Structural Characterization and Analyses of the Biological Properties of New Amphiphilic Block Copolymers

! ! ! ! ! Colloid!and!Polymers!Physic!Group.! Department!of!condensed!matter.! Faculty!of!Physics.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!!!!!Adriana!Cambón!Freire!!!!!!!!! ! ! Structural Characterization and Analysis of the Biological Properties of New Amphiphilic Block Copolymers ! ! PhD!!Thesis!!!!!!![2014]! ! ! ! ! ! ! ! !! ! Facultad!de!Física!-!Campus!Vida! !!15782!Santiago!de!Compostela! ! ! ! ! Víctor! Mosquera! Tallón,!Catedrático!del!Departamento!de!Física!de! la!Materia!Condensada!de!la!Universidad!de!Santiago!de!Compostela;!Pablo! Taboada!Antelo,!Profesor!Titular!del!Departamento!de!Física!de!la!Materia! Condensada!de!la!Universidad!de!Santiago!de!Compostela;!y!Silvia!Barbosa! Fernández,! Investigador! Doctor! del! Programa! Ramón! y! Cajal! en! el! Departamento! de! Física! de! la! Materia! Condensada! de! la! Universidad! de! Santiago!de!Compostela! ! Informan:! ! Que!el!trabajo!de!investigación!titulado!“Structural!Characterization! and! Analyses! of! the! Biological! Properties! of! New! Amphiphilic! Block! Copolymers”!ha!sido!realizado!bajo!nuestra!dirección!por!Adriana!Cambón! Freire!en!los!laboratorios!del!Grupo! de!Física!de!Coloides!y!Polímeros!del! Departamento! de! Física! de! la! Materia! Condensada! de! la! Universidad! de! Santiago!de!Compostela,!y!reúne!los!requisitos!de!calidad!y!rigor!científicos! necesarios!para!optar!al!Grado!de!Doctor!en!Ciencias!Físicas.! ! Para!que!así!conste!a!los!efectos!oportunos,! ! Santiago!de!Compostela,!10!de!Abril!de!2014.! ! ! ! ! ! ! ! ! ! ! Dr.!!V.!Mosquera!Tallón! Dr.!P.!Taboada!Antelo! Dr.!S.!Barbosa!Fernández! ! !!! ! Index ! ! Agradecimientos!!!!!!!!vii! Summary!!!!!!!!!!ix! Resumen!!!!!!!!!!xvii! Listado!de!artículos!!!!!!!!!!!!!!!!!!!xxvii! ! ! Chapter(1:(Introduction(((((((1( ( (1.1!Nanotecnhology!!!!!!!3! ! ! 1.1.1!Nanopharmaceutics!and!nanomedicine! ! ! 5! ! ! 1.1.2!References! ! ! ! ! ! ! 8! !1.2.!Nanoparticles!as!drug!carriers!!!!!10! ! ! 1.2.1!Influence!of!NP´s!properties!on!pharmaceutical!! !!!!!!!!!!!!performance! ! ! ! ! ! ! 10! ! ! ! 1.2.1.1!Drug!solubility!! ! ! ! ! 11! ! ! ! 1.2.1.2!Blood!circulation!time!! ! ! ! 12! !!!1.2.1.3!Targeting! ! ! ! ! ! 12! ! ! ! 1.2.1.4!Cellular!uptake! ! ! ! ! 13! ! ! ! 1.2.1.5!Release!rate! ! ! ! ! ! 13! ! ! ! 1.2.1.6!Clearance!mechanisms!and!excretion! ! 14! ! ! 1.2.2!NPs!used!in!pharmaceutics! ! ! ! ! 16! ! ! 1.2.3!References! ! ! ! ! ! ! 20! !1.3.!Polymeric!micelles!!!!!!!23! !!1.3.1!Block!copolymers!!!!!!24! ! ! 1.3.2!Block!copolymers!classification!! ! ! ! 25! !!1.3.3!Block!copolymer!micelles!!!!!27! ! ! 1.3.4!Polymeric!micelles!as!drug!reservoirs! ! ! ! 28! ! ! 1.3.5!References! ! ! ! ! ! ! 31! ! Chapter(2:(EOmSOnEOm(copolymers(as(nanocarriers(of( ((hydrophobic(drugs((((((33( ( (2.1!Aim!of!the!work!!!!!!!35! ! ! 2.1.1!Aim!of!the!work!! ! ! ! ! ! 35! !!2.1.2!Methodology! ! ! ! ! ! ! 35! ! ii! ! ! 2.1.3!References! ! ! ! ! ! ! 37! !2.2.!EOmSOnEOm!copolymers:!Micellization,!drug!solublization! !!!!!!!!!and!gelling!features!!!!!!!39! ! ! 2.2.1!Abstract!! ! ! ! ! ! ! 39! ! ! 2.2.2!Introduction! ! ! ! ! ! ! 39! ! ! 2.2.3!Experimental!section! ! ! ! ! ! 41! !!!2.2.3.1!Materials! ! ! ! ! ! 41 ! ! ! 2.2.3.2!Methods! ! ! ! ! ! 42 ! ! 2.2.4!Results!and!discussion! ! ! ! ! ! 45 ! ! ! 2.2.4.1!Characterization!of!the!copolymer!micelles! ! 45! ! ! ! 2.2.4.2!Phase!behavior!and!rheological!properties! ! 48 ! ! ! 2.2.4.3!Solubilization!capacity!studies! ! ! 53 ! ! ! 2.2.4.4.!In!vitro!release!of!griseofulvin! ! ! 55 !!2.2.5!Conclusions!!!!!!!57 ! ! 2.2.6!References!!!!!!!57! !2.3.!Supporting!information!!!!!!61! ! ! 2.3.1!Theoretical!estimation!of!polymeric!micelle!shape! ! 62! ! ! 2.3.2!Rheological!properties!of!copolymer!EO38SO10EO38!! !!!!!!!!!!Temperature!and!concentration!scans!!!!63! !!2.3.3!References! ! ! ! ! ! ! 64! !2.4.!EOmSOnEOm!copolymers:!From!“classical”!! !!!!!!!!!chemotherapeutic!nanocarriers!to!active!! !!!!!!!!!cellZresponse!inducers!!!!!!65! ! ! 