Deve lopm en t o f PLGA m icro-an d nano rod s w ith h igh capacity o f su rface ligan d con jugation fo r enhan ced targeted delivery

Jifu Co,Jin-Seok Choi,Murtd A.Oshi,Juho Lee,Nurhsni Hsn,Jihyun Kim ,c,Jin-W ook Yoo,∗

a College ofPharm acy,Pusan Nationa lUniversity,Busan 46241,Sou th Korea

b Departm ent ofMedicalManagem ent,Chodang University,Muan-gun 58530,South Korea

c College ofNanoscience&Nanotechnology,Pusan Nationa lUniversity,Busan 46241,Sou th Korea

Keyw ords:Particle shape PLGA nanoparticles Film-stretch ingm ethod Su rfacem od if ication Targeted d rug delivery

A B S T R A C TParticle shape has been recogn ized as one o f the key p roperties o f nanoparticles in biom edica l app lication s in clud ing targeted d rug de livery.Targeting ability o f shape-engineered particles depen ds largely on targeting ligands con jugated on the particle su rface.However,poo r capacity fo r su rface ligan d con jugation rem ain s a p rob lem in an iso trop ic nanoparticlesm ade w ith biodegradab le po lym ers su ch as PLGA.In th is study,w e p repared an isotrop ic PLGA nanoparticles w ith abundan t con jugatable su rface functionalgroups by a f ilm stretch ing-based fab rication m ethod w ith po ly(ethy lene-a lt-m a leic acid)(PEMA).Scann ing e lectron m icroscopy im ages show ed thatm icro rods and nano rods w ere successfu lly fab ricated by the PEMA-based f ilm stretch ing m ethod.The p resence o f su rface carboxy lic acid groups w as con f irm ed by con foca lm ic roscopy and zeta po ten tia lm easu rem en ts.Using the im p roved f ilm-stretch ing m ethod,the am oun t o f p ro tein con jugated to the su rface o f nanorods w as in creased th ree-fo ld.Tran sferrin-con jugated,nano rods fabricated by the im p roved m ethod exh ibited h igher bin d ing an d in terna lization than unm od if ied counterparts.Therefo re,the PEMA-based f ilm-stretch ing system p resen ted in th is stu dy w ou ld be a p rom ising fabrication m ethod for non-spherica l biodegradab le po lym eric m icro-and nanoparticles w ith h igh capacity o f su rfacem od if ication s for enhan ced targeted delivery.

1. In trodu ction

Over the past few decades,particu late system s such as m icroparticles(MPs)an d nanoparticles(NPs)have been ex tensively stud ied fo r biom ed ica l app lications in clud ing targeted d rug delivery,im aging,and d iagnostics[1-4].In design ing the MPs and NPs,a m a jo rity o f stud ies has focused on the particle size,su rface chem istry,and m ateria l com position to contro lparticle p roperties[5-9].In recen t years,the particle shape has em erged as a critica l pa ram eter thatm odu lates an in terp lay betw een a particle and a bio logica lm ilieu[10,11].In th is regard,atten tion has been given to the use o f non-spherica l shapes in the design o f d rug delivery carriers as a strategy to overcom e certain lim itations associated w ith spherica lsystem s.In particu lar,shapes insp ired by the d iverse,evo lu tionarily conserved shapes of pathogen s and cells are being used to eva luate the e ffects o f ca rrier shapes on ce llu lar up take,in vivo transport,and o rgan d istribu tion[12].

Non-spherica l particles su ch as rods and d isk have been show n to enable superior bio logical perform an ce compared to their spherica l coun terparts.Severa l theo retica l and in vitro m odels have p red icted in creased m argination o f nonspherica l particles com pared to spherica l partic les to b lood vessel w a lls due to hyd rodynam ic in teractions that p ropel non-spherica l particles tow ards the w a ll du ring b lood f low[13,14].In add ition,non-spherica l pa rticles show ed reduced imm une clearan ce as com pared to spherical ones[15].Other un ique p roperties o f non-spherica l particles in clude their bind ing ability to target ce lls and rap id in terna lization,compared w ith spherica l ones[16].Non-spherica l particles a lso exh ibited longer b lood-circu lation tim es and im p roved tum or accum u lation in so lid tum o rs in com pa rison w ith their spherica l coun terparts[17-19].

