Yang Pan,XiahuiWang,Zongning Yin

Key Laboratory ofDrug Targeting and Drug Delivery System s,W est China School ofPharm acy,Sichuan University,Chengdu 610041,China

Keywords:Targeted th rom bo lysis Lum brokinase Po lym eric m ice lles ATRP ROP

A B S T R A C T

1. Introduction

Amphiphilic po lym erm ice lles,as exce llen t nanocarriers,have received trem en dous atten tion in research on d rug delivery system(DDS),especia l fo r chem otherapy d rug[1],gene[2]and p rotein[3].For in stan ce,the am ph iph ilic po lym er constructing the structu re of co re-she ll has been app lied in so lubilization fo r hyd rophobic d rug[4].The cation ic po lym ers,su ch as po ly(L-lysine)(PLL)[5],po lyethyen im ine(PEI)[6]po lyam idoam ine(PAAM)[7],po ly(2-(d im ethy lam ino)ethy lm ethacrylate)(PDMAEMA)[8],have been ex tensive ly stud ied in gene delivery.Com pared to nucleic acids and sm a ll-m o lecu le d rugs,the d irect app lication of p ro tein-based therap ies in c lin ic is lim ited due to the in stability and short shelf-life of p rotein sam p les[9].The lack of su itab le carriers fo r effective de livery to the lesion is another m a jo r hu rd le for p rotein-based therap ies.In recen tly stud ies,p rotein w as m ain ly de livered by carriers th rough the electron ic in teraction[10]or the form ation of cova len t bond w ith po lym er[11].Pro tein-po lym er con jugates(PPC)are m o re stab le than p ro tein/po lym er comp lexes fo rm ed in electron ic in teraction in vivo,w h ich p rone to be used in the con tro lled release system[12]or im p rove the stability of p ro tein[12,13].In add ition,p ro tein can be m od if ied to be stim u lus-responsive d rug th rough con jugating environm en t sen sitive cross-lin ker or polym er[14,15].W h ile p ro tein/po lym er com p lex can be easily fo rm ed in a m ild cond ition th rough e lectrostatic in teraction,it can be easier and m ore conven ien t ob tained than PPC[16].

Schem e 1-The illu stration of p reparation on LK loaded RGDfk-con jugated m ixed m icelles.

Th rom bosis,a com m on card iovascu lar d isease in clin ic,is th reaten ing peop le's(especia lly the aged peop le)hea lth[17].Fibrino ly tic enzym e is necessary for th rom bo lysis.How ever,the f ib rinogen level w ou ld be reduced a fter the adm in istration of th rom bo lytic agen t,lead ing to an in crease of b leeding risk[18-20]Hem o rrhage is a com m on side e ffect of f ibrino ly tic enzym e.In o rder to redu ce the hem o rrhagic risk,m any stud ies have been perfo rm ed to develop targeted delivery of th rom boly tic d rug to th rom bi[21-23].Notably,p latelets p lay an im portan t ro le in b lood c lo tting.Prior to clo t fo rm ation,the p latelets becom e activated upon the form ation of the glycop ro tein com p lex GPIIb/IIIa,w h ich is the bind ing target of A rg-Gly-Asp(RGD)-con tain ing pep tides,su ch as RGD fk[24,25].Previous repo rts show ed that RGD-con jugated nanoparticles can e ff icien tly bin d to GPIIb/IIIa[26-28].Therefore,w e choose RGD-con jugated nanoparticles as the de livery veh ic le fo r f ibrino ly tic agen ts in th isw ork so that the f ib rino ly tic agen ts can be effectively delivered to th rom bus sites.

Cation ic po lym ers have been w idely used as the d rug de livery carrier for gene and p ro tein d rug via electrostatic in teraction.Lum brokinase(LK),a f ibrino lytic enzym e originated from earthw o rm,had been used as them odeld rug in th isw o rk[29].The isoe lectric poin t of LK is 3-5[29],a llow ing fo r strong e lectrostatic in teractionsw ith cation ic po lym ers under physio logical cond itions.In ou r p revious study[30],polycap ro lactoneb lock-po ly(2-(d im ethy lam ino)ethy lm ethacry late)-b lock po ly(2-hyd roxyethy l m ethacry late)(PCL-PDMAEMA-PHEMA)w as used as the carrier of LK.How ever,the syn thesis of PCLPDMAEMA-PHEMA is cha llenging,especia lly for the po lym erization of PHEMA b lock using PCL-PDMAEMA asm acroin itiato r.The d iff icu lt syn thesis techno logy is no t easily sca led up and app lied in industry.Add itionally,m ethoxy po lyethy lene g lyco l-b lock-po lycap ro lactone(m PEG-PCL)w as used to enhan ce the co lloida l stability and biocom patibility of carriers.

