ll tum o rs and o rgans w ere f ixed in fo rm a lin fo r im m unoh istochem ica l assay.The experim en ts w ere app roved by the Institu tiona l An im a l Eth ica l Com m ittee,Yeungnam Un iversity,Sou th Ko rea[33].

2.11.1.Histopathological characterization

The specim ens ex tracted from m ice bearing HCT-116 tum o rs w ere cu t(3-4μm)and stud ied using an op tical m icroscope(Nikon Co rporation,Tokyo,Japan)a fter stain ing w ith hem atoxy lin and eosin(H&E).Fu rtherm ore,com pu ter-based au tom ated im age ana lyzer(iSo lu tion FL ver 9.1;IMT i-so lu tion In c.;Van couver,Quebec,Canada)w as used to estim ate tum or cell vo lum es from in tact tum o r cell-occup ied regions(%m m-2o f tum o rm ass)[42,43].

2.11.2.Immunohistochem istry

The levels o f CD 31,Ki-67,caspase-3,and PARP in ex tracted tum ors w ere determ ined using a com bination o f specif ic antibod ies,an avid in-biotin-perox idase com p lex(ABC),and a perox idase substrate k it(Vector Labs,Bu rlingam e,CA,USA).Brief ly,tissue sections w ere in cubated w ith m ethano l and 0.3%H2O2and then b locked by in cuba tion w ith nonspecif ic im m unog lobu lins an d endogenous perox idase.Sam p les w ere then in cubated fo r 1 h in a hum id if ied cham ber at 95-100°C,w ashed w ith 10m M citrate bu ffer(pH 6.0),and blocked using norm a l horse serum.Subsequen tly,the sam p les w ere treated w ith p rim ary an tisera and in cubated at 4°C overn igh t.A ll sam p les w ere incubated for 1 h at room tem peratu re w ith biotiny lated un iversa l secondary an tibod ies an d ABC reagen ts and rinsed 3 tim es w ith PBS betw een each step.The sam p les w ere noted as positive fo r apop tosis if the sections w ere covered by 20%o rm o re o f each m arker(caspase-3 and PARP)fo r apop tosis.An au tom ated im age ana lyzer w as used to ca lculate the area(%m m-2o f tum orm ass)occup ied by caspase-3 and PARP-positive ce lls located in the tum o rm ass[44,45].

2.11.3.In vivo im aging and biodistribu tion analysis

Qua litative an d quan titative d istribu tion o f Cy 5.5-loaded targeted nanoparticles and Cy 5.5-loaded non-targeted nanoparticles w ere stud ied in m ice bearing HCT-116 tum o rs a fter tail vein in jections(100μl o f sam p les con tain ing 1μg/m l Cy 5.5).The f luorescen ce in tensity o f Cy 5.5 w as determ ined at 1,6,12,and 24 h using FOBIf luorescen ce im aging system(Neoscien ce Co.Ltd).A fter24 h,the f luorescence in tensity in healthy organs(sp leen,heart,lung,liver,an d kidneys)an d tum o rs w as a lso estim ated[46].

2.12. Statisticalana lysis

To determ ine the statistica lly sign if ican t d ifferen ces betw een the groups in a ll experim en ts,Studen t’s t-test(fo r pairs o f groups)and one-w ay ana lysis o f varian ce(ANOVA)w ere used.A ll data are p resen ted as the m ean±standard deviation(SD)and P＜0.05 w as considered statistica lly sign if ican t.