2.4.1!Abstract!! ! ! ! ! ! ! 65! ! ! 2.4.2!Introduction! ! ! ! ! ! ! 65! ! ! 2.4.3!Experimental!section! ! ! ! ! ! 67! ! ! ! 2.4.3.1!Materials! ! ! ! ! ! 67 !!!2.4.3.2!Methods! ! ! ! ! ! 68 ! ! 2.4.4!Results!and!discussion! ! ! ! ! ! 70 ! ! ! 2.4.4.1!Solubilization!capacity! ! ! ! 70! ! ! ! 2.4.4.2!Size!distribution!and!physical!stability!of!! ! ! ! ! DOXOZpolymeric!micelles!!!!71 ! ! ! 2.4.4.3!In!vitro!release! ! ! ! ! 72 ! ! ! 2.4.4.4!Cytocompatibiity!of!EO33SO14EO33!and!! !!!!EO33SO14EO33!!!!!!74! ! ! ! 2.4.4.5!Inhibition!of!PZgp!efflux!pump!and!intracellular! ! ! ! ! DOXO!accumulation! ! ! ! ! 75! ! ! ! 2.4.4.6!Cellular!uptke!and!in!vitro!cytotoxicity!of!! ! iii! ! ! ! ! DOXOZloaded!polymeric!micelles!(PZgp!evasion)! 77 !!2.4.5!Conclusions!!!!!!!81 !!2.4.6!References!!!!!!!81! !!!!!!!!! Chapter(3:(BOnEOmEOn(copolymers(as(nanocarriers(of( ((hydrophobic(drugs((((((95( ( !3.1.!Aim!of!the!work!!!!!!!97! ! ! 3.1.1!Aim!of!the!work!!!!!!!98! !!3.1.2!Methodology! ! ! ! ! ! ! 98! ! ! 3.1.3!References! ! ! ! ! ! ! 100! !3.2.!Micellisation!of!triblock!copolymers!of!ethylene!oxide!and! !!!!!!!!!1,2Zbutylene!oxide:!Effect!of!BOZblock!length!!!101! ! ! 3.2.1!Abstract!! ! ! ! ! ! ! 101! ! ! 3.2.2!Introduction! ! ! ! ! ! ! 101! ! ! 3.2.3!Experimental!section! ! ! ! ! ! 103! ! ! ! 3.2.3.1!Materials! ! ! ! ! ! 103 ! ! ! 3.2.3.2!Methods! ! ! ! ! ! 104 !!3.2.4!Results!and!discussion! ! ! ! ! ! 105 !!!3.2.4.1!Determination!of!the!critical!micelle!concentration! 105! !!!3.2.4.2!Influence!of!temperature!on!cmc! ! ! 105 !!!3.2.4.3!Correlation!of!cmc!with!hydrophobicity! ! 109! !!3.2.5!Conclusions!!!!!!!111 ! ! 3.2.6!References!!!!!!!111! !3.3.!Complex!selfZassembly!of!reverse!! !!!!!!!!!poly(butylene!oxide)Zpoly(ethylene!oxide)Zpoly(butylene! !!!!!!!!!oxide)!triblock!copolymers!with!long!hydrophobic!and! !!!!!!!!!extremely!lengthy!hydrophilic!blocks!!!!114! !!3.3.1!Abstract!! ! ! ! ! ! ! 114! !!3.3.2!Introduction! ! ! ! ! ! ! 115! !!3.3.3!Experimental!section! ! ! ! ! ! 116! ! ! ! 3.3.3.1!Materials! ! ! ! ! ! 116 ! ! ! 3.3.3.2!Methods!!!!!!117 !!3.3.4!Results!and!discussion!!!!!!119 !!!3.3.4.1!Clouding!!!!!!119! !!!3.3.4.2!Population!size!distributions!!! ! ! 120 !!!3.3.4.3!Micellar!properties! ! ! ! ! 122 !!!3.3.4.4!Rheological!behavior! ! ! ! ! 124! ! iv! !!3.3.5!Conclusions!!!!!!!133 !!3.3.6!References!!!!!!!133! !3.4.!Solution!behavior!of!reverse!! ! !!!!!!!!poly(butylene!oxide)Zpoly(ethylene!oxide)Zpoly(butylene! !!!!!!!!!oxide)!triblock!copolymers!with!lengthy!hydrophilic!blocks!136! ! ! 3.4.1!Abstract!! ! ! ! ! ! ! 136! ! ! 3.4.2!Introduction! ! ! ! ! ! ! 137! ! ! 3.4.3!Experimental!section!!!!!!138! ! ! ! 3.4.3.1!Materials! ! ! ! ! ! 138 ! ! ! 3.4.3.2!Methods!!!!!!139 !!3.4.4!Results!and!discussion! ! ! ! ! ! 141 !!!3.4.4.1!Clouding!!!!!!141! !!!3.4.4.2!Population!size!distributions!!! ! ! 142 !!!3.4.4.3!Micellar!properties! ! ! ! ! 145 !!!3.4.4.4!Rheological!behavior! ! ! ! ! 148! !!3.4.5!Conclusions!!!!!!!156 ! ! 3.4.6!References!!!!!!!157! !3.5.!Supporting!information!!!!!!160! !3.6.!DoxorubicinZloaded!micelles!of!reverse!! !!poly(butylene!oxide)Zpoly(ethylene!oxide)Z! !!poly(butylene!oxide)!block!copolymers!as!efficient!! !!“active”!chemotherapeutic!agents!!!!162! !!3.6.1!Abstract!! ! ! ! ! ! ! 162! !!3.6.2!Introduction! ! ! ! ! ! ! 163! !!3.6.3!Experimental!section! ! ! ! ! ! 165! ! ! ! 3.6.3.1!Materials! ! ! ! ! ! 165 ! ! ! 3.6.3.2!Methods! ! ! ! ! ! 165 !!3.6.4!Results!and!discussion! ! ! ! ! ! 169 !!!3.6.4.1!Cytocompatibility!of!BOnEOmBOn!copolymers!170! !!!3.6.4.2!Solubilization!capacity! ! ! ! 170 !!!3.4.4.3!Size!and!stability!of!the!DOXOZloaded!polymeric! !!!!micelles!!!!!!172 !!!3.6.4.4!In!vitro!release! ! ! ! ! 173! ! ! ! 3.6.4.5!Intracellular!DOXO!accumulation!by!inhibition!of!! !!!!PgZP!efflux!pump!!!!!174! ! ! ! 3.6.4.6.Cellular!uptake!of!DOXOZloaded!polymeric!! !!!!micelles!(PZgp!evasion)! ! ! ! 175! !!3.6.5!Conclusions!!!!!!!180 !!3.6.6!References!!!!!!!181! !3.7.!Supporting!information!!!!!!184! ! v! ! Chapter(4:(Linear(copolymers((((((187( ( !4.1.!Aim!of!the!work!!!!!!!189! ! ! 