Co-de livery o f resveratro l an d docetaxe l v ia po lym ericm ice lles to im p rove the treatm en t o f d rug-resistan t tum o rs✩

Xiong Guo,Zhiyu Zho,Dw i Chn ,M ingxiQio,∗,Fng Wn ,Dongm iCun,Yi Sun ,M ingsh i Yng,,∗∗

a Wuya College of Innovation,Shenyang Pharm aceu ticalUniversity,Wenhua Road No.103,Shenyang 110016,China

b School ofGraduate,Liaoning University of Traditional Chinese Medicine,Chong Shan Road No.79,Shenyang 110847,China

c School ofPharm acy,Shenyang Pharm aceu tical University,Wenhua Road No.103,Shenyang 110016,China

d Departm ent ofPharm acy,Facu lty ofHealth and M edica l Sciences,University ofCopenhagen,Universitetsparken 2,DK-2100 Copenhagen O,Denm ark

e Departm ent ofM icro-and Nanotechnology,Technical University ofDenm ark,Ørsteds Plads,Kgs.Lyngby 2800,Denm ark

Keyw ords:Resveratro l Docetaxel Methoxy l po ly(ethy lene glyco l)-po ly(D,L-lactide)copo lym er(m PEG-PDLA)M ice lles Drug resistance tum or

A B S T R A C TCo-de livery o f an ti-can cer d rugs is p rom ising to im p rove the e ff icacy o f can cer treatm en t.Th is stu dy w as aim ing to investigate the poten tia lo f con cu rren t delivery o f resveratro l(RES)and docetaxel(DTX)via polym eric nanocarriers to treatbreast cancer.To th is end,m ethoxy l po ly(ethy lene glyco l)-po ly(D,L-lactide)copo lym er(m PEG-PDLA)w as p repared an d characterized using FTIRan d 1H NMR,an d theirm o lecu lar w eigh tsw ere determ ined by GPC.Isobo logram analysis an d com bination in dex ca lcu lation w ere perform ed to f in d the op tim al ratio betw een RESand DTX to against hum an breast adenocarcinom a cell line(MCF-7 cells).Subsequen tly,RES an d DTX w ere loaded in the m PEG-PDLA m ice lles sim u ltaneou sly,and the m o rpho logy,particle size d istribu tion,in vitro release,pharm acokinetic p ro f iles,as w e ll as cytotox icity to the MCF-7 ce lls w ere characterized.IC50 o f RES an d DTX in MCF-7 cells w ere determ ined to be 23.0μg/m l and 10.4μg/m l,respectively,w h ile a low er IC50 of 4.8μg/m l of the com bination o f RESan d DTX w as ob tained.The com bination o f RESand DTX at a ratio o f 1:1(w/w)generated stronger synergistic effec t than o ther ratios in the MCF-7 ce lls.RES and DTX loaded m PEG-PDLA m icelles exh ibited p ro longed release p ro f iles,and enhanced cy totoxicity in vitro against MCF-7 cells.The AUC(0→t)o f DTX an d RES in m PEG-PDLA m ice lles a fter i.v.adm in istration to rats w ere 3.0-fo ld an d 1.6-fo ld h igher than that o f i.v.in jection s o f the in d ividual d rugs.These f in d ings in d icated that the co-delivery o f RES an d DTX using m PEG-PDLA m icelles cou ld have better treatm en t o f tum ors.

1. In trodu ction

The e ffect o f chem o d rug therapy fo r can cer is con f ined by the deve lopm en t o f viru len tm u lti d rug resistan t(MDR)pheno types,w h ich is a m a jo r im ped im en t to can cer treatm en t.MDR is associa ted w ith acqu ired de fen se m echan ism s,fo r instan ce blocked apop tosis and in creased d rug eff lux[1].Com bination chem o therapy is one o f the stra tegies used to overcom e the can cerm u ltid rug resistan ce,w h ich cou ld o ffer advan tages such as targeting d ifferen t signa l pathw ays in can cer cells,and im p roving the therapeu tic index either by in creasing therapeu tic eff icacy o r redu cing tox icity[2-5].