3. Resu lts an d d iscussion

3.1. Prepara tion and characterization of Bor/Cs/Chs-FA

In th is study,w e deve loped Bor-loaded po lym eric nanoparticles by using d ifferen t ratios o fnatu ralpolym ers(ch itosan and chond roitin su lfate)w ith fo late targeting DSPE-PEG.The op tim ized nanopartic les had Chs:Cs at a ratio o f 1:6,an d a particle size o f 165.2±0.9 nm.A fter d rug load ing,the nanoparticle size w as 174.7±0.7 nm;Bor/Cs/Chs-FA w as larger w ith a size o f 186.5±1.2 nm.A llnanoparticles had a narrow size d istribution as show n by po lyd ispersity index(PDI)va lues(Fig.2A).In add ition,excess o f the am ine groups o f ch itosan as com pared to the su lfate group o f chon d roitin su lfate resu lted in the overa ll positive charge,as show n by zeta po ten tia l(Fig.2B).

The m o rpho logy o f Cs/Chs NPs,Bor/Cs/Chs,and Bor/Cs/Chs-FA w as characterized by TEM(Fig.2C).Resu lts show ed that the nanoparticles w ere successfu lly coated by fo late-targeted DSPE-PEG.M o reover,the nanoparticles had sm a ll particle sizes(∼200 nm)and w ere d istin ct an d d iscrete spherica l particles[40].

To determ ine the e ffects o f d rug loaded in Bo r/Cs/Chs-FA,d rug load ing capacity and en capsu lation e ff icien cy are very im portan t param eters.Bor/Cs/Chs-FA w as p repared w ith differen t ratios o f Chs and Cs to study the en capsu lation e ff icacy o f d rugs[47].In th is study,w e selected Ch s/Cs ratio o f 1:6 fo r p reparation o f nanopa rticle because it has h igh Bo r load ing capacity(98.5%)and encapsu lation eff iciency(21.4%)(Fig.2D).In add ition,to characterize the chem ica l in teraction o f d rugs w ith fo late targeting or po lym eric com ponen ts,FTIR ana lysis w as perfo rm ed(Fig.2E).The spectra o f Bo r/Cs/Chs-FA show ed characteristic peak at 1275 cm-1(C=N stretch),1401 cm-1(strong B-O stretch ing o f bortezom ib),and 1720 cm-1(C=O,PEG layer).Borw as thus su ccessfu lly loaded in Bor/Cs/Chs-FA.

Fig.2-Physicochem ica l characterization o f Bo r/Cs/Chs-FA(Bo r/Cs&Ch s/FA w eigh t ratios,1:2:1).(A)Particle size an d PDI,(B)Zeta po ten tia l o f b lank po lym e ric nanoparticle w ith d ifferen t ratios o f Ch s an d Cs,Bo r/Cs/Ch s,and Bo r/Cs/Ch s-FA.(C)TEM im ages o f Bo r/Cs/Ch s an d Bo r/Cs/Ch s/-FA.(D)Percen tage en trapm en t e ff icien cy an d load ing capacity o f bo rtezom ib in Bo r/Cs/Chs-FA w ith d ifferen t ratios o f Ch s an d Cs.(E)FTIR spectra o f Bo r,Cs,Ch s,an d fo rm u lation w ith fo late ta rgeting ligand an d w ithou t fo late targeting ligan d.(F)In vitro release p ro f iles o f Bo r from Bo r/Cs/Chs and Bo r/Cs/Chs-FA at p H 5.0 an d p H 7.4.Data a re show n asm ean±SD(n=6)(∗P＜0.05,∗∗P＜0.01,∗∗∗P＜0.001).

The release rate o f Bor from Bor/Cs/Chs and Bor/Cs/Chs-FA w as eva luated at pH 7.4(PBS)and pH 5.0(ABS)using d ia lysis m em brane(Fig.2F).The re lease o f Bo r from bo th Bo r/Cs/Ch s and Bor/Cs/Chs-FA show ed sign if ican t in crease at acid ic pH as com pared w ith physio logica l pH.M oreover,the re lease rate from Bo r/Cs/Chs-FA w as sligh tly slow er than that from Bor/Cs/Chs.Th is m ay be due to rap id d istribu tion and en zym atic degradation o f free d rug from Bo r/Cs/Chs in system ic circu lation;m o reover,de livery o f d rugs loaded in targeted nanoparticles resu lts in con tro lled re lease at tum o r site.Fu rtherm ore,ch itosan is easily d isso lved in acid ic m ed ia,resu lting in the release o f d rug and con jugate layer in acidic environm en t o f tum o r site,lead ing to in creased an titum or e ff icacy and redu ced un tow ard e ffects to hea lthy tissue[48].