4.1.1!Aim!of!the!work!!!!!!!189! !!4.1.2!Methodology! ! ! ! ! ! ! 189! ! ! 4.1.3!References! ! ! ! ! ! ! 191! ! 4.2.!Cytocompatibility!and!PZglycoprotein!inhibition!of!block! !!!!!!!!!copolymers:!StructtureZactivity!relationship!!!193! ! ! 4.2.1!Abstract!! ! ! ! ! ! ! 193! ! ! 4.2.2!Introduction! ! ! ! ! ! ! 193! ! ! 4.2.3!Experimental!section! ! ! ! ! ! 195! ! ! ! 4.2.3.1!Materials! ! ! ! ! ! 195 ! ! ! 4.2.3.2!Methods! ! ! ! ! ! 196 ! ! 4.2.4!Results!and!discussion! ! ! ! ! ! 198 !!!4.2.4.1!Cytocompatibility!of!the!block!copolymers! ! 198! !!!4.2.4.2!Block!copolymers!as!inhibitors!of!the!PZgp!efflux!! !!!!pump!!!!!!!203! !!4.2.5!Conclusions!!!!!!!209 !!4.2.6!References!!!!!!!209! !4.3.!Supporting!information!!!!!!213! ! Chapter(5:(Experimental(techniques(((((217( ( ! 5.1.!Spectroscopy!!!!!!!!219! !!5.1.1!EMR!and!light! ! ! ! ! ! ! 219! !!5.1.2!Structure!of!matter! ! ! ! ! ! 220! !!5.1.3!Light!interaction!with!matter! ! ! ! ! 221! ! 5.2.!Nuclear!magnetic!resonance!spectroscopy!!!223! ! ! 5.2.1!Nulear!magnetic!properties! ! ! ! ! 223! ! ! 5.2.2!Nuclear!magnetic!resonance!spectroscopy! ! ! 223! !!5.2.3!Proton!RMN!spectrscopy!(1H)!!!!!225! ! 5.3.!UVZVis!spectroscopy!!!!!!!227! !5.4.!Fluorescence!spectroscopy!!!!!!231! !!5.4.1!The!pyrene!method! ! ! ! ! ! 234! !5.5.!References!!!!!!!!235! !5.6.!Gel!permeation!chromatography!!!!!237! ! ! 5.6.1!References! ! ! ! ! ! ! 238! !5.7.!Light!scattering!!!!!!!239! ! xii! mainly!focused!on!the!micellar!systems!and!the!advantages!these!possess!as!drug!delivery! vehicles!.! ! Chapter'2!presents!an!analysis!of!copolymers!EO33SO14EO33!and!EO38SO10EO38!(where! So! denotes! styrene! oxide,! and! the! subscripts! the! block! lengths).! Their! structure! and! aggregation!process!in!aqueous!solution!was!tested!as!a!preliminary!step!to!be!evaluated!as! drug! carriers.! These! copolymers! have! been! chosen! as! an! alternative! to! the! previous! mentioned! Pluronics,! provided! that! they! have! the! same! molecular! structure! but! a! more! hydrophobic!central!block,!which!should!confer!the!micellar!core!with!a!higher!solubilizing! ability! for! hydrophobic! drugs.! The! block! length! has! been! chosen! to! obtain! the! optimal! compromise! between! chain! solubility,! micelle! formation! and! micellar! core! size! based! on! previous!studies.!These!copolymers!were!synthesized!by!sequential!anionic!polymerization! and! their! composition! and! block! architecture! was! characterized! by! nuclear! magnetic! resonance! (NMR)! and! gel! permeation! chromatography! (GPC).! Evaluation! of! ! polymeric! micelles! as! nanocarriers! for! drug! administration! was! performed! by! a! detailed! physicoB chemical!characterization!in!order!to!structural!details,!as!well!as!their!behavior!in!aqueous! solution!in!a!wide!range!of!concentrations!and!temperatures,!followed!by!in1vitro!studies!to! evaluate! their! ability! to! encapsulate! hydrophobic! drugs! providing! a! suitable! stability! and! protection!to!the!cargo!molecules,!their!biocompatibilyt!and!cytotoxic!action.! ! The! selfBassociation! process! and! subsequent! micelle! formation! was! followed! by! fluorescence!spectroscopy,!using!the!pyrene!method.!It!was!verified!that!the! aggregation! process!starts!at!lower!concentrations!than!for!Pluronics!having!similar!block!lengths!due!to! to!the!larger!hydrophibicity!of!the!SO!blocks.!! ! Using!static!and!dynamic!light!scattering!techniques,!