Given the aforem en tioned un ique p roperties o f non-spherica l shapes,severa l d ifferen t shape-engineering techn iques have been developed;these m ethods in c lude non-w etting tem p lates,m icro f lu id ic system s,tem p late assem blies,and a f ilm stretch ing[20-22].Am ong the fabrication m ethods,the f ilm stretch ing m ethod has been considered a sim p le,versatile,inexpensive,and h igh-th roughpu t m ethod[23,24].In the f ilm stretch ingm ethod,d ifferen t non-spherica l shapes can be fabricated by im m obilization o f po lym eric particles in a po lyviny l a lcoho l(PVA)f ilm,heating the f ilm above the g lass transition tem peratu re o f po lym eric particles,fo llow ed by stretch ing to deform the particles.The f ilm stretch ing m ethod has been w ide ly used fo r the investigation o f shape e ffects on d rug de livery app lications such as particle-cell in teractions,d rug re lease,vaccine de livery,cellu lar tra ff ick ing[24-26].

Recen tly,shape e ffect on targeting eff icacy o f NPs in the p resen ce o f su rface ligands has gained m u ch atten tion.Several studies found that in terp lay betw een shape and su rface targeting ligand in NP is o f im po rtan ce in particle-cell interaction in c lud ing NP bind ing to cells and ce llu lar up take[27].Barua et a l.dem onstrated that trastuzum ab-coated rodshaped po lystyrene NPs exh ibited h igher specif ic and low er nonspecif ic up take in b reast can cer ce lls than d id spherica l coun terparts[28].In ano ther study,Poo rn im a et a l.dem onstrated that the shape o f ligand-d isp laying nanoparticles enhan ces the specif icity o f endo thelia l targeting[29].In the stud ies,po lysty rene-based nanoparticles w ere used to fab ricate non-spherica l NPs by a PVA f ilm stretch ing m ethod and the targeting ligands w ere coated on the su rfaces by simp le physica l adso rp tion.How ever,the po lysty rene is neither biodegradab le no r biocom patib le,thus lim iting clin ica l app lications.One p rom ising alternative to po lystyrene is po ly(lactic-co-g lyco lic acid)(PLGA),an FDA-app roved biodegradab le po lym er,that has long been used fo r d rug delivery due to its biocom patibility,sa fety for hum an use,and ability to release encapsu lated d rugs in a con trolled m anner[30].However,physica ladso rp tion o f targeting ligands to the negative ly charged PLGA partic les is ine ff icien t due to the d iff icu lties associated w ith con tro lling the m olecu lar orien tation o f physica lly boun d ligands[31].M o reover,use o f PVA f ilm fo r the stretch ing p rocess resu lts in the residua l PVA on the su rface o f PLGA NPs,w h ich rep resen ts a barrier against the cova len t ligand con jugation because it sh ields the functional group o f PLGA(i.e.carboxy lic group)[32,33].

Fig.1-Fab rication o f su rface-con jugatab le,m icro rods an d nano rod s by PEMA f ilm stretch ing.

In th is study,su rface-con jugatab le non-spherica lPLGA NPs w ith a su ff icien t num ber o f carboxy lic acids on the su rface w ere developed.To in crease the num ber o f carboxy lic acid groups im m obilized to the su rface o f non-spherica l PLGA particles,the PVA f ilm w as rep laced w ith po ly(ethy lenealt-m aleic acid;PEMA)f ilm bearing carboxy lic acid side chains(Fig.1).Rod-shaped PLGA MPs(m icro rods)an d NPs(nanorods)w ere fabricated by the PEMA f ilm-based stretch ing m ethod.Tw om ode l ligands,f luorescein iso th iocyanate(FITC)-con jugated album in an d transferrin(Tf),w ere con jugated on the partic le su rfaces.The p resen ce o f su rface carboxy lic acid groups w as determ ined by con foca lm icroscopy and zeta poten tial m easu rem en ts.Cellu lar up take of Tf-con jugated nano rods by KB hum an carcinom a cells w as a lso eva luated.