Stability of Bor/Cs/Chs and Bor/Cs/Chs-FA w as exam ined in physio logica l state,p lasm am ed ia an d at tw o d ifferen t sto rage tem peratu res(4°C an d 40°C)by m easu ring partic le sizes,PDI,zeta po ten tia land am oun t o f d rug con ten ts in nanoparticles[49].Accord ing to the resu lts o f hyd rodynam ic d iam eter,PDIand zeta po ten tia lo f Bo r/Cs/Ch s and Bo r/Cs/Ch s-FA at tw o d ifferen t tem peratu res(Fig.S1),created nanoparticles,bo th targeted and non-targeted nanopartic les have e ff icien t stability at d ifferen t tem peratu res because there w as no sign if ican t d ifferen ce in m easu rem en t o f hyd rodynam ic d iam eter,PDI and zeta po ten tia l.The electrostatic attraction betw een Cs and Chs w as su ccessfu lly developed to fo rm po lye lectro ly te com p lex.Sim ilarly,the op tim ized stability o f Bor/Cs/Chs-FA in physio logica l cond ition had been p roved by estim ating the con ten ts o f d rug in nanoparticles and hyd rodynam ic d iameter,PDIand zeta poten tia l o f Bo r/Cs/Chs and Bo r/Cs/Chs-FA after load ing w ith p lasm a.The resu ltsw ere show n in(Fig.S2).En trapm en t e ff icien cy o f Bo r in Bor/Cs/Chs-FA w asm o re than that o f Bo r/Cs/Chs an d it w as supported that DSPE-PEG in fo late targeted ligand used in created form u lation p rotected the d rug from unw an ted re lease before reach ing the targeted area and RES clearan ce.

Fig.3-(A)In vitro cy to tox icity e ffect o f Bo r,Cs/Ch s NPs,Bo r/Cs/Ch s,an d Bo r/Cs/Chs-FA(Bo r/Cs&Chs/FA w eigh t ratios,1:2:1)at d ifferen t con cen tration on fo la te recep to r positive co lo recta l can cer ce lls,HCT-116 an d HT-29,an d fo late recep to r negative lung can cer cell line,A 549.Data are show n asm ean±SD(n=6)(∗P＜0.05,∗∗P＜0.01,∗∗∗P＜0.001).(B)Con foca l im ages fo r ce llu lar u p take o f Coum arin-6-loaded ta rgeted nanoparticles w ith lyso tracker red in HCT-116,HT-29,and A 549 ce lls(i)w ith p retreatm en t o f FA,(ii)w ithou t p retreatm en t o f FA.Sca le ba rs rep resen t 100μm(Fo r in terp retation o f the re feren ces to co lo r in th is f igu re legend,the reader is re ferred to the w eb ve rsion o f th is a rticle.).