In this work,RGD fk-con jugated m ixed m ice lles w ere constructed as the carrier of LK for targeted th rom bo lysis.The p reparation p rocedu re of LK-loaded targeted m icelles(LKTM)w as show n in Schem e 1.W e em p loyed PCL-PDMAEMA as the co re of ou r d rug delivery carrier.LK can be absorbed to the cation ic po lym er via electrostatic in teraction s.In order to cova len tly lin k the RGD fk pep tide to the carrier,po lycap ro lactone-b lock-po lyethy lene g lyco lw ith a carboxy lic group(PCL-PEG-COOH)w as used as a fun ctiona lized po lym er.The p reparation p rocess of the po lym er carrier con tain ing PCL-PDMAEMA,m PEG-PCL and RGD fk-con jugated PCL-PEGCOOH(PCL-PEG-RGD fk)w as m u ch sim p ler than ou r po lym er carrier deve loped p rev iously[30].The p repared LKTM w as characterized by various spectroscop ic techn iques and chemica l assays to exam ine its chem ica l and physica l p roperties.Hem o lysis and cy totox icity assays w ere perfo rm ed to determ ine the sa fe con cen tration range of th is po lym er carrier.In vivo th rom bo lysis assays w ere perfo rm ed to assess the f ibrino ly tic capacity of LKTM.Fina lly,the tail b leed ing assay w as taken to eva luate the b leed ing risk of LKTM.(Schem e 2).

2. M ateria ls and methods

2.1. M aterials

Dodecano l,stannous octoate(Sn(Oct)2),cap ro lactone,N,N,N”,N”,N’”-Pen tam ethy ld iethy lenetriam ine(PMDETA),2-Brom oisobu ty ry l brom ide (2-BiBB), 2-(d im ethy lam ino)ethy l m ethacry la te(DMAEMA)and cup rous brom ide w ere pu rchased from A ladd in corpo ration (Shanghai,Ch ina).Cup rous brom ide w as w ashed by glacial acetic acid,ethy l a lcoho l,and acetone th ree tim es fo r pu rif ication.M ethoxy po lyethy lene glyco l(m PEG,Mn:5000Da)and alpha-hyd roxyom ega-carboxy po ly (ethy lene glyco l) (HO-PEG-COOH,M n:5000Da)w ere pu rchased from Toyongbio corporation (Shanghai,Ch ina).The po lym eriza tion inh ibito r in DMAEMA w as rem oved by passing an alum ina co lum n.3-(4,5-Dim ethy lth iazo l-2-y l)-2,5-d ipheny ltetrazo lium brom ide(MTT)w as pu rchased from Biosharp corpo ration(Hefei,Ch ina).Py rene w as pu rchased from J&K chem ica l co rporation(Beijing,Ch ina).RGDfk(Mw:603.69)w as pu rchased from Synpep tide Co.,Ltd(Shanghai,Ch ina).Lum b rok inase(28 000 IU/m g)w as ob tained from Xi'an Rea lin Biotechno logy Co.,Ltd(Xi'an,Ch ina).The o ther agen ts in clud ing N,N-Dim ethy lfo rm am ide (DMF),d im ethy lsu lfox ide (DMSO),tetrahyd rofu ran(THF),to luene,triethy lam ine(TEA),g lacia l acetic acid,ethy l a lcoho l,and acetone w ere ob tained from Cdkelongchem corporation(Chengdu,Ch ina).Kunm ing m ice and sp rague daw ley(SD)rats w ere pu rchased from Chengdu Dashuo Bio logica l Institu te(Chengdu,Ch ina).A ll an im a l experim en ts w ere com p lied w ith the gu idelines of the Laboratory Protocol of An im al Care and Use Comm ittee,Sichuan Un iversity.

2.2. Syn thesis and characterization of PCL-PDMAEMA

The syn thesis and characterization of PCL-PDMAEMA re feren ced ou r p revious study[30].