(SLS!and!DLS,!respectively)!and! analyzing!a!wide!range!of!concentrations,!information!about!the!properties!of!the!polymeric! micelles! formed! (hydrodynamic! radius,! micellar! aggregation! number,! micellar! molecular! weight,! shell! volume! and! hydration! extent)! was! extracted.! It! was! found! that! in! aqueous! solution! EOmSOnEOm!copolymers! selfBassemble! at! low! concentrations! to! form! micelles! of! sizes!around!ca.!15!nm,!making!them!suitable!for!parenteral!administration.!Such!micelles! are!spherical!and!have!a!hydrophobic!core!and!a!hydrophilic!shell!of!PEO.!To!confirm!the! reliability! of! the! DLS! analysis! regarding! the! micelle! size,! copolymer! samples! were! also! visualized! by! transmission! electron! microscopy! (TEM)! verifying,! at! the! same! time,! the! spherical!symmetry!of!the!micelles.!The!behavior!of!these!block!copolymers!in!a!wide!range! of! concentrations! and! temperatures! was! also! analyzed! by! rheometry,! gaining! a! detailed! knowlede!about!the!phase!behavior!and!associated!flow!properties.! ! Next,!the!solubilization!ability!of!poorly!aqueous!soluble!drugs!within!EO33SO14EO33! and! EO38SO10EO38!copolymer! micelles! was! tested! by! varying! the! drug/copolymer! ratio! at! polymer! concentrations! above! their! critical! micelle! concentration.! To! do! that,! two! poorly! ! xiii! waterBsoluble!drugs!were!tested:!the!antifungal!griseofulvin!and!the!anticancer!doxorubicin.! It!was!observed!that!the!increase!in!solubility!for!both!drugs!was!higher!for!EO33SO14EO33!as! a!result!of!its!larger!and!more!hydrophobic!core!being,!therefore,!more!compatible!with!the! drug.!Stability!tests!show!that!the!polymeric!micelles!formed!by!these!copolymers!maintain! their!initial!size!after!one!freezeBdryingBreconstitution!cycle,!which!makes!them!suitable!for! storage.!The!temporal!evolution!of!both!empty!and!drugBloaded!micelles!under!high!dilution! conditions!show!that!these!nanosystems!are!physically!stable!for!at!least!12!days,!keeping! almost!invariable!their!size!and!cargo!loading!during!first!5!days.! ! Another!important!point!for!the!potential!use!of!EOmSOnEOm!copolymers!as!delivery! systems! is! the! drug! release! rate! once! the! nanocarrier! reachs! its! target! site.! DrugBloaded! micelles!exhibited!a!rapid!cumulative!release!in1vitro!at!short!incubation!times,!to!become! more!sustained!at!later!stages.!It!was!also!verified!that!the!release!rate!depends!on!the!ph! of! the! surrounding! medium! in! which! the! vehicle! is! located.! To! verify! this! point! buffered! solutions!of!pH!4.0,!5.5!and!7.4!were!used,!which!mimic!the!acidic!environment!of!cancer! cells,!lysosomes!and!cytoplasm,!respectively.!Thus,!it!was!observed!that!griseofulvin!release! was! higher! and! faster! as! more! alkaline! the! medium!is.! GriseofulvinBloaded! EO33SO14EO33! copolymer!micelles!released!the!largest!amount!at!pH!4.0,!whereas!those!of!EO38SO10EO38! released!faster!at!pH!7.4,!reaching!80!%!drug!released!before!10!hours!of!incubation.!In!the! case! of! doxorubicin,! the! observed! behavior! was! just! the! opposite:! the! drug! release! was! smaller!at!pH!7.4!than!at!pH!5.5!as!a!result!of!the!reprotonation!of!the!amino!group!of!the! anticancer! drug! at! acidic! pH,! which! increases! its! aqueous! solubility! and! accelerates! the! release;! in! this! case,! amount! released! was! larger! for! EO38SO10EO38! than! for! EO33SO14EO33! copolymer.! ! To! test! the! potential! viability! of! these! copolymers,! biological! tests! in1 vitro!were! performed! in! order! to! ascertain! the! biocompatibility! of! the! bare! and! drugBloaded! nanocarriers! and! their! cytotoxicity! into! different! cell! lines.! The! nonBtoxic! nature! of! the! present!EO38SO10EO38! and! EO33SO14EO33!copolymers! was! revealed! by! testing! their! cytotoxicity! by! means! of! the! lactate! dehydrogenase! (LDH! kit)! assay! and! the! cellular! proliferation! by! measuring! the! formation! of! formazan! crystals! (MTT! assay)! in! a! BALBB3T3! fibroblast!mouse!cell!line.! These!copolymers!showed! viabilities! of!ca.!100%! except!at!the! highest! polymer! concentrations! tested! (1.66! wt.