3.2. Cytotoxicity of Bor/Cs/Chs-FA

The cy to toxicity o f Bor/Cs/Chs-FA w as eva luated in both folate recep tor exp ressing co lo recta l can cer cell lines(HCT-116 and HT-29)and fo late recep to r def icien t lung can cer cell line(A 549)[30](Fig.3A).Cs/Chs NPs show ed cy to tox ic effect on all th ree cell lines at con cen trations＞50μg/m l.These resu lts are in line w ith those o f som e p revious stud ies,w h ich reported that ch itosan has sign if ican t bio logical activities,includ ing im m uno-enhan cing e ffects,an tim icrobia l activities,w ound hea ling,and an titum o r activities[50].As expected,Bo r/Cs/Chs-FA w as m o re cy to tox ic in fo la te recep to r positive cell lines(HCT-116 and HT-29)than free d rug an d Bor/Cs/Chs w ere.In con trast,the cy to tox icity o f Bo r/Cs/Ch s-FA in A549 cell line w as sim ilar to that o f non-targeted nanopa rticles Bo r/Cs/Chs,because A549 ce ll line lacks fo late recep tors.IC50values o f Bor and Bor/Cs/Chs-FA con f irm ed these resu lts.To determ ine the IC50va lues,standard cu rvesw ere p lo tted by using the cy to tox icity p ro f iles(Fig.S3).IC50va lues o f Bo r/Cs/Chs-FA in both fo late recep to r exp ressing co lo recta l can cer cell lines w ere low er than in folate recep tor def icien t lung cancer cells(Tab le S1).

3.3. In tracellu lar uptake and targeting potency of Bor/Cs/Chs-FA

In th is study,co loca lization o f coum arin-6-loaded fo late targeted nanoparticles in a ll ce lls w ere observed using confoca l m icroscopy.M ost coum arin-6-loaded fo late targeted nanoparticles(green)w ere localized around the perinuc lear region o f bo th fo late recep tor-exp ressing cell lines,suggesting that these nanopartic les w ere d istribu ted in to the cy top lasm.There w asm in im um up take in FA p retreatm en t group.M oreover,the up take o f FA-targeted nanoparticles in these cell lines w as m a rked ly inh ibited by the p resen ce o f excess am oun ts o f free folic acid.It suggested that the up take o f targeted nanoparticles depended on the fo late recep to r m ed iated endocy tosis.Fu rtherm o re,the ro le o f fo late ligand in cellu lar up take m echan ism w as con f irm ed in A549 ce lls,w h ich show ed no sign if ican t change of up take w ith or w ithou t FA p retreatm en t(Fig.3B)[38].

Fig.4-(A)Quan titative cellu lar u p take o f coum arin-6-loaded targeted nanoparticles in d ifferen t cell lines determ ined by using FACS;ce lls w ere either p retreated w ith FA o r w ere w ithou t FA p retreatm en t.(B)Fluo rescen ce m icroscop ic im ages o f fo late recep to r positive hum an co lo recta l can cer ce lls,HCT-116 an d HT-29,an d fo late recep to r nega tive lung can cer ce ll line,A 549,in cubated w ith(i)con tro l,(ii)Bo r,(iii)Bo r/Cs/Ch s,and(iv)Bo r/Cs/Chs-FA(Bo r/Cs&Ch s/FA w eigh t ratios,1:2:1)fo r 24 h w ere taken after stain ing w ith Live/Dead assay k it con ten ts;green o r red f luorescen ce is def ined as live or dead cells,respective ly.Sca le ba rs rep resen t 100μm(Fo r in terp reta tion o f the re feren ces to co lo r in th is f igu re legen d,the reader is re ferred to the w eb version o f th is a rticle.).

Mo reover,qua litative tim e-and con cen tration-dependen t up take p ro f ile o f coum arin-6-loaded fo late-targeted nanoparticles w as exam ined in bo th fo late recep tor exp ressing and def icien t ce ll lines by using FACS ana lysis(Fig.S4).The f luorescen ce in tensity w as in creased w hen the dose(1-5μg)and treatm en t tim e(30-90m in)w ere in creased.How ever,there w as no sign if ican t change in f luo rescen ce in ten sity in A 549 cell line,a lthough treatm en t tim e and con cen tration w ere increased.Fu rtherm o re,the ro le o f fo late ligan d in cellu lar uptake o f targeted nanopartic les w as con f irm ed by low up take in fo lic acid p retreatm en t group o f fo late recep tor exp ressing HCT-116 and HT-29 cells(Fig.4A)[51].