It has been reported that the co-delivery o f com bined an tican cer d rugs to tum or cells using nanocarriers is a hopefu l strategy fo r treating MDR[6-7].Various nanocarriers have been investigated,in clud ing lip id nanoparticles[8],po lym eric nanopartic les,po lym eric m ice lles[9,10],liposom es[11],and dend rim ers[12],etc.Am ong them,self-assem b ly polym eric m icelles are very attractive for co-delivery o f m u ltip le an tican cer agen ts.Th is is because(i)the hyd rophobic co re cou ld a llow for load ing hyd rophobic d rug com pounds such as antican cer d rugs;(ii)the ou ter hyd roph ilic she ll cou ld p ro long circu lation tim e;and(iii)they cou ld p rovide sustained d rug re lease p ro f iles.In ou r p reviousw o rk,resveratro l(RES)and docetaxel(DTX)have been loaded in m PEG-PDLA m icelles separate ly[13].The ob tained m PEG-PDLA m icelles exh ibited p rolonged re lease p ro f iles fo r the tw o d rug com pounds.In th is study,w e in tended to study the po ten tia lo f co-delivery o f RES and DTX usingm PEG-PDLAm icellesw ith respect to the treatm en t o f b reast can cer.

DTX is a w idely used an tineop lastic agen t.How ever,it o ften su ffers from d rug resistan ce,resu lting in a decrease in the an ti-can cer e ffect.RES is a so rt o f phy tochem ica l,w h ich can be ob tained from the roo ts o f w h ite he llebore and d ifferen t food sou rces.It has been reported that RES can dow n regulate the exp ression o fm u ltid rug resistan tgenes encoded for Pgp,and inh ibit the m am m a lian target o f rapam ycin via py ruvate k inase isoenzym e type M 2,thereby p reven ting can cer ce ll m etabo lism[14].It has been observed that RES rein fo rced cytotox ic p roperties o f both an ti-can cer d rugs th rough in creasing their in trace llu lar level base on P-gp inh ibition and dow nregu lation o f MDR1[15].W e hypothesized that co-de livery o f RES and DTX cou ld p rovide synergistic an ticancer effect and generate better treatm en t effecto f can cer than the an ti-tum o r w ith each d rug a lone.In th is study,the synergistic e ffects o f co-adm in istra tion o f RESand DTXw ere f irst investiga ted.Subsequen tly,co-delivery o f RES and DTX using m PEG-PDLA m icelles w as eva lua ted w ith respect to in vitro re lease,k illing d rug-resistan ce tum o r ce lls,and pharm acok inetic p ro f iles in rats.

2. M ateria ls an d m ethods

2.1. Materia ls

D,L-lactide w as pu rchased from GLACO Ltd.(Ch ina).Po lyethylene g lyco l(m PEG2000),stannous octoate(Sn(Oct)2)and 3-[4,5-d im ethy l-2-th iazo ly l]-2,5-d ipheny l-2-tetrazo lium brom ide(MTT)w ere pu rchased from Sigm a(USA).Docetaxelw as pu rchased from Shanghai Jinhe Bio-tech Co.,Ltd.(Ch ina).Resveratro l w as obtained from Guanyu Bio-tech Co.,Ltd.(Ch ina).A ll other chem ica ls and so lven ts w ere o f the h ighest reagen t grade and used w ithou t fu rther pu rif ication.

2.2. Preparation and characterization ofm PEG-PDLA block copolym er

m PEG-PDLA b lock copo lym er w as p repared by ring-open ing po lym erization o f app rop riate am oun t o f D,L-lactide m onom er in the p resence o f po lyethy lene glyco l(m PEG,Mn=2000)using stannous octoate as the cata lyst in a sea led tube(Fig.1).The sea led tube w asm ain tained under 130°C fo r 12 h w ith con tinuousm agnetic stirring.A fter that,the syn thesized po lym erw as collected by add ing som e d ich lorom ethane in to the tube an d then fo llow ed by p recip itation in ice-coo l ether fo r 48 h.The p recip itate w as f iltered,d ried at room tem peratu re under vacuum[16].