%),! at! which! the! viabilities! were! slightly! lower.! ! To!achieve!efficient!nanocarriers,!in!particular!for!their!use!in!antineoplasic!therapy,! it!is!necessary!to!accumulate!the!required!amount!of!drug!inside!the!cell!in!order!to!exert!its! cytotoxic!activity.!This!concentration!depends!on!efflux!pump!mechanisms!through!the!cell! membrane,!which!are!those!that!cells!use!to!expel!foreign!substances.!In!tumor!cells!these! mechanisms!are!much!more!active,!ususally!giving!rise!to!subBoptimal!drug!concentrations! inside!cells!which!derive!in! inefficient!therapeutic! actions.!In! previous!studies!it!has!been! ! xiv! demonstrated! that! certain! copolymers! modify! the! cellular! response! inhibiting! the! efflux! pump! mechanisms! in! some! cases.! The! NCIBADRBRES! cell! line! corresponds! to! drug! multiB resistant!ovarian!tumor!cells,!that!is!a!suitable!model!to!test!the!effect!of!efflux!pumps!on! the!accumulation!of!anticancer!drugs,!in!this!case,!of!the!PBglycoprotein!pump!(PgBP)!which! is!highly!overexpressed!in!this!cell!line.!For!comparison!and!control,!a!MCF!B7!cell!line!was! used!which! corresponds! to! a! breast! cancer! cell! line! highly! sensitive! to! doxorubicin,! and! which!shows!no!overexpression!of!PgBP.!To!verify!whether!the!present!copolymers!have!a! potential!inhibitory!effect!of!the!PgB!P!efflux!pump,!several!copolymer!concentrations!were! added!to!cell!cultures.!!Strikingly,!the!inhibition!of!the!PgBP!efflux!pump!in!the!NCIBADRBRES! cell!line!was!found!whilst!for!the!MCFB7!cell!line!no!variations!in!doxorubicin!were!found! compared! to! the! administration! of! free! drug.! To! corroborate! these! results,! other! wellB known! PgBP! inhibitors! were! also! tested! as! verapamil! and! Pluronic! P85,! which! do! not! increase!the!levels!of!the!antiBcancer!drug!on!MCF!B7!cells,!but!duplicate!their!levels!in!the! NCIBADRBRES!cell!line.!The!greater!accumulation!of!doxorubicin!in!the!NCIBADRBRES!cell!line! when!administered!within!polymeric!micelles!was!also!confirmed!by!confocal!microscopy,!! also!revealing!a!slower!drug!accumulation!inside!cells!as!a!consequence!of!the!time!delay! associated! with! its! release! from! micelle! cores.! The! fluorescent! pattern! found! for! drugB loaded! micelles! was! typical! of! cytoplasmatic! cargo! relase,! that! is,! the! loaded! micelles! accumulate!in!the!cytoplasm!where!the!acidic!environment!favoured!drug!release,!allowing! free!doxorubicin!to!enter!the!nucleus!and!subsequently!bind!DNA!strands.! ! ! Finally,!to!complete!the!study!as!well!as!to!verify!the!suitability!and!efficacy!of!the! present! copolymers! as! drug! delivery! vehicles,! the! cytotoxicity! of! doxorubicinBloaded! polymeric!micelles!of!the!present!copolymers!was!evaluated!using!the!crystal!violet!method.! MCFB7!and!NCIBADRBRES!tumor!cell!lines!were!used!to!test!the!effect!of:!the!free!drug,!and! empty!and!drugBloaded!micelles!of!the!two!copolymers!and!Pluronic!P85!used!as!a!control,.! It!was!found!that!the!effect!of!the!loaded!nanocarriers!is!exclusively!due!to!the!release!of! the!drug!inside!the!cell,!since!the!cell!inhibition!was!not!originated!from!empty!micelles.!Cell! growth! inhibition! levels! obtained! for! doxorubicinBloaded! micelles! in! the! NCIBADRBRES! cell! line! (that! overexpresses! the! PgBP! efflux! pump)! were! higher! than! for! the! free! drug,! approximately!twice.!Growth!inhibition!on!the!MCFB7!cell!line!(that!not!overexpresses!the!PB Pg! efflux! pump)!showed! similar! results,! which! additionally! confirms! the! sustained! drug! release!over!time!after!14!h!incubation!previously!observed.!! ! In!Chapter'3,!reverse!triblock!copolymers!bearing!long!poly(butylene!oxide)!and!very! lengthy! poly(ethylene! oxide)! blocks!were! evaluated! (PBO! and! PEO,! respectively).! These! copolymers!are!of!great!interest!due!to!the!shortage!of!copolymers!of!this!family!possessing! such! structures,! which! can! largely! influence! their! solution! properties.! Their!high! hydrophobicity!and!their!block!length!allows!the!formation!of!more!polymeric!micelles!at! lower!concentrations,!with!the!subsequent!increase!in!drug!solubilization!ability.!Hence,!five! copolymers! were! synthesized! using! PBO! side! blocks! with! a! central! PEO! block.! They! were! ! xv! designed!having!different!block!lengths,!allowing!to!compare!their!properties!as!a!function! of! block! length! or! the! block! ratio.! As!EOnSOmEOn,! these! copolymers! were! obtained! by! oxyanionic!sequential!polymerization!and!their!molecular!structure!was!determined!using! GPC! and! NMR! techniques.! Their! selfBassembly! properties! in! aqueous! solution! were! also! studied.!Micellization!curves!were!obtained!by!fluorescence!spectroscopy!using!the!pyrene! method.! Critical! micelle! concentration! values! obtained! for! the! present!BOnEOmBOn! copolymers! are! lower! as! the! BO/EO! ratio! increased.! As! in! Chapter! 