2. M ateria ls an d m ethods

2.1. Materia ls

PLGA(50:50 DLG 5E,in trinsic viscosity 0.47 d L/g;PLGAester(w ith ester term inated en d)and 50:50 DLG 4A,intrinsic viscosity 0.38 d L/g;PLGA-acid (w ith acid term inated end))w as pu rchased from Lakeshore Biom ateria ls(Birm ingham,AL,USA).PVA,PEMA,N-hyd roxysuccin im ide(NHS),N-(3-d im ethy lam inop ropy l)-N′-ethy lcarbod iim ide hyd roch lo ride(EDC),and FITC-a lbum in w ere pu rchased from Sigm a-A ld rich(St.Lou is,MO,USA).Tf w as pu rchased from Athens Research&Techno logy(Athens,GA,USA).A ll other chem ica ls used w ere o f ana ly tica l grade.

2.2. Particle preparation

PLGA nanospheres an d m icrospheres w ere fab rica ted by the oil-in-w ater em u lsion so lven t-evapo ration m ethod[34].Fo r nanospheres p reparation,50m g o f PLGA w as d isso lved in 5m l o f m ethy lene ch loride.For the up take study,0.1m g of coum arin-6(C6)w as added to th is so lu tion.The o rgan ic phase w as m ixed w ith 1%(w/v)PVA or 1%(w/v)PEMA so lu tion(20m l)and then w as em u lsif ied using a p robe son icator(KFS-300N u ltrason ic p rocesso r,Ko rea Process Techno logy,Ko rea)at a pow er o f 100W fo r 1m in in an ice bath.The resu ltan t em u lsion w as m echan ica lly stirred fo r 3 h at 500 rpm in a fum e hood to rem ove residual organ ic so lven ts.Nanospheres w ere co llected by cen trifugation at 20 000×g fo r 1 h an d then w ashed th ree tim es w ith doub le-d istilled w ater.Fo r m icrospheres p reparation,100m g o f PLGA w as d isso lved in 3m l o f m ethy lene ch loride.Th is organ ic phase w as m ixed w ith 1%(w/v)PVA o r 1%(w/v)PEMA so lu tion(20m l)and then em u lsif ied fo r 5m in,using a hom ogen izer w ith a stirring rate o f 2,000 rpm in an ice bath.The resu ltan tem u lsion w asm echanica lly stirred fo r 3 h at500 rpm in a fum e hood to rem ove residua l organ ic so lven ts.M icrospheres w ere co llected by cen trifugation a t 200×g for 30m in and w ere w ashed 3 tim es using double-d istilled w ater.

2.3. Surface-conjugatablem icrorods and nanorods preparation

M icrorods w ere p repared by the p reviously reported f ilmstretch ing m ethod w ith som e m od if ication(Fig.1)[23].To fab ricate the stretch ing f ilm,10%(w/v)PVA and 10%(w/v)PEMA w ere in itia lly b lended at d ifferen t ratios,a fter w h ich PLGA-acid/PVA(po lym er:PLGA-acid,su rfactan t:PVA),PLGAacid/PEMA(po lym er:PLGA-acid,su rfactan t:PEMA),PLGAester/PVA(po lym er:PLGA-ester,su rfactan t:PVA),an d PLGAester/PEMA(po lym er:PLGA-ester,su rfactan t:PEMA)w ere added to them ix tu res.The f ilm w as d ried on square d ishes fo r 15 h in an in cubato r at 37°C.The d ried f ilm w as cu t in to 2.5×5 cm2sections and m oun ted on a custom ax ia l-m ade stretcher.The f ilm w as stretched in m inera l oil at 58-64°C at a rate o f 0.5-1.0m m/s.A fter stretch ing,each f ilm w as sto red in co ld m inera l oil for 5m in to low er the tem pera tu re and w ashed w ith ethy l ether to rem ove residua l oil.Each f ilm w as then disso lved in doub le-d istilled w ater and the stretched particles w ere co llected by cen trifuging fo r 1 h at 200×g,fo llow ed by w ash ing 5 tim es w ith doub le-d istilled w ater.Nano rods w ere a lso p repared acco rd ing to the f ilm-stretch ing m ethod(80%PEMA f ilm)w ith the excep tion that the nanopartic les w ere added to the m ix tu res to m ake the f ilm.