The m o lecu lar stru ctu re o f the syn thesized m ateria l w as determ ined by using FTIR(Bruker,Sw itzerland),an d p roton nu clear m agnetic resonan ce(1H NMR,Bruker ARX-300,Sw itzerland).FTIR spectrum s w ere co llected using a Bruker vecto r 22 ana ly tica l FTIRSpectrom eter over the region o f 400-4000 cm-1.The stru ctu re,average m o lecu lar w eigh t and the m PEG con ten t o f the copo lym er w ere determ ined by gel perm eation ch rom atography(GPC,W aters,USA)and1H NMR in CDCl3at 300Hz.The GPC m easu rem en ts w ere conducted w ith a W aters 2414 GPC detecto r in strum en t equ ipped w ith Sty rage l®HR THF(7.8m m×300m m)co lum n.THF w as used as eluen t at a f low rate of 1.0m l/m in and co lum n therm ostat w as set at 35°C.

2.3. Screen ing the ratio ofRES to DTX

The op tim a l ratio o f RES to DTX w ith respect to an titum or effect w as determ ined by in cubating the tw o com pounds w ith MCF-7 cells as repo rted p reviously[17].Brief ly,MCF-7 tum o r cellsw ere seeded in a 96-w ells p late(6000 cells/w e ll)12 h p rio r to the experim en t.Then,the ce lls w ere treated w ith veh icle Du lbecco’s m odif ied Eagle m ed ium(DMEM)(con tro l group),DTX(0-50.0μg/m l,DTX group),RES(0-100.0μg/m l,RESgroup),and com bination o f DTX and RES(com bination group),respective ly.Fo r the com bination group,1.0m l o f DTX and RES w ith various con cen trations w ere com bined in the schem e of 2.5+2.5,5+5,10+10,20+20,and 50+50μg/m l and added in to the w ells.Tw o days a fter d rug stim u lation,the op tica l density(OD)o f each w e llat 490 nm w asm easu red using a fu llw ave length m u lti-fun ction m icrop late reader(Therm o Scientif ic Co rp.,W a ltham,USA).Each cond ition com p rised six rep licate w ells w ith a t least th ree independen t dup licates.

Fig.1-Syn thesis o fm PEG-PDLA.

2.4. Evaluation ofsynergistic effect ofdrug com binations using the CIm ethod

The com bination index(CI)w as used to eva luate the synergistic,an tagon istic o r add itive effects o f d rug com bina tions[18],w h ich is ca lcu lated by using the fo llow ing fo rm u la:

w here IC50ab1,IC50ab2are the IC50va lues w hen the d rugs a re adm in istered in com bination,w h ile IC50a,IC50bare the IC50va lues w hen the d rugs are adm in istered as sing le agen ts.CI＞1 ind icates an tagon ism,CI＜1 ind icates synergy and CI=1 in d icates an add itive effect.

2.5. Prepara tion and characterization ofdrug-loaded m PEG-PDLA m icelles

DTX and RES loaded m PEG-PDLA m icelles w ere p repared by using the repo rted so lven t casting m ethod[19].Brief ly,DTX,RES and m PEG-PDLA w ere d isso lved in aceton itrile and then added in to a round-bo ttom f lask.A th in and hom ogenous f ilm con tain ing the tw o d rugs and po lym er w as form ed after the so lven tw as rem oved w ith a ro tary evapo rator un der vacuum in a 50°Cw ater bath.A fter the f ilm w as coo led dow n to room tem peratu re,it w as rehyd rated in 2.0m l o f deion ized w ater w ith gen tle agitation to form the DTX/RES m ice lles spon taneously.The resu ltan t m icellar co lloida l system w as f iltered th rough 0.22μm f ilter.