2,! the! aggregation! properties!in!aqueous!solution!were!obtained!using!SLS,!DLS!and!TEM!techniques.!Also,!the! formation! of! both! unimolecular! and! polymolecular! micelles! was! confirmed! at! concentrations! below! their! critical! concentration,! as! well! as! the! existence! of! micellar! bridging! as! observed! by! atomic! force! microscopy! (AFM)! and! rheometric! analysis.! Their! phase!and!rheological!behavior! was! studied!at!high!concentrations! due! to!their!potential! use! as! associative! thickeners.! First,! the! tube! inversion! method! enabled! to! visualize! the! macroscopic! phase! behavior! under! temperature! increases,! allowin! to! identify! three! different!regions! (sol/soft! gel/hard! gel).! To! complete! the! phase! diagram,! the! cloud! temperature! was! obtained! by! UVBvis! spectroscopy.! The! copolymers´! behavior! under! controlled!stress!or!strain!was!also!analyzed!and!master!curves!constructed!that!enabled!to! analyze! the! flow! behavior! in! a! range! of! frequencies! not! achievable! using! conventional! instrumentation.!It!was!also!observed!that!the!thickening!character!of!these!copolymers!is! present! despite! the! storage! moduli! (G')! is! lower! than! the! loss! moduli! (G'')! in! a! relatively! narrow! concentration! range,! as! opposed! to! other! widely! ised! thickeners! used! as! HEUR.! Furthermore,!their!behavior!does!not!conform!to!that!of!Maxwell!fluids.! ! Evaluation!of!polymeric!micelles!of!this!type!of!copolymers!as!nanocarriers!for!drug! administration!was!performed!by!the!physical!characterization!of!the!drugBloaded!micelles,! followed! by! cargo! release! in1 vitro! studies! in! the! dilute! concentration! regime.! In! aqueous! solution!these!copolymers!form!micelles!of!10B40!nm!in!diameter!with!a!hydrophobic!core! (BO)!and!a!hydrophilic!shell!(EO),!which!makes!them!suitable!for!parenteral!administration.! Their!ability!to!encapsulate!hydrophobic!drugs!was!tested!by!varying!the!drug/copolymer! ratio,! being! doxorubicin! the! drug! used.! The! encapsulation! efficiency! of! these! copolymers! was! higher! than! that! found! for! other! copolymers! such! as! Pluronic,! but! smaller! than! the! EOnSOmEOn!systems!previously!reported!in!Chapter!2.! ! Concerning! the! stability! and! protection! of! their! loading! cargo,! these! copolymers! maintain! the! initial! size! after! one! freeze! B! drying! B! reconstitution! cycle;! their! temporal! evolution! in! solution! showed! that! these! systems! are! physically! stable! up! to! 20! days! of! incubation,!keeping!the!micellar!size!virtually!unchanged!and!the!loaded!cargo!at!90%!of!the! initial!value.!Like!EOnSOmEOn!copolymers,!BOnEOmBOn!ones!exhibited!a!burst!release!phase! in1 vitro! at! short! incubation! times,! becoming!more! sustained! later.! Drug! release! also! depended! on! the! surrounding! medium,! that! is,! of! the! solution! pH:! the! lower! the! pH! the! higher!the!release!was!as!in!the!case!of!EOnSOmEOn!copolymers.! ! xvi! ! Biological! assays! were! performed! following! the! same! scheme! commented! in! the! previous! chapter.! Three! of! the! copolymers! with! different! block! lengths! were! selected! in! order!to!correlate!their!effectiveness!with!the!molecular!structure.!Cytocompatibility!assays! for!bare!copolymers!were!made!using!the!BALBB3T3!cell!line!by!means!of!the!MTT!and!LDH! tests! which! revealed! cell! viabilities! up! to! 100%! at! the! studied! concentrations,! except! for! copolymer! BO8EO90BO8,! which! was! 75%.! In! any! case,! all! of! the! copolymers! verified! their! nonBtoxicicity!and!cytocompatibility!