Fu rtherm o re,quan titative ce ll death w as exam ined in a ll cells treated w ith free d rug and fo rm u lations w ith and w ithou t fo late targeting ligand by sequen tia l treatm en t w ith acrid ine orange(AO)an d p rop id ium iod ide(PI)to d istinguish the live and dead ce lls[52].A ll cells treated w ith free d rug show ed m o re dead cells as com pared to in the con tro l group.HCT-116 and HT-29 ce lls treated w ith Bo r/Cs/Chs-FA had h igher cell death rate than A 549 cells had(Fig.4B).Th is cou ld be attribu ted to the h igher nanoparticle in terna lization in fo late recep to r overexp ressing co lo recta l can cer cells as com pared to fo late recep tor def icien t cells.

Ce ll cycle ana lysis w as perfo rm ed to study the cell d ivision and p ro liferation o f a ll th ree cell lines fo llow ing treatm en t w ith Bo r,Bo r/Cs/Chs,an d Bor/Cs/Chs-FA using a redox dye,w h ich changes co lor accord ing to the cell cycle phases(G1,S,G2,and M)[39].Bo r/Cs/Chs-FA treated group in HCT-116 and HT-29 cell lines on ly had ce ll popu la tion w ith G1 phase,w hereas cell popu lation w as observed in all phases in A549 cells.How ever,Bo r and Bor/Cs/Chs treated groups show ed cells in M and S phase(Fig.5A,S5).Th is show s that cell cycle arrest a fter treatm en tw ith Bor/Cs/Chs-FA is h igh ly p rom o ted by folate recep torm ed iated up takem echan ism.These resu lts a lso h igh ligh t the e ffect o f fo late targeting ligand in eff icacy o f fo rm u la tions.

Fig.5-(A)Distribu tion o f ce lls in ce ll cycle phases,G1,G2,M,and S phase a fter 24 h treatm en tw ith Bo r,Bo r/Cs/Ch s,and Bor/Cs/Ch s-FA(Bor/Cs&Ch s/FA w eigh t ratios,1:2:1)in fo late recep tor positive hum an co lo recta l can cer cells,HCT-116 an d HT-29,an d fo late recep to r negative lung can cer ce ll line,A 549,w as v isua lized by f luo rescen ce m icroscopy.Sca le ba rs rep resen t 100μm.(B)Ce llm ig ration e ff icien cy a fter 24 h treatm en tw ith(i)con tro l,(ii)Bo r,(iii)Bo r/Cs/Ch s,an d(iv)Bo r/Cs/Ch s-FA in fo late recep to r positive hum an co lo recta l can cer ce lls,HCT-116 an d HT-29,and fo late recep to r negative lung can cer cell line,A 549.(C)W estern b lo t ana lysis in stated cells fo llow ing incubation w ith(i)con tro l,(ii)Bo r,(iii)Bo r/Cs/Ch s,an d(iv)Bo r/Cs/Ch s-FA(v)by using specif ic p ro teins,P53 an d Ubiqu itin.GAPDH w as used as a con tro l.

To observe the effect o f Bo r/Cs/Chs-FA on the m igration ability o f co lo recta l can cer cells,w ound hea ling assay w as perfo rm ed.The resu lts show ed that the cell popu lation o f HCT-116 and HT-29 treated w ith Bor/Cs/Chs-FA w as sign if ican tly low er in the area betw een the scratched edges than tha t in the con tro l and free d rug cond ition s(Fig.5B).How ever,area betw een the scratched edges o f A 549 ce lls a fter treatm en t w ith Bo r/Cs/Chs-FA w as sim ilar to that a fter treatm en tw ith non-targeted nanoparticles(Bor/Cs/Chs).Th is show s that Bo r/Cs/Chs-FA w as ab le to inh ibit m igration and invasion m ed iated by FA recep tor in vitro.