The particle size and zeta poten tial o f DTX/RES m icelles w ere m easu red using M a lvern Zetasizer Nano-ZS(M a lvern Instrum en ts Ltd.,Eng lan d).The m o rpho logy o f DTX/RESm icellesw as observed by using a JEM-2100 transm ission electron m icroscopy(TEM,JEOL Ltd.,Tokyo,Japan).In sam p le p reparation p rocedu re,certain am oun t o f DTX/RESm icelles w ere attached to copper grids and negative ly stained w ith 2%phosphotungstic acid for 15 s.After the rem ovalo fexcessive liquid,the sam p les w ere d ried in air and sub jected to TEM exam ination.

2.6. In v itro release kinetics ofDTX and RES from m icelles

The re lease p ro f ile o f DTX and RES from the m PEG-PDLA m icelles w as evaluated by using a p reviously reported m ethod w ith sligh t m od if ication[20].In b rie f,1.0m l o f DTX ethano l so lu tion(1.0m g/m l),RES ethano l so lu tion(1.0m g/m l)and DTX/RES m PEG-PDLA m ice lles w ere p laced in d ia lysis bags w ith the cu tting off MWof 3000Da.These d ialysis bags w ere in cubated in 100m l o f pH 7.4 phosphate bu ffer con tain ing 0.5%(w/w)Tw een 80 to ach ieve the sink cond ition and w ere shaken in a ho rizon ta l shaking w ater bath(37±0.5°C)at 100 rpm for 72 h.At p redeterm ined tim e in tervals,1.0m l o f sam p le w as w ithd raw n and rep laced w ith an equa l vo lum e o f the fresh released m ed ium.The co llected sam p les w ere f iltered th rough 0.22μm f ilter and quan tif ied by HPLC.In detail,the ana lysis w as perform ed w ith Hitach i HPLC system(UV Detector L-2400,Pum p L-2130,Hitach i,Tokyo,Japan)and a reversed-phase co lum n(Diam onsil,C18,4.6m m×250m m,5μm).The m obile phase m ade up o f aceton itrile and w ater(50:50;v/v)w as in jected at a f low rate o f 1.0m l/m in,and RES and DTX w as detected at a w ave length o f 228 nm.

2.7. Pharm acokinetics studies

2.7.1. Anim als

Sp rague-Daw ley rats(m ale,body w eigh ing abou t 200 g)obtained from the Experim en ta l An im a l Cen ter at Shenyang Pharm aceu tica l Un iversity(SPU)w ere em p loyed in pharm acok inetic stud ies.A ll an im a l experim en ts w ere perfo rm ed strictly in linew ith gu ide lines app roved by the Life Scien ce Research Cen ter a t SPU.A ll e ffo rts w erem ade to lim it the number o f an im a l used and to m in im ize an im a l su ffering.

2.7.2. Experim ent design

The rats w ere ran dom ly d ivided in to th ree groups(n=4),i.e.DTX ethano l so lu tion group,RES ethanol so lu tion group and DTX/RES m PEG-PDLA m ice lles group.Each sam p le w as intravenously adm in istered a t a dose o f 10m g/kg body w eigh t,respectively.At given tim e poin ts(0.083,0.5,1,2,4,6,8,10,12 h post in jection),0.5m l o f b lood w as d raw n from the orbit venous p lexus o f the rats and p laced in heparin ized tubes.Plasm a w ere im m ed iate ly separated by cen trifugation at 5000 rpm for 10m in and stored at-20°C un til fu rther analysis.