2.4. Imm obiliza tion of Tfand FITC-album in to the surfaces ofm icrorods and nanorods

Tf and FITC-a lbum in w ere con jugated on to the su rfaces of m icrorods or nanorods using carbod iim ide chem istry.Partic les (20m g) w ere d ispersed in 5m l o f 2-(Nm orpho lino)ethanesu lfon ic acid (MES)bu ffer(0.1M,pH 5.5)and then reacted w ith 200μlo f EDC(10m g/m l)and 200μl o f NHS(10m g/m l)fo r 2 h.Excess un reacted EDC and NHS w ere rem oved by 3 rounds o f w ash ing w ith MES bu ffer and cen trifugation.The partic les w ith activated carboxy l groups w ere d ispersed in 6m l o f phosphate-bu ffered sa line(PBS,pH 7.4),and then reacted w ith 300μl o f FITC-a lbum in(1.0m g/m l in PBS)o r Tf(10.0m g/m l in PBS)fo r 4 h in the dark at room tem peratu re.Excess un con jugated Tf and FITC-a lbum in w ere rem oved by th ree rounds o f cen trifuga tion at 20,000×g fo r 30m in in PBSbu ffer.Tf and FITC-a lbum in adsorp tion to particle su rfaces w ere perfo rm ed as described fo r the con jugation experim en ts,excep t that MES bu ffer w as used instead of the EDC and NHS so lu tions.

2.5. Determ ination of Tfand FITC-album in on the surfaces ofm icrorods and nanorods

The con jugation o f FITC-a lbum in to the su rfaces o f m icro rods w as visua lized by con foca l m icroscopy(Fluoview FV10i,Olym pus,Tokyo,Japan).The f luorescence in tensities o f FITC-a lbum in-con jugated nano rods w ere m easu red on a m u ltim ode m icrop late reader(TriStar LB 941,Bertho ld Techno logies,Bad W ildbad,Germ any),using excita tion and em ission w avelengths o f 460 nm and 540 nm,respectively.Un con juga ted free Tf p ro tein s in the supernatan ts w ere quan tif ied using a BCA Assay Kit(Fisher Scien tif ic,W a ltham,MA,USA).The quan tities o f Tf on the nanorods su rfacesw ere ca lcu lated by sub tracting the free p ro tein in the supernatan ts from the o rigina l am oun t added.

2.6. Scann ing-electron m icroscopy(SEM)

M icrorods an d nanorodsw ere characterized by f ield-em ission SEM(FE-SEM,S4800,Hitach i,Japan).The particles w ere d ispersed in double-d istilled w ater,d ropped on to a carbon tape,and then d ried in a fum e hood and desiccato r.The carbon tapesw ith PLGA partic lesw ere coated w ith p latinum fo r 2m in under a vacuum.The sam p les w ere view ed by FE-SEM,under acce leration vo ltages o f 5 kV and 35 kV.Partic le sizes w ere m easu red using Im ageJ so ftw are(N IH,Bethesda,MD,USA;n=100).

2.7. Zeta poten tial

The zeta po ten tia l d istribu tion w as m easu red using a Zetasizer Nano ZS(Malvern Instrum en ts,Malvern,UK).Sam p les w ere d ilu ted in doub le-d istilled w ater and p laced in a zeta ce ll.The average o f th reem easu rem en ts o f each sam p le w as used to derive the average zeta po ten tia l.