Fu rtherm ore,the effect o f Bor/Cs/Chs-FA on the exp ression o f d ifferen t apop to tic p ro teins w as investigated in bo th fo late recep to r exp ressing and def icien t ce ll lines by w estern b lo t ana lysis(Fig.5C,S6).The exp ressions o f apop to ticm arker,P53,and Bor specif ic m arker,ubiqu itin,in folate recep tor exp ressing co lo recta l can cer cells trea ted w ith Bo r/Cs/Chs-FA w ere sign if ican tly enhan ced as com pared w ith con tro l,free d rug,and Bor/Cs/Chs trea tm en ts.How ever,no such e levation in apop to tic p ro tein levels w as observed in fo late recep to r def icien t ce ll line A 549.These data show ed that Bo r/Cs/Chs-FA cou ld in crease the num ber o f cells in late apop to tic phase via fo late targeted DSPE-PEG of nanoparticles.

3.4. In vivo an titum or effects

HCT-116 tum o r bearing xenogra ftm ice m ode lw as developed to ana lyze the in vivo an titum o r e ffect o f Bo r/Cs/Chs-FA on co lo recta l can cer.Bo r/Cs/Chs-FA cou ld inh ibit the tum o r p rogression by early DNA fragm en tation coup led w ith apop tosis,w h ich enhan ced its an titum o r e ffect on fo late recepto r overexp ressing co lorecta l can cer(Fig.6A).Th is sign if ican t im p rovem en t in an titum o r e ffect suggested tha t the re lationsh ip betw een fo late targeted nanoparticles and fo late recepto rs on can cer cells cou ld p lay a vita l ro le in chem o therapy.In add ition,a fter check ing tum o r vo lum e and body w eigh t in the con tro l and treatm en t groups,w e can conclude that fo late targeted delivery o f Bo r/Cs/Chs to can cer ce llsw ith ability o f a p ro longed system ic circu lation resu lted in m in im ization o f un tow ard e ffects(Fig.6B).M o reover,tum o r ce ll apoptosis w as in creased by inh ibition o f tum o r ce ll p ro liferation and angiogenesism echan ism s w ithou t toxico logica l signs on the f ive p rin cipa l o rgans at h isto logica l leve l,as show n by H&E stain ing(Fig.6C,Table S2).The levels of CD31 and Ki-67,cellp ro liferation and angiogenesism arkers,w ere sign if ican tly decreased in tum or tissues treated w ith Bo r/Cs/Chs-FA than in those treated w ith free d rug,and non-targeted nanoparticles.Fu rtherm ore,h igh level of apop totic m arkers,caspase-3 and PARP,w h ich p rom o te can cer ce ll apop tosis an d angiogenesis,and p ro liferation w ere observed in ce lls treated w ith Bo r/Cs/Chs-FA(Fig.6D,Tab le S3).Th is in vivo an titum o r study p roved that Bo r/Cs/Ch s-FA cou ld successfu lly de liver Bo r to can cer cells via fo late targeted nanoparticles and exert the antitum or e ffects by inh ibiting tum o r ce ll p ro liferation and indu cing cellu lar apop tosis by activating im m une cells.

Fig.6-In vivo an titum or stud ies on(A)tum or vo lum e,(B)body w eigh t,an d(C)h istopatho logica l changes o f organ s(heart,liver,sp leen,lungs,an d k idneys)fo llow ing in travenous adm in istra tion o f d ifferen t fo rm u lations(con tro l,Bo r,Bo r/Cs/Ch s,and Bo r/Cs/Ch s-FA(Bo r/Cs&Ch s/FA w eigh t ratios,1:2:1))to HCT-116 tum o r bea ring BALB/c nude m ice.Each fo rm u lation w as adm in istered fou r tim es at th ree-day in terva ls(∗P＜0.05,∗∗P＜0.01,).(D)Im m uno-h istopatho logica l ana lyses.Rep resen tative changes in tum o r h istopatho logy an d im m uno reactiv ity to caspase-3,PARP,Ki-67,an d CD31 in tum o r m asses o f nude m ice fo llow ing treatm en tw ith the ind icated fo rm u lation s a re show n.Caspase-3 an d PARP a re m a rkers fo r apop tosis and CD 31 and Ki-67 a re m arkers fo r angiogenesis.Sca le ba r is 120μm.