2.7.3. Quantitative analysis ofDTX and RES in plasm a samples

For the f irst step of extraction p rocedu re,200μl p lasm a w as m ixed w ith 20μl o f DTX(10.0μg/m l)and carbam azep ine(10.0μg/m l)that w as used as the in terna l stan dard.DTX and RES w ere ex tracted from p lasm a w ith 2.0m l o f m ethy l tertbu ty lether by vigorousm ixing for 5m in fo llow ed by cen trifugation at 12,000 rpm for 5m in to separa te the organ ic phase.The organ ic phase w as co llected in to a clean tube and evaporated to d ryness under n itrogen gas f low at 35°C.The residue w as d isso lved w ith 100μl o f aceton itrile by vo rtex-m ix ing fo r 5m in and cen trifuged fo r 5m in at 12,000 rpm.The superna tan t w as ana lyzed using the HPLC ana lysis w h ich w as carried ou t w ith Hitach i HPLC system(UV Detector L-2400,Pum p L-2130,Hitach i,Tokyo,Japan).A reversed-phase co lum n(Diam onsil,C18,4.6m m×250m m,5μm)w as used.The m obile phase for detecting DTX w asm ade up o f aceton itrile and w ater(50:50,v/v)e lu ted at a f low rate o f 1.0m l/m in,and fo r RES m ethano l an d w a ter(50:50)w as used at a f low rate o f 1.0m l/m in.The DTX detection w as perform ed ata w avelength o f 228 nm w h ile fo r RES a w ave length o f 306 nm w as used.

2.7.4. PK data analysis

The pharm acok inetic param eters in clud ing m ax im um concen tration(Cmax),area under the d rug con cen tration-tim e cu rve(AUC),and tota l clearan ce(CL w as ca lcu lated by using DAS2.0 so ftw a re(M athem atica l Pharm aco logy Pro fessiona l Com m ittee o f Ch ina,Shanghai,Ch ina).

2.8. Evaluation of the cytotoxicity ofDTX/RES m PEG-PDLA m icelles

The cy to tox icity o f DTX/RES m PEG-PDLA m icelles to MCF-7 cellsw as eva luated by MTT assay[21].Free d rugs(i.e.DTX and RES),DTXm PEG-PDLAm ice lles,and RESm PEG-PDLA m icelles w ere used as con tro l.The MCF-7 cellsw ere seeded in to sterile 96-w e lls p late(6000 cells/w e ll)and cu ltu red in a ce llcu ltu re incubato r at 37°C under 5%CO2fo r 16 h.Then them ed ium w as rep laced w ith 200μl o f fresh cu ltu re m ed ium con tain ing the sam e am oun to f free DTX,free RES,DTXm PEG-PDLAm icelles,RES m PEG-PDLA m ice lles o r DTX/RES m PEG-PDLA m icelles.In add ition,d rug-freem PEG-PDLA m icelles w as a lso added as con tro l.Fo r the group o f free DTX and RES,DMSO have to be used in o rder to d isso lve the d rugs,bu t the f ina l DMSO concen tration in cu ltu re m ed ium w as strictly con tro lled and limited to 0.02%to m in im ize its effects on cell viability.A fter fu rther in cubation for 48 h,them ed ium w as rem oved and 100μl o f MTT so lu tion(0.5m g/m l)w as added in to each w ell.A fter 4 h,them ed ium w as rem oved and 100μl o f DMSO w as added to d isso lve p recip itated fo rm azan crysta ls an d the p late w as shaken for 1m in.The viability o f ce lls w as m easu red w ith a m icrop late reader,and p resen ted as a percen tage o f the viability ra tio.

2.9. Statistical ana lysis

Data w ere genera ted in trip licates and exp ressed as m ean±S.D.Statistical analysis w as perform ed using Studen t’s t-test w ith the SPSS 17.0 so ftw are.Sign if ican ce w asdeterm ined by a P-va lue o f 0.05(dem oted by∗),P o f 0.01(denoted by∗∗)and P o f 0.001(deno ted by∗∗∗).

Tab le 1-CI va lues o f d ifferen t DTX an d RES ratios.Data rep resen t m ean±SD o f th ree in depen den t experim en ts from f ive sam p les fo r each g rou p.

3. Resu lts an d d iscu ssion

The syn thesis and cha racterization o f them PEG-PDLA can be found in the supp lem en tary m ateria l.