2.8. Confocalm icroscopy

Hum an ep iderm oid cancer KB cells w ere grow n in RPM I 1640supp lem en ted w ith 10%(v/v)feta l bovine serum and antibio tics(100 IU/m l o f pen icillin G sod ium and 100μg/m l o f strep tom ycin su lfate).Ce lls w ere m ain tained in an in cubato r supp lied un der a hum id if ied 5%CO2/95%air atm osphere at 37°C.KB cells w ere seeded on cover glasses in a 6-w e ll p late at an in itia l density o f 5×104cells/w ell.The ce lls w ere in cuba ted w ith Tf-con jugated and C6-loaded nano rods fab ricated using PVA an d PEMA.After 2 h,the cells w ere w ashed severa l tim es w ith co ld PBS an d f ixed w ith 4%pa ra fo rm a ldehyde fo r 10m in.The ce lls w ere w ashed tw ice w ith co ld PBS and then in cubated w ith 0.1%Triton X-100 in 0.2N NaOH for 5m in.The nu cleiw ere stained w ith 4′,6-d iam id ino-2-pheny lindo le(DAPI;2μg/m l)fo r 5m in,the ce lls f ixed w ith Perm oun tM oun ting M ed ium(Fisher Scien tif ic),and then observed by con foca l m icroscopy(Fluoview FV10i).

Fig.2-SEM im ages o fm icro rods fab ricated u sing d ifferen t p ropo rtion s o f PEMA and PVA w ith fou r d ifferen tly fo rm u lated m icrospheres.The sca le ba rs a re 10μm.

2.9. Statistical ana lysis

Statistica l ana lysis w as perfo rm ed using the paired t-test in Sigm aPlo t 12.0(SYSTAT,In c.,Ch icago,IL,USA).Resu lts are exp ressed as m ean s±SD.Statistical sign if ican ce w as accep ted fo r P va lues＜0.05.

3. Resu lts an d d iscu ssion

3.1. Preparation of PLGA m icrorods and nanorods

The pu rpose o f th is study w as to develop a m od if iab le su rface o f non-spherical biodegradable NPs p repared by a f ilm stretch ing m ethod fo r use as a d rug delivery system.In troduction o f a fun ctiona l group,su ch as a carboxy late group,to non-spherical particles m ade by the conven tional PVA f ilm stretch ing m ethod,has been lim ited due to the residua l PVA on the su rface.In th is study,how ever,w e u tilized the p roperty o f residua l f ilm com position to fabricate a su rfacecon jugatable non-spherical NPs.PEMA has been used as a su rfactan t du ring particles fabrication to in crease the number o f carboxy l groups on the su rface o f PLGA particles fo r su rface m od if ication[35].If PEMA w as used as a stretching f ilm to fab ricate non-spherica l NPs,non-spherica l NPs w ith abundan t con jugatab le su rface fun ctiona l groups can be fabricated.Fab rication o f su rface-m od if iab le,m icro rods and nanorods using PEMA f ilm w as illustrated in Fig.1.In order to see w hether w e can in crease the am oun t o f carboxy l groups on the su rface o fm icro rods by in creasing the am oun t o f carboxy l groups on the su rface o f m icrospheres,fou r d ifferen tm icrospheres(PLGA-acid/PVA,PLGA-acid/PEMA,PLGAester/PVA and PLGA-ester/PEMA)w ere fab ricated using the oil-in-w ater em u lsion so lven t-evapo ration m ethod.It isw o rth to note that there are tw o types o f PLGA end groups(acidend and ester-end).Un like PLGA-ester,PLGA-acid has en d carboxy lic groups,w h ich cou ld poten tia lly in crease the availability o f carboxy lic groups in the su rface o f non-spherica l particles after the f ilm stretch ing[36].Likew ise,w e a lso com pared tw o su rfactan ts,PVA and PEMA.As com pared to PVA,PEMA has carboxy lic groups,thus it m ay a lso in crease the availability o f carboxy lic groups at the f inal non-spherical particles.Fou r d ifferen tm icrospheresw ere converted tom icro rods by the f ilm stretch ing m ethod and their aspect ratios w ere eva luated.

Fig.3-Con foca l im ages o fm icro rod s w ith su rface-con jugated FITC-a lbum in.Sca le ba rs in b righ t f ie ld(BF)im ages co rrespon d to 5μm.