2.3. Syn thesis and characterization ofm PEG-PCL

m PEG-PCL w as syn thesized via ROP using m PEG as the in itiator.Brief ly,0.5 g ofm PEG(0.1mm o l)w as added in to a 10m l sch len k f lask and d isso lved by 1.0m l to luene.A fter add ition of 0.456 g of cap ro lactone(4mm o l)and 76μl of to luene con taining 4μm o lof Sn(Oct)2,the f lask w as vacuum ed and recharged w ith n itrogen fo r th ree tim es.A fter heated at 100°C fo r 24 h,the reaction m ix tu re w as exposed to air to quen ch the reaction.The toluene in m ix tu re w as rem oved under vacuum in ro ta ry evapo ra to r.The crude p rodu ct w as d isso lved in THF and then p recip itated in excess co ld m ethano l.The p recip itation w as iso lated and d isso lved in THFagain.A fter th ree tim es of d isso lu tion/p recip itation,the p roductw as d ried under vacuum at room tem peratu re.The pu rif ied p rodu ct w as characterized by hyd rogen nu clear m agnetic resonan ce(1H NMR,400MHz,Va rian In c.,USA),fu rier transform in frared spectroscopy(FTIR,Perk in Elm er,USA)and gel perm eation ch rom atograph(GPC,Tosoh co rporation,Japan).

2.4. Syn thesis and characterization of PCL-PEG-RGDfk

PCL-PEG-RGD fk w as syn thesized via tw o steps.First,PCLPEG-COOH w as syn thesized via ROP using HO-PEG-COOH as in itiato r.The m o lar ratio of m onom er and in itiato r w as 40:1.The p ro toco ls of syn thesis and characteriza tion w ere as sam e as m PEG-PCL.Second,PCL-PEG-RGD fk w as syn thesized via EDC/NHS chem istry accord ing to the literatu re[31].0.15 g PCL-PEG-COOH(15.69m m o l),14m g RGD fk(23.54m m o l),4.5m g EDC·HCl(23.54m m o l),3m g NHS(23.54m m o l)and 1.5m l DMSO w ere added in to 10m l f lask.A fter degassed by th ree evacuation/n itrogen recharge cyc les,the m ix tu re w as reacted at room tem peratu re fo r 24 h.The p rodu ct w as d ia lyzed(M n:2500Da)against deion ized w ater fo r 2 d to rem ove the EDC,NHSand un reacted RGD fk.A fter lyoph ilization,the obtained p roduct w as characterized by FTIR and1H NMR.

The content of RGD fk w as determ ined bym easu ring the argin ine content acco rd ing to the p revious repo rt[32].

2.5. M easurem en t ofCMC

Considering the com p lex po lym ers w ere used as carriers,the CMC ofm ixed m ice lle of PCL-PDMAEMA and m PEG-PCL w as m easu red using f luorescen ce spectroscopy.As the content of RGDfk in carriersw as low,the RDGfk-con jugated PCL-PEGw as no tadded in th is assay.The ratio of PCL-PDMAEMA andm PEGPCLw as 1:1.The con cen tration ofm ixed m icelles ranged from 0.01-1000m g/l.The con cen tration of py rene stock so lu tion w as 6μM in acetone.The pyrene stock so lu tion(0.3m l)w as added in to a 4m l cen trifuge tube.A fter evaporation of acetone,3m l ofm ixed m ice lles w ith d ifferen t con cen tration w as added,fo llow ed by in cuba tion at room tem peratu re fo r 24 h.The f inal con cen tration of pyrene in m icelles w as 0.6μM.The excitation spectrum of py rene w as scanned from 300 to 350 nm by using RF5301 PC spectrof luo rom eter(Sh im adzu,Japan)at an em ission w avelength of 393 nm.The excitation w avelength at 333 nm sh ifted to 336 nm w ith the increasing of con cen tration of m ixed m ice lles.The CMC va lue w as the con cen tration ofm ixed m icellesw hen the ra tio of I336/I333w as in creased w ith an ex trem e ra te,w h ich determ ined accord ing to the ratio of I336/I333against to the logarithm of concen tration ofm ixed m icelles.

2.6. Preparation ofcharacteriza tion of LKTM

Be fo re the p reparation of LKTM,the b lank m ixed m ice lles w ere p repared.Brief ly,10m g PCL-PDMAEMA,6m gm PEG-PCL and 4m g PCL-PEG-RGD fk w ere d isso lved by 1m l THF.A fter add ition of 5m l of deion ized w a ter,THF w as rem oved under vacuum by ro tary evapo rato r.The vo lum e of ob tained b lank m ixed m icelles w as supp lem en ted to 5m l by deion ized w ater.Nex t,0.75m l of 2.0m g/m l b lank m ixed m ice lles,0.188m l of 4.0m g/m l LK so lu tion and 0.562m l deion ized w ater w ere m ixed and stirred toform the LK loaded m ixed m icelles.Considering the instability of LK as p rotein,the f ina l p reparation(LKTM)w as p repared as lyoph ilized pow der.30m g ofm an ito l and 15m g of su crose w ere added as lyop rotectan t in p rescription.The hyd rodynam ic d iam eter,po lyd ispersity index and zeta po ten tia l of LKTM w ere m easu red by Zetasizer Nano ZS(M a lvern instrum en t).The m o rpho logy of LKTM w as characterized by transm ission e lectron m icroscope(TEM).