In the fo llow ing section the co-de livery o f RES/DTX loaded m PEG-PDLA m icelles w as reported.

3.1. Evaluation ofsynergistic effect ofDTX and RES

The poten tial synergistic effects o f DTX and RES w ere investigated by m easu ring IC50va lues o f DTX so lu tion,RES so lu tion,and their com bination at d ifferen t ratio w ith MTT assay in MCF-7 ce lls.The IC50o f DTX,RES and their com bination at d ifferen tm ass ratio listed in Tab le 1 suggested that co-adm in istra tion o f DTX and RES cou ld enhan ce their cell p ro liferation inh ibition e ff icien cy.Fu rtherm o re,in o rder to f ind the op tim alm ass ratio betw een DTX and RES,CIvalues o f DTX and RES at the ra tio o f 2:1,1:1,1:2 w ere ca lcu lated.The resu lts show ed that CI va lues varied w ith the w eigh t ratio o f DTX to RES and the low est one w as obtained at the ratio o f 1:1.It ind icated the com bination at am ass ratio o f 1:1 generated stronger synergistic effect than o thers.As them ass ratio in creased from 1:1 to 1:2,the synergistic e ffect f lipped and becam e an an tagon ism effect.Therefore,the m ass ratio o f 1:1 w as chosen as the op tim a l ratio and w as used in the subsequen t p reparation o f d rug-loaded m PEG-PDLA m ice lles.

In add ition,the isobo logram ana lysis w as used to eva luate the synergistic effect o f the d rug com pounds(Fig.2).As a m athem atica l app roach,isobo logram has been used to eva luate the d rug in teractive e ffects[22].A particu lar e ffect level,su ch as 50%o f them axim um w as selected,and doses o f d rug A and B(each d rug a lone)that gave sam e effect w ere p lo tted.The straigh t line connecting A and B a llow s a com parison w ith the actua l dose pair that p roduces th is e ffect level experim en tally.The best com binations can be d istinguished.As show n in Fig.2,the poin ts below the straigh t line suggested that the com bination o f the tw o d rugs(DTX:RES=2:1 and 1:1)cou ld generate a synergistic effect in MCF-7 ce lls.It w as reported that RES cou ld exh ibit chem osensitization effect[23].RES has been observed to be a chem osensitizer in docetaxel chem o therapy b locks up regu lation and activation o f hum an ep iderm a l grow th facto r recep to r-2(HER-2)fu rtherm ore b lock ing dow n stream signa ling pathw ays fo r instan ce Ak t[24].The possib le exp lanation fo r the synergistic e ffects betw een DTX and RES cou ld be that RES dow n regu lated the exp ression o f m u ltid rug resistan t gene en cod ing the m embrane transporter P-gp[25]w h ich inh ibits the m am m alian ta rget o f rapam ycin th rough py ruvate k inase isoenzym e type M 2,thus p reven ting can cer ce llm etabo lism[26].The reported treatm en t o f com bin ing RES up regu lated the p ro-apop to tic genes(BAX,BID,and BAK),cleaved PARP and dow n regu lated the an ti-apop to tic genes(MCL-1,BCL-2,BCL-XL)p rom o ting apop tosis[27].

Fig.2-The isobo logram o f DTX an d RES at d iffe ren t ratios.The straigh t line(add itive ly line)connects the IC50 va lues o f DTX an d RESw hen the d rugs w e re used a lone,and the poin ts p lo tted on the isobo logram s w ere based on the resu lt o f Tab le 1.