Fig.2 show s the SEM im ages o fm icrorods fab ricated w ith d ifferen t ratios o f PEMA and PVA w ith fou r d ifferen tly fo rm u lated m icrospheres.First,the m icro rods w ere fab ricated by the 100%PVA f ilm(ca lled herea fter PVA f ilm)as a con tro l fo r com parative stud ies.Fou r PLGA m icro rods w ith the sam e aspect ratio(rod length d ivided by d iam eter)o f 5 w ere observed by using PVA f ilm.The PLGA and su rfactan ts types have no effect on the aspect ratio o f m icro rods.Second,w e attem p ted to fabricatem icro rods using 100%PEMA f ilm;however,the use o f 100%PEMA f ilm resu lted in an in com p lete stretch ing as determ ined by SEM in Fig.2,ind icating the d iff icu lty in fab ricating m icro rods using 100%PEMA f ilm.It cou ld be because the force ho ld ing the particles in the PEMA f ilm w as low er than that operating in the PVA f ilm.At last,PEMA w as b lended w ith PVA at d ifferen t ratios.W e found that 50%PEMA f ilm and 80%PEMA f ilm resu lted in m icro rodsw ith similar aspect ratiosw ith PVA f ilm,ind icating that su ccessfu l fabrication o fm icrorods.For fu rther stud ies,w e selected the 80%PEMA f ilm(ca lled herea fter PEMA f ilm)because h igh carboxylate group densities are requ ired on the partic le su rface fo r m od if ication o f the nano rods by ligand con jugation.

3.2. Determ ination of carboxylgroups on the surface ofm icrorods

Fo r detection o f the su rface carboxy l groups,w e con jugated FITC-a lbum in to the su rface o f m icro rods using a carbod iim ide chem istry-based m ethod.Using EDC and NHSas chemica l crosslinkers,the su rface carboxy l groups o f the PLGA m icro rods w ere activated so that they cou ld bind cova len tly to the term ina l am ine group o f FITC-a lbum in.The am oun t o f FITC-album in coating on the su rface o f m icrorods w as assessed by m on ito ring the f luorescen ce changes in con foca l m icroscopy.Fig.3 show s con foca l im ages o f m icro rods w ith FITC-a lbum in con jugated to their su rfaces.W e fabricated 8 kinds o fm icrorods using PVA f ilm and PEMA f ilm.The FITC channel revea led the green f luorescen ce o f FITC-a lbum in.The m icro rods fabricated by using PEMA f ilm exh ibited h igh f luorescen ce levels,w hereas none o f them icrorods fabricated using the PVA f ilm exh ibited f luorescence.PLGA-acid and PEMA su rfactan t d id not in crease the f luorescen ce leve ls,ind icating using PLGA-acid as a po lym er and PEMA as a su rfactan t do not in crease the availability o f su rface carboxy l groups.These resu lts dem on strated tha t,w hen using PEMA f ilm,w e cou ld fab ricate m icro rods w ith su rface carboxy l groups regard less o f end groups o f PLGA an d su rfactan t types.

3.3. Zeta potentials

W e ana lyzed the zeta poten tia ls o f m icro rods using a Zetasizer Nano ZS system to fu rther con f irm the p resen ce o f su rface carboxy l groups on them icro rods.It is w o rth no ting that the term ina l carboxy l groups on the su rface decrease the zeta poten tials.As show n in Fig.4,w hen PEMA w as used as a su rfactan t to p repa re m icrospheres,the zeta poten tia l o fm icrospheres w as nearly-40m V.W hereas the zeta poten tia l o f m icrospheres p repared w ith PVA as a su rfactan t w as app roxim ately-20m V.The zeta po ten tia l o fm icrospheres fabricated using PLGA-esterw as h igher than those fab ricated using PLGA-acid.In genera l,su rfactan t-free PLGA NPs exh ibit a negative zeta poten tial(-49m V)due to the p resence o f term ina l carboxy lic groups in PLGA[37].A residua l su rfactan t on the particle su rface can a ffect the zeta po ten tia l.M icrospheres p repared using PVA as a su rfactan t show a low negative charge due to hyd roxy lgroups(-OH)o f PVA,w hereas ones p repared using PEMA show h igh negative charge due to carboxy l groups(-COOH)o f PEMA[38].The PLGA type had little effect on the zeta poten tial o f m icrospheres although the va lue obtained using PLGA-acid w as som ew hat h igher than that ob tained using PLGA-ester.A fter f ilm-stretch ing using PVA f ilm,the zeta po ten tia lo f a llo f them icro rods becam e app roxim ately-20m V;how ever,the zeta poten tialo fm icrorods a fter f ilm-stretch ing w ith PEMA f ilm w as nearly-40m V.W e found that the facto r that determ ines the zeta po ten tia lo fm icrorods w as the f ilm com position du ring particle stretch ing,not the type o f PLGA nor su rfactan ts used du ring the m icrosphere p repara tion.