2.7. Study on drug loading and release

The free LK in LKTM w as isolated via u ltraf iltration(300KDa).The content of free LK w as determ ined by Brand ford m ethod.The d rug load ing content(DLC)and en capsu lation e ff icien cy(EE)w ere ca lcu lated acco rd ing to the content of free LK w ith the fo llow ing fo rm u las.

In vitro re lease of LK w as stud ied in 0.01M pH 7.4 PBS at 37°C.Each sam p le con tain ing 0.5m l of LKTM and 4.5m l releasing m ed ium w as set at every sam p ling tim e poin t.0.4m l of sam p le w as p ipetted to u ltracen trifuge tube(Mw:300KDa)at every p redeterm ined tim e poin t.A fter cen trifugation at 10 000 rpm fo r 20m in,the re leased content of LK in f iltrate w as m easu red by Brand ford m ethod.

2.8. Colloida l stability assay in v itro

The enough co lloida l stability is the base fo r the targeted delivery of therapeu tic d rug to site of lesion.The co lloida l stability of LKTM in PBS con tain ing 10%feta l bovine serum(FBS)w as evaluated via the variation of tu rbid ity,partic le size and PDI in th is study.The in crease of tu rbid ity ref lects w hether there is an aggregate phenom enon of nanoparticles in FBS[33,34].In tu rbid ity assay,the absorp tion cu rve of LKTM d ilu ted 5 tim es by FBSw as scanned by u ltravio let-visib le spectrophotom eter(Varian Cary 100 Con c,Agilen t,U.S.A.)from 400-700 nm to ob tain the m ax im um abso rp tion peak(λmax).Then,the abso rban ce of LKTM in FBS bathed in w ater at 37°C w ith shak ing at 100 rpm w as m easu red a tλmaxat the p reset tim e poin t.The sam p le treated acco rd ing to the p rotoco lm entioned above w as a lso characterized by dynam ic ligh t scattering(DLS)to assess the variation of size and PDIof LKTM.

2.9. Biocom pa tibility study

2.9.1. Hem olysis assay

The laten t hem o ly tic risk is a com m on ly p rob lem fo r kinds of cation ic po lym ers.In order to en su re a good biocom patibility,the concen tration cation ic po lym er shou ld be restricted.Acco rd ing to the literatu re,the accep tab le hem o lysis level of d rug carriersis10%or low er[35].In th is assay,the con cen tration of LKTM ranged from 0.1-1.0m g/m l.A fter the co llection of def ibrinated blood from sp rague-daw ley(SD)m ouse,the red b lood cell(RBC)w as sepa rated by cen trifuge at 1200 rpm for 15m in.The iso lated RBCsw ere d ilu ted by PBS to ob tain the 2%cell suspension.100μl of 2%RBC suspension w asm ixed w ith d ifferen t vo lum e of LKTM(po lym er:2.0m g/m l).The m ix tu re w as supp lem en ted to 200μl by PBS.A fter in cubation at 37°C for 1 h,the m ix tu re w as cen trifuged at 1500 rpm for 5m in.Then 100μl of supernatan t w as p ipetted to a 96-w e ll p late for the m easu rem en t of abso rban ce at 540 nm by m icrop late reader.The PBS and 1%triton X-100 treated RBCw ere used as negative and positive group,respective ly.

2.9.2. Cell viability assay

PCL-PDMAEMA,as a cation ic po lym er,can be absorbed by cell in vivo as the ce llm em b rane is a negatively charged su rface.The in teraction betw een the cation ic po lym er and the cell m em b rane can change the m em brane porosity and poten tial[36].Cytotoxicity of cation ic po lym er has been w idely stud ied to eva luate its biocom patibility.It has been show n that m PEG-PCL exh ibits an exce llen t com patibility and low im m unogen icity,w h ich has been w idely used as d rug delivery carriers[37,38].The low cytotoxicity ofm PEG-PCL on L929 ce ll had been p roved in ano ther group[39].Considering the in fo rm ation m en tioned above,w e m easu red the cy to toxicity of PCL-PDMAEMA on L929 cell using MTT assay.The p rotoco l of MTT assay re feren ced p revious repo rt[40].Brie f ly,0.1m l L929 cellsw as seeded in to a 96-w ellp late(1.0×104ce lls per w ell).A fter in cubation w ith 5%CO2at 37°C fo r 24 h,the o ld cu ltu ralm ed ium w as renew ed by 140μl fresh Du lbecco's m od if ied Eagle'sm ed ium(DMEM)con tain ing 10%feta l bovine serum.Then 60μl d ifferen t con cen trations of PCL-PDMAEMA in 0.01M pH 7.4 PBS w ere added.A fter in cubation fo r 24 h,20μl of 5m g/m l MTT(in PBS)w as added and fo llow ed w ith a cu ltu ra l fo r 4 h.Fina lly,the fo rm azan crysta l generated by live cell w as d isso lved by 200μl DMSO a fter rem ova l of the m ed ium.The abso rban ce w asm easu red at 490 nm using am icrop late reader.PBSand 1%triton X-100 treated ce llunder the sam e cond ition w ere set as the negative and positive con tro l group,respective ly.The cellviability w as ca lcu lated as the fo llow equation.