(viability!≥!50%)!.! ! As! the! previous! chapter,! the! ability! of! BOnEOmBOn!copolymers! to! behave! as! biological!response!modifiers!was!tested!and,!in!particular,!as!inhibitors!of!the!PBPg!efflux! pump! in! the! NCIBADRBRES! cell! line.! Their! inhibitory! capacity! was! confirmed,! allowing! a! greater! accumulation! of! the! antitumor! drug! inside! cells,! as! observed! directly! by! confocal! microscopy.! ! Chapter'4!extends!the!study!of!block!copolymers!as!drug!delivery!systems!in!cancer! therapy!to!a!wide!range!of!linear!block!copolymers,!with!diblock!and!triblock!architecture!as! well! as! copolymers! with! the! same! constituent! blocks! and! different! block! lengths.! The! objective!was!to!determine!the!role!played!by!copolymer!structure!and!composition!on!the! viability!and!cellular!response!in!different!cell!lines.!Over!30!polymers!with!similar!structures! possessing! PEO! as! the! common! hydrophilic! unit! were! analyzed! in! terms! of! their! cytocompatibility!and!their!influence!on!the!inhibition!of!the!PgBP!efflux!pump.!An!empirical! threshold!of!the!ratio!EO/POeffective!=!1.5!for!cell!viability!was!found!for!triblocks,!whereas! the! inherent! larger! cytotoxicity! enables! precluded! such! finding! at! the! concentrations! studied.! Furthermore,! it! was! observed! that! some! of! the! studied! polymers! such! as! EO57PO46EO57!or!C16EO455C16!acted!as!inhibitors!of!the!PgBP!efflux!pump,!promoting!a!greater! accumulation!of!doxorubicin!within!NCIBADR–RES!cells.! ! Chapter' 5! is! a! summary! of! the! experimental! techniques! used! along! this! thesis.! Particular!emphasis!on!operation!principles!and!experimental!accessible!data!was!made!in! order! to! clarify! the! results.! The! different! equipments! are! also! shown,! in! some! cases! accompanied!by!diagrams!of!their!most!relevant!parts/configurations.! ! ! ! ! ! ! ! xvii! RESUMEN' ! La!nanotecnología!es!un!área!emergente!en!la!que!ya!se!han!obtenido!y!se!esperan! conseguir! nuevos! avances! revolucionarios! mediante! la! manipulación! de! materiales,! sus! propiedades!y!los!procesos!de!obtención!a!nivel!molecular,!abarcando!dimensiones!entre!1! nm!hasta!los!100!nm!aproximadamente.!Pero!lo!más!interesante!de!la!nanotecnología!no!es! la!posibilidad!de!trabajar!con!materiales!de!reducidas!dimensiones,!sino!el!cambio!radical! que! sufren! las! propiedades! físicas! y! químicas! de! la! materia! cuando! se! trabaja! a! escala! nanométrica:!la!conductividad!!eléctrica,!el!color,!la!resistencia!o!la!elasticidad,!entre!otras! propiedades.!Así,!los!nanomateriales!se!comportan!de!manera!diferente!a!como!lo!hace!el! mismo!material!a!escala!macroscópica,!por!lo!que!estos!cambios!en!las!propiedades!de!los! materiales!pueden!ser!aplicados!en!diferentes!campos!entre!los!que!destacan,!por!ejemplo,! la! producción! de! nuevos! materiales,! la! electrónica,! la! medicina! o! la! producción/almacenamiento/! recuperación! de! energía.! Ya! existen! productos! nanotecnológicos!en!el!mercado!como!cosméticos!más!eficaces!y!protectores,!raquetas!de! tenis!más!flexibles!y!resistentes,!ropa!que!no!se!arruga!ni!se!mancha,!o!gafas!y!cristales!que! no!se!rayan,!por!citar!algunos!ejemplos.! ! La!irrupción! de! la! nanotecnología! en! las! ciencias! de! la! salud! ha! dado! lugar! a! una! nueva!disciplina!denominada!Nanomedicina,!cuyo!objetivo!es!el!desarrollo!de!sistemas!que! permitan! diagnosticar,! prevenir! y! tratar! enfermedades! en! el! inicio! de! su! desarrollo! o! en! estados!poco!avanzados.!La!nanomedicina!agrupa!tres!