3.2. Prepara tion and characterization ofDTX/RES loaded m PEG-PDLA m icelles

The effect o f the p rocess an d form u lation param eters inc luding the f ilm-fo rm ing tem peratu re,the hyd ration tem peratu re,and the d rug to po lym er ra tio on the d rug load ing/en trapm en t eff icien cy,size,zeta po ten tia l o f DTX/RES-loaded m PEG-PDLA m icelles w ere investigated and reported in one o f ou r p revious artic le[28].In the p resen t study,DTX to RESw as f ixed at 1:1 and loaded in to m PEG-PDLA m ice lles w ith the op tim ized p rocess and form u lation param eters obtained in the p revious article,and the obtained m icelles w ere cha racterized in term s o f size,m o rpho logy,en capsu lation e ff icien cy and d rug load ing e ff icien cy.As show n in Fig.3A,DTX/RES-loadedm PEGPDLA m icelles had a m ean particle size o f 17.1±3.2 nm,and PDIo f 0.27±0.01.The load ings o f RES and DTX in them ice lles are 16.89%an d 16.87%,respectively.The TEM im ages dep icted in Fig.3B revea led that DTX/RES-loaded m PEG-PDLA m ice lles w ere spherica land un ifo rm,and the partic le sizew as app rox im ately in the range o f 20-50 nm,w h ich are sligh tly larger than the value determ ined by Marlven Nano ZS Zetasizer Instrum en t.

3.3. In vitro drug release ofm icelles

The in vitro release resu lts are show n in Fig.4.Both RES and DTX exh ibited a con tinuous and fast release in the f irst 12 h.A fter that,the re lease rate w as slow ed dow n an d f ina lly the cum u lative re lease o f bo th DTX and RES reached nea rly 80%at 72 h.Fu rtherm ore,it w as noticed that although the absolu te re lease percen tage o f RES w as low er than tha t o f DTX,the re lease rate o f these tw o d rugs w as n ice ly kep t to be synch ronous,w h ich w as very im portan t because it cou ld ensu re the ratio o f DTX to RES(1:1)consisten t in tissue to ach ieve the synergistic effect.In con trast to the sustained re lease p rof iles o f DTX/RES loaded m PEG-PDLA,ca.80%o f DTX an d RES disso lved w ith in 5 h in the releasem ed ium.Th is ex ten ded release o f RES and DTX from the m icelles cou ld be attribu ted to the hyd rophobic in teraction betw een the d rugs and hyd rophobic core.The re lease p ro f iles w ere f itted w ith a few equa tions in c lud ing Higuch i,Peppas and Sah lin m odel.Itw as found that the re lease o f RES an d DTX from the m ice lles w as a com bined effect o f d iffusion an d d isin tegration.Itm igh t be exp lained to the d iffusion o f the d rugs from the core and the d isin tegration o f the ou ter po lym eric shell[29].

3.4. Cytotoxicity ofDTX/RES-loaded m PEG-PDLA m icelles on MCF-7 cells

The cy to tox icity o f DTX/RES loaded m PEG-PDLA against MCF-7 ce lls w as assessed w ith MTT study.The resu lts in Fig.5 exh ibited that the b lank po lym eric m icelles d id no t have any cytotoxic effect on the MCF-7 cells,w h ich suggested that the syn thesized m PEG-PDLA po lym er is sa fe and biocom patib le w ith MCF-7 cell line.The DTX/RES loaded m PEG-PDLA show ed the h ighest cy totox ic activity again st MCF-7 ce lls am ong the a ll treatm en t groups and led to the death o f 70%cells,w h ich w as h igher than the effect o f m ice lles con tain ing either the sam e con cen tration o f DTX or RES.The in creased sensitivity o f DTX/RES loaded m PEG-PDLA m icelles in MCF-7 cells can be attribu ted to the p resen ce o f PEG shell on the su rface w h ich enhan ced the up take m ed iated th rough en docy tosis.The copo lym er m icelles have been dem onstrated to be poten tia l nanocarriers fo r e ffective in trace llu lar de livery d rug to reverse tum o r MDR[30-31].In add ition,co-de livery o f the tw o d rugs acted on d ifferen t pathw ays to evade cell resistan ce to DTX[32].

Fig.3-Pa rticle size d istribu tion(A)and TEM im age(B)o f RES/DTX m PEG-PDLA m ice lles.

Fig.4-Release p ro f iles o f DTX/RESm PEG-PDLA m icelles(n=4).