2.10. Study on targeted poten tial ofRGDfk-conjugated m ixed m icelles in vivo

The targeted po ten tia l of RGD fk-con jugated m ixed m icelles w as stud ied by f luorescen t trace techn ique.m PEG-PCL w as labeled by f luo rescein iso th iocyana te(FITC)using m ethod described p reviously[41].The p rodu ct w as characterized by u ltravio let-visib le spectrum.LKM(LK-loaded m icelles w ithou t RGDfk)and LKTM w ere set as experim en tal groups.Notab ly,the LKTM sam p le used in th is set of experim en ts con tained FITC-labe led m PEG-PCL(m PEG-PCL-FITC).In the LKM sam p le,there w as no PCL-PEG-RGD fk;add itiona lly,FITClabeled m PEG-PCL instead of m PEG-PCL w as p resen t.FeCl3-induced carotid artery th rom bosis in Kunm ing m ouse(18-22 g)w as con stru cted acco rd ing to the repo rted p ro toco l[42].FITC-con tain ing LKM and LKTM w ere in jected in to the m ice th rough the tail vein at a f inal dosage of 0.012m l/g(168U/g).A fter 2 h of in jection,the caro tid artery w ith th rom bus in anestheticm ouse w as excised fo r H&E stain ing.The targeted ability of d ifferen tm ateria lsw as com pared by the f luorescen t d istribu tion.

2.11. Throm bolysis assay in vivo

There w ere fou r groups in th rom bo lysis in vivo,in clud ing PBS,LK,LKM,LKTM.Five m a le Kunm ing m ice have been used in each group.After 5 h of form ation of th rom bus in carotid artery,d ifferen tm ateria ls w ith the sam e dose in“2.10′′w ere in jected via tail vein.A fter 24 h,the caro tid artery w as excised for H&E stain ing.The effect of th rom bo lysis in d ifferen t groups w as evaluated according to the residual th rom bi in vesse l.

2.12. Determ ination ofbleeding tim e

M ice-tail b leed ing m odelw as used to assess the hem o rrhagic risk of th rom bo ly tic agen t.The tail of the treated laborato ry m ouse w as transected abou t 5mm from the tip.A f ilter paper m PEG-PCL:1H NMR(400MHz,CDCl3)δ(ppm):1.38(m,OCOCH2CH2CH2CH2CH2O of PCL b lock),1.65(m,OCOCH2CH2CH2CH2CH2O of PCL b lock),2.31(t,OCO CH2CH2CH2CH2CH2O of PCL block),3.38(s,CH3O),3.64(m,O CH2CH2,O CH2CH2OCO of PEG and OCOCH2CH2CH2CH2CH2OH of PCL),4.07(t, OCOCH2CH2CH2CH2CH2O of PCL b lock), 4.22 (t,OCH2CH2OCO).w as used to touch the in cision every 15 s.The b leed ing tim e w as determ ined as the b lood no longer b lo tted in tof ilter in 30 s.

2.13. Sta tistics

A ll data in th is study w ere show n as m ean±standard deviation.One-w ay ana lysis of varian ce(ANOVA)w as used to determ ine the statistical sign if icance by SPSS softw are.In th is study,?P＜0.05 and??P＜0.01 w ere used to show statistica lsign if ican ce.

3. Resu lts and d iscussion

3.1. Syn thesis and characterization ofm PEG-PCL and PCL-PEG-RGDfk

PCL-PEG-COOH:1H NMR(400MHz,DMSO-d6)δ(ppm):1.29 (m,OCOCH2CH2CH2CH2CH2O of PCL b lock),1.54(m,OCOCH2CH2CH2CH2CH2O of PCL b lock),2.27 (t,OCO CH2CH2CH2CH2CH2O of PCL b lock),3.51(m,O CH2CH2,O CH2CH2OCO of PEG b lock and OCOCH2CH2CH2CH2CH2OH of PCL),3.98(t,OCOCH2CH2CH2CH2CH2O of PCL b lock),4.11(t,OCH2CH2OCO and HOOC CH2OCH2CH2).