Fig.4-The su rface charge o f PLGA MPs.Resu lts a re ex p ressed as the m eans±SD(n=3).

Fig.5-SEM im ages o f nano rods.The d im en sions o f nano rod s fab ricated using PVA f ilm w ere 373.2±97.5 nm(leng th)by 98.7±18.5 nm(w id th),w h ile those o f nanorods fab ricated using PEMA f ilm w ere 385.8±88.2 nm(length)by 105.9±25.7 nm(w id th).The sca le bars a re 1μm.

Tab le 1-Physicochem ica l characteristics o f nanorods.

3.4. Characterization ofnanorods

PLGA NPs have been used as d rug delivery system s in various biom ed ica l app lications invo lving can cer,in f lam m ation,and o ther d iseases,because o f their versatility in term s o f biocom patibility,d rug-load ing ability,and con tro lled-d rug release[39].A w ide range of pharm aceu ticals such as hyd roph ilic or hyd rophobic sm a llm o lecu les o rm acrom o lecu les has been delivered via PLGA NPs[40].By the PEMA-based f ilm stretch ing m ethod,su rface-m od if iab le nano rods w ere a lso successfu lly fabricated.Fig.5 show s SEM im ages o f nanorods fabricated using bo th PVA f ilm and PEMA f ilm.SEM im ages show ed that the nanorods exh ibited a regu lar rod shape and un ifo rm size w ithou t any noticeab le p inho les o r cracks and w ith a narrow size d istribu tion.The p roperties o f nano rods are sum m arized in Tab le 1.The m ean size o f the nano rods w as 373.2 nm×98.7 nm fo r PVA f ilm an d 385.8 nm×105.9 nm for PEMA f ilm,resu lting in an average aspect ratio o f 4.Their m ean zeta poten tia ls w ere-19.5 and-38.5m V,respective ly,w h ich are consisten tw ith resu lts from m icro rods.

3.5. Determ ina tion of Tfand FITC-album in on the surfaces ofnanorods

To determ ine the feasibility o f nano rods for su rfacem od if ication,the capacity fo r p rotein im m obilization on the su rface o f nanorods fabricated using either PVA f ilm o r PEMA f ilm w as tested using T f and FITC-a lbum in.Tw o strategies for a ttach ing p rotein s to nanorods w ere used:con jugation and adso rp tion.Fig.6A show s the am oun t o f su rface-con jugated Tf and FITC-a lbum in on nanorods.The am oun t o f p ro tein conjugated to the su rface o f nanorods generated using PEMA f ilm w as∼70μg/m g NPs.How ever,w hen PVA f ilm w as used,the am oun t o f Tf an d p rotein on the su rface o f nano rods w as on ly∼20μg/m g NPs regard less o f bin d ing m ethods.Fig.6B show s the f luorescen ce in tensities o f nano rods fab ricated using d ifferen t f ilm types.The f luorescen ce cam e from FITCa lbum in,w h ich w as con jugated on the su rface o f nano rods.Nano rods fab ricated using PEMA f ilm show over 3-fo ld h igher f luorescen ce in ten sities than ones fabricated using PVA f ilm,w h ich is consisten t w ith resu lt o f the am oun t o f p rotein on the nano rods,ind icating that the Tf an d FITC-a lbum in on the su rface o f nano rods fabricated using PVA w ere no t con jugated bu tm erely adso rbed,p roving that residua l PVA from PVA f ilm is a po ten tia lbarrier against ligand con jugation to the particle su rface.Thus,PVA f ilm is not app rop riate fo r su rface ligand con jugation o f non-spherical particles.Overall,PEMA f ilm is the op tim a l f ilm for fab ricating non-spherica l particles w ith a h igh capacity o f su rface ligand con juga tion.