Fig.5-Cell viabilities o f DTX/RES loaded m PEG-PDLA in MCF-7 ce lls.Sign if ican ce w as determ ined by P＜0.05(dem o ted by∗),P＜0.01(deno ted by∗∗),P＜0.001(deno ted by∗∗∗)as com pared to the con tro l g roup.

3.5. In vivo pharm acokinetic study ofDTX/RES-loaded m PEG-PDLA m icelles

The PK param eters ob tained from the in vivo study(Tab le 2)show ed thatm PEG-PDLAm ice lles large ly ex tended the expos-ing tim e o f RES and DTX in the b lood circu lation(Fig.6).The ha lf-lives o f RES and DTX in m PEG-PDLA m ice lles w ere abou t 9 tim es and 4 tim es longer than the so lu tions.In add ition,the AUC(0→t)value o f RES and DTX w hen adm in istered asm PEGPDLA m ice lles w as abou t 1.6 tim es and 3 tim es h igher than the so lu tion s.Hyd ro-PEG cou ld p ro long the ha lf-life o f d rug in rats an d enhan ce the targeting and residen ce tim e in tum o r site[33].The p ro longed b lood circu lation of the m PEG-PDLA m ice lles can be attribu ted to its hyd roph ilic su rface com posed by PEG chain,w h ich endow ed the m PEG-PDLA m icelles the ability of escap ing from the reticu loendothelial system,exh ibited a slow er clearan ce.Sin ce tum o r tissue possess leaky b lood vessels and poo r lym phatic d rainage,m ice llesm ay p re feren tia lly accum u late in the so lid tum o rs due to the enhan ced perm eability and p rolonged circu lation tim e[34]an d are expected to passive targeting to can cer cells to exert the an tican cer effect.

Tab le 2-Pharm acokinetic param eters o f RES so lu tion,DTX so lu tion,an d RSE/DTXm PEG-PDLA m ice lles a fter in travenous in jection in ra ts at a dose o f 10m g/kg(n=3).

Fig.6-Plasm a con cen tration-tim e cu rves o f RES so lu tion,DTX so lu tion an d RSE/DTX m PEG-PDLA m ice lles a fter i.v.adm in istration to rats(n=3).Plasm a con cen tra tion-tim e cu rve o f RES(A);Plasm a con cen tration-tim e cu rves o f DTX(B).The inserts a re zoom-in o f the p lasm a con cen tra tion at f irst 2 h.

4. Con clusion s

Th is study dem onstrated that the com bination o f resveratro l and docetaxel cou ld generate synergistic effect against MCF-7 tum o r ce lls.Co-delivery o f resveratro l and docetaxel via m ethoxy l po ly(ethy lene glyco l)-po ly(D,L-lactide)m icelles cou ld no t on ly ex tend the exposing tim e o f the d rug compounds in b lood circu lation in rats bu t a lso enhan ced their AUC.In addition,the d rug com bination exh ibited im p roved an tican cer effect as com pared to w hen each d rug com pound used a lone in vitro.Ou r f in d ings suggested that the co-delivery o f resveratro l and docetaxel using m ethoxy l po ly(ethy lene glyco l)-po ly(D,L-lactide)m icelles cou ld be p rom ising to treat d rug-resistan t tum o rs.

Con f licts o f in terest

The au tho rs declare that there is no con f licts o f in terest.The au thors alone are respon sible for the con ten t and w riting of th is artic le.

Acknow ledgm en ts

We are gratefu l to the Liaon ing Province Pan Deng Xue Zhe Gran t(M.Yang),Liaon ing Prov in cia l Education o ff icer’s Exce llen t Ta len ts Suppo rting Plan(D.Cun)and Nationa lNatu ra lScien ce Foundation o f Ch ina(No.81302720 and 81573380)fo r f inan cia l suppo rt.

Su pp lem en ta ry m ateria ls

Supp lem en tary m aterial associated w ith th is article can be found,in the on line version,at doi:10.1016/j.a jps.2018.03.002.