PCL-PEG-RGDfk:1H NMR(400MHz,DMSO-d6)δ(ppm):0.93(m,R14 of RGD fk),1.29-1.38(m,OCOCH2CH2CH2CH2CH2O of PCL b lock and R10-R12 of RGD fk),1.55-1.66 (m,OCOCH2CH2CH2CH2CH2O of PCL b lock and R13 of RGD fk),2.27(t,OCO CH2CH2CH2CH2CH2O of PCL block),2.65-3.07(m,R7-R9 of RGD fk),3.51(m,O CH2CH2,O CH2CH2OCO of PEG b lock and OCOCH2CH2CH2CH2CH2OH of PCL),3.98(t,OCOCH2CH2CH2CH2CH2O of PCL b lock and R4 of RGD fk),4.11-4.34(t,OCH2CH2OCO,HOOC CH2OCH2CH2and R5-R6 of RGD fk),4.58-4.66(m,R2-R3 of RGD fk),7.16-7.22(m,R1 of RGD fk).As the low content of RGD fk in po lym er,the effect of hyd rogen in RGD fk on peak shape of hyd rogen in PCL-PEG w as very poor.

m PEG-PCL and PCL-PEG-COOH w ere syn thesized via ROP usingm PEG and HO-PEG-COOH as in itiato r,respectively.PCLPEG-RGD fk w as syn thesized by EDC/NHS chem istry.The1H NMR and FTIR spectra of syn thesized po lym er are show n in Fig.S1 and S2.The GPC resu lts of po lym ers are show n in Tab le S1 and Fig.S3.As show n in Fig.S1A,the trip let atδ2.31,4.07 and m u ltip let atδ1.38,1.65 p roved the p resence of PCL.The m u ltip let atδ3.38 is attribu ted to the term ina lm ethy l in PEG.M u ltip let a tδ3.64 is attribu ted to them ethy lene in m PEG and m ethy lene ad jacen t to hyd roxy l in PCL.The signa l atδ4.22 is attribu ted to them ethy lene ad joined the PCL b lock.Its low chem ica l sh iftw as resu lted from the strong e lectronegativity of ester bond in PCL,w h ich suggested the successfu lsyn thesis of m PEG-PCL.The degree of po lym eriza tion(DP)of PCL w as calcu lated accord ing to the ratio of peak areas atδ3.64 andδ 2.31.In1H NMR spectrum of PCL-PEG-COOH,the ana lysis of chem ica l sh ift w as sim ilar to m PEG-PCL.The peaks atδ1.29,1.54,2.27 and 3.98 in d icated the p resen ce of PCLb lock.The DP of PCLw as ca lcu lated by the ratio of peak areas atδ3.51 andδ 2.27.

Fig.1-(A)Plo t of in ten sity ratio(I336/I333)versus logarithm of con cen tra tion ofm ixed m ice lles.(B)TEM im age of LKTM.(C)Percen tage of cum u lative re lease of LK from LKTM.

The FT-IR resu lts of the po lym ers a re consisten t w ith the1H NMR resu lts.A fter po lym erization ofε-cap ro lactone in itiated by m PEG,the ex trem ely strong carbony l peak from PCL w as observed in the FT-IR spectrum,ind icating the successfu l syn thesis of m PEG-PCL.A sim ilar resu lt w as observed in the IR spectrum of PCL-PEG-COOH.

To con f irm the su ccessfu l con jugation of RGD fk to PCLPEG-COOH,the1H NMR spectrum of RGD fk w as also characterized.In1H NMR spectrum of PCL-PEG-RGD fk,the peaks at δ7.16-7.22,4.34-4.64,2.65-3.04,0.93 p roved the p resen ce of RGD fk in po lym er.As show n in Fig.S2F,the w avenum bers at 1533 cm-1and 1647 cm-1attribu ted to benzene ring in RGD fk fu rther supp lem en ted the eviden ce fo r the above view.Accord ing to the resu lt of determ ination on the content of argin ine,abou t 55%of PCL-PEG-COOH w as labeled by RGD fk.The ca lib ration cu rve of argin ine is show n in Fig.S4.