Fig.6-Am oun t o f p ro tein on the particle su rface(A)and f luo rescen ce in tensities(B)o f nano rod s fab rica ted using d iffe ren t f ilm types.The resu lts a re ex p ressed as the m ean±SD(n=3).∗P＜0.05 vs.nano rods fab ricated u sing PVA f ilm.

3.6. Cellu lar uptake

The delivery o f d rugs in to specif ic cell types can sign if ican tly reduce d rug toxicity and in crease their therapeu tic effects[41,42].Various targeting ligan ds have been u tilized to increase the in tracellu lar up take o f NPs[43].Am ong them,T f is a w idely used targeting ligand for tum or-targeted delivery.The ex ten t o f the cellu lar up take o f nanorods con jugated w ith the Tf w as investigated in KB ce lls w h ich overexp resses Tf recep tors.Fig.7 show s the cellu lar-up take of Tf-con jugated coum a rin-6-loaded nano rods(fab ricated using PVA and PEMA)in KB cells.The b lue f luo rescen ce by the nuclear dye DAPI,w h ich d id no t co-loca lize w ith green f luorescen ce rep resen ting the nanorods in ternalized to the cytop lasm.Nano rods fab ricated using PEMA f ilm exh ibited even h igher in trace llu la r accum u lation as com pared to those fabricated using PVA f ilm.The cellu lar up take o f NPs can be view ed as a tw o-step p rocess:a bind ing step on the ce ll m em b rane an d an in terna lization step[44]In teractions betw een ligand-m od if ied NPs and cellu lar recep to rs depend on the m o lecu lar o rien tation and ligand density on the NPs su rface.The nano rods fab ricated using PEMA f ilm have m o re ligands con jugated to the su rface o f nanorods.As show n in the Fig.7,Tf-nano rods(PEMA f ilm)have strong bind ing a ff in ity to the Tf recep to r on the ce llm em b rane com pare to Tf-nanorods(PVA f ilm)because o f m u ltip le recep tor-ligand com p lexes at the ce ll su rface.Recep tor-m ed iated endocy tosis has been show n to facilitate and p rom o te particle penetration in to cells w hen Tf w as con jugated on the su rface o f NPs[45].These resu lts dem onstrated the advan tage o f the su rfacem od if iab le nano rods in targeted d rug delivery.

Fig.7-Con foca lm icroscopy im age o f ce llu la r-up take stud ies o f T f-con jugated,coum a rin-6-loaded,nano rods(fabricated using PVA and PEMA)in KB cells a fter a 2-h in cubation.The sca le bars a re 10μm.

4. Con clusion

In th is study,su rface-con jugatab le,m icro-an d nanorodsw ere su ccessfu lly fab ricated by the PEMA-based f ilm-stretch ing m ethod.The f ilm type w as iden tif ied as the key facto r facilitating the fab rication o f su rface-con jugatab le non-spherica l partic les.FITC-a lbum in and Tf w ere su ccessfu lly con jugated to the su rfaces o f nanorods,dem onstrating that the system has the ability to enhan ce su rface ligand con jugation.Fu rtherm ore,Tf-con jugated nano rods fab ricated using PEMA f ilm exh ibited h igher bin d ing an d cellu lar up take in KB ce ll than those fabricated using PVA f ilm.Therefore,the PEMA-based f ilm-stretch ing system p resen ted in th is study w ou ld be a p rom ising fab rication m ethod fo r non-spherica l biodegradab le po lym eric m icro-and nanopartic les w ith h igh capacity o f su rface m od if ications fo r enhan ced targeted de livery.

Decla ration o f in terest

The au tho rs repo rt no con f licts o f in terest.The au tho rs a lone are responsib le for the con ten t and w riting o f th is artic le.

Acknow ledgm en ts

Th is research w as supported by a gran t from the Korean Hea lth care Techno logy R&D Pro ject,M in istry fo r Hea lth and W elfare A ffairs,Repub lic o f Ko rea(HI15C2558).