Fig.7-In vivo biod istribu tion f luorescen ce im aging o f subcu taneous HCT-116 tum or-bearing BALB/c nude m ice a fter iv in jection o f 100μl cyan ine 5.5 loaded Bo r/Cs/Ch s-FA(Bo r/Cs&Chs/FA w eigh t ratios,1:2:1)and Bo r/Cs/Chs(bo th con tain ing 1μg/m l cyan ine 5.5).

3.5. Biodistribu tion studies

In vivo f luorescen ce im aging w as carried ou t to estim ate the d istribu tion ability o f targeted nanoparticles in tum o r area an d other vital organs.Cy 5.5 w as used as the f luorescen ce m arker.A fter in jection o f Cy 5.5 loaded targeted nanoparticles and Cy 5.5 loaded non-targeted nanoparticles in to the tail veins o fm ice m odels,f luo rescen ce w as im m ed iately d istribu ted th roughou t the body in both in jected groups(Fig.7).How ever,the f luo rescen ce in tensity in the tum o r area o f group in jected Cy 5.5 loaded targeted nanopa rticles w as m u ch h igher than that o f non targeted nanopartic les,suggesting that the fo late targeted nanoparticles cou ld enhan ce the d rug de livery an d accum u la tion o f nanoparticles in to targeted tum or areas.A fter 24 h,f luo rescen ce in ten sity o f d issected organs(heart,liver,sp leen,lung,an d kidney)and tum or tissue w ere checked.If nanoparticle cou ld d istribu te in vivo for an accep tab le tim e to release the d rug in the tum or area,an enhan ced perm eability and reten tion(EPR)effect is like ly to be obtained w ith redu ction o f card io tox icity.Thus,w e suggest that Bo r/Cs/Chs-FA h igh ly accum u lates in tum o r area and show s low non-specif ic up take in reticu loendothelialsystem(RES).Th is in tu rn facilitates up take of fo late ta rgeted nanoparticles to fo late recep tor overexp ressing cancers via the recep to r-m ed iated endocy tosis[53].

4. Con clusion

In con clusion,Bor w as su ccessfu lly en capsu lated in folateta rgeted Cs/Chs com p lex nanoparticles.Bo r/Cs/Chs-FA had h igh d rug load ing capacity w ith nano-sized particles.The e ff icacy o f inducing apop tosis and ce llu lar up take e ff icien cy o f Bo r/Cs/Chs-FA in fo late recep to r overexp ressing can cer cells w ere enhan ced by using biocom patib le and biodegradab le po lysaccharides com p lexed nanocarriersw h ich a llow the sustained release and im p rove load ing capacity of Bor.In vivo an titum or stud ies in HCT-116 xenogra ft m ice revea led that Bo r/Cs/Chs-FA m ed iated a greater supp ression o f xenogra ft tum o rs w ith lesser toxicity to norm a l o rgans and tissues as com pared to those of con tro l,free d rugs,and form u lation w ithou t fo late targeting ligand.Hen ce,ou r op tim ized form u lation m ay be bene f icia l in fo late-recep to r-targeted chem otherapy,lead ing to be poten tial cand idates for treatm en t of fo late recep to r overexp ressing co lorecta l can cers.

Decla ration o f in terest

The au tho rs report no con f licts o f in terest.The au tho rs a lone are responsible for the con ten t and w riting o f th is article.

Acknow ledgm en t

Th is research w as suppo rted by the Yeungnam Un iversity research gran t in 2017.

Su pp lem en ta ry m ateria ls

Supp lem en tary m ateria l associated w ith th is a rticle can be found,in the on line version,at doi:10.1016/j.a jps.2018.09.004.