3.2. M easurem en t ofCMC

The CMC value of am ph iph ilic copo lym er closely related to its ability against d ilu tion in b lood in vivo.A low CMC va lue(1-5m g/l)can ensu re an enough stability ofm icelles to avoid the d issociation before a rriving the site of lesion[43].The CMC value ofm ixedm icelles con tain ing PCL-PDMAEMA and m PEGPCLw as 1.27m g/laccord ing to the in tersection poin t in Fig.1A.It is eviden t tha t the therm odynam ic stability of the m ixed m icelles is excellen t.

Tab le 1-Resu lts of particle size,PDI,Zeta po ten tia l,DLC and EE of LKTM.

3.3. Characterization of LK loaded m ixed m icelles

As show n in Tab le 1,the particle size,PDIand Zeta po ten tia l of LKTM m easu red by DLSw ere 193.8±0.75 nm,0.176±0.020 and 38.8±2.42m V,respective ly.These param eters in d icated that the p repared LKTM is com posed of un ifo rm and stab le nanoparticles.The m orpho logy of LKTM is near spherica l as illustrated in the TEM im age show n in Fig.1B.

3.3. Study on drug loading and release

The determ ined values of DLC and EE are show n in Table 1.The EE ofm ixed m ice lles w as abou t 80%,w h ich suggested LK can be e ff icien tly abso rbed by m ixed m ice lles.The low DLC va luew as due to the add ition of lyop ro tectan t.The cum u lative release cu rve of LK is show n in Fig.1C.The rest of LKm igh tbe ether adso rbed on the su rface or loaded in to m ice lles.M eanw h ile,it a lso m igh t be ex isted in the tw o type situation m entioned above.The large standard deviation m igh t be resu lted from the com p lex ity of p ro tein and the low accu racy of Brandfo rd m ethod.

Fig.2-Resu lts of co llo ida l stability assay.(A)UV scann ing cu rve of LKTM,(B)The abso rban ce of LKTM in 10%FBS,(C)The va ria tion of size and PDIof LKTM in 10%FBS at d ifferen t tim e.

3.4. Colloidal stability assay in vitro

Theλmaxof LKTM in PBS supp lem en ted w ith 10%FBS w as at 408 nm acco rd ing to the UV scann ing cu rve.As show n in Fig.2B,the absorban ce of LKTM decreased sligh tly in 4 h.The sligh t decrease of abso rban ce ind icated there w as no aggregation phenom enon of LKTM in stud ied m ed ium.The particle size and PDIw ere keep a t abou t 110-120 nm and 0.30 respectively,w h ich exh ibited an excellen t stability under the sam e cond ition.The few adso rp tion of FBSm igh t due to the sh ie lding effect of PEG chain.PEG located on the su rface of nanopa rtic les can form a m ush room o r hair b rush shape structu re to im pede the adsorp tion betw een polym erm icelles and FBS.The resu lts above p roved the co lloida lstability of LKTM in 10%FBSw as excellen t.

3.5. Biocom patibility assay

3.5.1. Hem olysis assay

The resu lts of hem o lysis assay are show n in Fig.3A.The hem o lysis w as low er than 10%w hen the con cen tration of po lym er w as 0.1-0.6m g/m l.A fter the con cen tration of po lym er reached at0.8m g/m l,the tox icity w as unaccep tab le.Compared w ith ou r p revious study[31],the biocom patibility of LKTM w as sign if ican tly better.The im p rovem en tm igh tbe due to the add ition ofm PEG-PCL.PEG and its ana logues have been w ide ly used to im p rove the biocom patibility for cation ic po lym er[36,44].The resu lts in th is w o rk are consisten t w ith p revious reports.

3.5.2. Cell viability assay

The sa fe con cen tration range of PCL-PDAEMA w as determ ined accord ing to the resu lts of cellviability.As show n in Fig.3B,the viability of PCL-PDMAEMA treated L929 cells w as decreased w ith the in crease of con cen tration.W hen the con cen tration of PCL-PDMAEMA w as 0.12m g/m l,the cell viability w as low er than 90%.To en su re a good biocom patibility of carriers,the con cen tra tion of PCL-PDMAEMA w as suggested to no m o re than 0.09m g/m l.

3.6. Study on targeted poten tial ofRGD-conjugated m ixed m icelles in vivo

3.6.1. Characterization ofm PEG-PCL-FITC

The UV-vis spectra of FITC,m PEG-PCL and m PEG-PCL-FITC w ere co llected.As show n in Fig.S5,m PEG-PCL exh ibited no abso rp tion in the region of 400-600 nm.FITC d isp layed absorp tion peaks at449 nm and 476 nm.m PEG-PCL-FITC show ed sim ilar abso rp tion peaks as those of FITC,ind icating that the FITC labe ling on m PEG-PCL w as su ccessfu l.The abso rp tion m ax im um w avelengths of m PEG-PCL-FITC w ere 459 nm and 487 nm.Com pared to the tw o absorp tion peaks of free FITC,there w as abou t 10 nm red-sh ift in bo th peaks.

3.6.2. Analysis off luorescent distribu tion

As show n in Fig.4A and B,bo th LKTM and LKM induced a decrease in the am oun t of th rom bi in the m ice.There w ere m ore residual th rom biin the LKM group than the LKTM group,suggesting that LKTM is a better th rom bo ly tic agen t.The f luo rescen t d istribu tion of LKTM and LKM in the caro tid artery is show n in Fig.4C and D,respectively.As show n in Fig.4C,there is very strong f luorescence in th rom bi,ind icating that the RGD fk-con jugated po lym er can e ffectively target th rombus.In con trast,there is nof lo rescen ce in Fig.4D,suggesting that LKM w as no t ab le to specif ica lly target th rom bus.Therefore,it can be con cluded that the better th rom bo lytic effect of LKTM com pared to LKM is due to its ability of targeted d rug delivery.

Fig.3-(A)Resu lts of hem o lysis assay.(B)Resu lts of ce ll viability in cy to tox icity assay.

Fig.4-Flo rescen t d istribu tion of d ifferen t experim en ta l g roup s(m agn if ication 50×,n=5).(A)LKTM group and(B)LKM group in brigh t f ield.(C)LKTM group and(D)LKM group in dark f ield.

Fig.5-The resu lts of th rom bo lysis in d ifferen t group s(m agn if ica tion 100×,n=5).(A)PBS group,(B)LK g rou p,(C)LKM g roup,(D)LKTM g roup.

Fig.6-Bleed ing tim e ofm ice in d ifferen t g roups(n=4-5).

3.7. Throm bolysis assay in vivo

The d ifferen t e ffects of th rom bo lysis are show n in Fig.5.PBS group w as set as blank con tro l.As show n in Fig.5A,the th rom bi rem ained una ltered a fter the adm in istration of PBS.In LK,LKM,LKTM groups,a certain ex ten t th rom bo lysis p roved the e ff icacy of LK.The rest of clo t in LK group w as sim ilar w ith LKM group,w h ich suggested the po lym er had little effect on th rom bo lysis.The LKTM group exh ibited the least residua l th rom bi,w h ich w as consisten tw ith the resu lts show n in“3.6′′.A ll the resu ltsm en tioned above p roved LK had been su ccessfu lly delivered to th rom bi.The cu rative effect of LK had been enhan ced a fter targeted delivery using RGD fk-con jugated m ice lles.

3.8. Determ ination of tailbleeding tim e

A long tail bleed ing tim e corresponds to a h igh hem orrhagic risk.As show n in Fig.6,the b leed ing tim e of LKTM group w as sign if ican tly sho rter than LK and LKM groups(P=0.036 and 0.020,respectively),w h ich ind icated the b leed ing risk had been redu ced due to the targeted d rug delivery.The app roxim ate b leed ing tim e in LK group and LKM group(P=0.476)ind icated the effect of ca rriers w ithou t RGD fk on b leed ing w as poor.Thus,RGD fk p layed a critica l ro le in reducing the b leeding tim e via the targeted de livery of LK to the lesion sites.

4. Con clu sion

In sum m a ry,a RGD fk con jugated hyb rid m icelles fo r targeted delivery of LK w as successfu lly developed in th is w o rk.The particle size ofp repared LKTM w as hom ogenous.The co lloidal stability and b lood com patibility of LKTM w ere bo th enhan ced than ou r p revious repo rted LKTM p repared by PCL-PDMAEMAPHEMA.Fluorescen t trace techno logy in vivo p roved that LKTM had been successfu lly delivered to th rom bi.Th rom bo lysis assay in vivo suggested the cu rative effectw as im p roved a fter the targeted delivery of LK.The resu lts of b leed ing tim e p roved the hem o rrhagic risk of LK had been redu ced,w h ich reached the pu rpose of th is p ro ject.Therefore,RGDfk con tained cation ic po lym ericm ice llesm ay be as p rom ising cand idate for the delivery of LK fo r targeted th rom bo lysis.

Con f licts of in terest

The au thors report no con f licts of in terest.The au thors alone are responsib le fo r the content and w riting of th is article.

Acknow ledgm en t

Th is w ork w as f inan cia lly suppo rted by Nationa l Natu ra l Scien ce Foundation of Ch ina(No.81673363).

Sup p lem en tary m ateria ls

Supp lem en tary m ateria l associated w ith th is article can be found,in the on line version,at doi:10.1016/j.ajps.2018.03.004.