áreas!principales:!el!nanodiagnóstico,! la! liberación! controlada! de! fármacos! (o! nanoterapia)! y! la! medicina! regenerativa.! El! nanodiagnóstico!consiste!en!el!desarrollo!de!sistemas!de!análisis!y!de!técnicas!de!imagen! para! la! detección! y! monitorización! de! enfermedades! en! los! estadíos! más! tempranos! posibles.!La!nanoterapia!pretende!dirigir!nanosistemas!activos!que!contengan!elementos!de! reconocimiento! para! obtener! respuestas! biológicas! y! transportar! y! liberar! medicamentos! exclusivamente! en! las! zonas! afectadas! a! fin! de! conseguir! un! tratamiento! más! efectivo,! minimizando! los! efectos! secundarios! adversos.! La! medicina/ regenerativa! tiene! como! objetivo! la! reparación! o! sustitución! de! tejidos! y! órganos! dañados! usando! herramientas! nanotecnológicas.! ! xviii! ! La!necesidad!de!encontrar!nuevos!métodos!diagnósticos!y!terapéuticos!para!diversas! dolencias!como!el!cáncer,!las!enfermedades!cardiovasculares,!la!diabetes!o!enfermedades! neurodegenerativas! para! las! que! no! existen! tratamientos! definitivos,! ha! generado! el! progresivo!aumento!del!número!de!investigaciones!en!Nanomedicina.!Uno!de!sus!grandes! retos! consiste! en! el! desarrollo! de! “nanoterapias”,! terapias! basadas! en! materiales! nanométricos! que! se! puedan! dirigir! de! forma! selectiva! a! los! tejidos! y! órganos! enfermos,! evitando! así! los! efectos! secundarios! inevitables! en! los! tratamientos! actuales.! Entre! ellas! caben!destacar!los!sistemas!de!liberación!controlada!de!fármacos,!que!consisten!en!utilizar! nanoestructuras! que! transporten! el! fármaco! hasta! la! zona! dañada,! y! sólo! cuando! la! han! reconocido,! lo! liberen.! Para! ello,! es! necesario! la! previa! encapsulación! o! protección! del! fármaco! para! que! este! sea! inerte! en! su! recorrido! por! el! cuerpo! y! mantenga! intactas! sus! propiedades.!Una!vez!que!el!nanosistema!ha!llegado!a!su!destino,!debe!liberar!el!fármaco!a! una! velocidad! apropiada! para! que! sea! efectivo,! y! luego! permitir! la! expulsión! del! nanotransportador!del!cuerpo!humano.!! ! En!esta!Tesis!Doctoral!nos!hemos!centrado!en!la!nanoterapia,!creando!sistemas!de! liberación! controlada! de! fármacos! enfocados! en! el! tratamiento! del! cáncer.! El! fármaco! empleado! ha! sido! la! doxorubicina,! que! es! uno! de! los! agentes! anticancerígenos! más! ampliamente!empleados!en!el!tratamiento!de!leucemias,!linfomas!de!Hodking,!así!como!en! diversos!cánceres!de!vejiga,!pecho,!estómago,!pulmón!u!ovario,!entre!otros.!El!fármaco!se! acumula!en!el!núcleo!celular,!donde!se!intercala!con!el!ADN!produciendo!que!se!escinda!y,! por!tanto,!se!produzca!la!muerte!celular.!! ! Para! trasportar! y! proteger! el! fármaco! anticancerígeno! se! han! empleado! nanotransportadores!basados!en!copolímeros!de!bloque!anfifílicos.!Esta!clase!de!polímeros! a! cierta! concentración! y/o! temperatura! se! agregan! de! forma! espontánea! en! estructuras! denominadas! micelas,! de! manera! que! los! bloques! hidróbobos! configuran! el! núcleo! y! los! hidrófilos!la!corona.!Alojar!los!fármacos!hidrófobos!en!el!núcleo!micelar!permite!aumentar! su!solubilidad,!además!de!protegerlo!frente!a!la!degradación!externa.!El!amplio!espectro!de! polímeros!disponibles!ofrece!un!amplio!campo!de!posibilidades,!aunque!se!prima!que!sean! sistemas! solubles! en! agua! para! que! se! pueda! asegurar! su! redispersión! en! disoluciones! tampón!biológicas.! ! La!estructura!molecular!de!un!copolímero!de!bloque!consiste!en!la!unión!química!de! dos! o! más! macromoléculas! de! homopolímeros! diferentes.! Atendiendo! al! número! de! bloques! estos! polímeros! se! denominan! dibloque,! tribloque! o! multibloque! (si! poseen! dos,! tres!o!más!bloques,!respectivamente),!denominándose!lineales!a!aquellos!que!forman!una! cadena! simple,! sin! ramificaciones,! y! anfifílicos! cuando! presentan! bloques! constituyentes! que!poseen!distinta!afinidad!por!el!medio!continuo!circundante.!Generalmente,!el!óxido!de! etileno! es! la! unidad! base! hidrófila! (si! consideramos! que! el! medio! circundante! del! ! xix! copolímero! en! cuestión! es! de! base! acuosa)! para! la! construcción! de! los! copolímeros! de! bloque.!Debido! a!este!carácter!hidrófilo,!los!otros!bloques!que!constituyen!el!copolímero! tendrán! un! carácter! más! hidrófobo,! dotando! al! copolímero! de!su! carácter! anfifílico.! El! carácter!hidrófobo!de!un!copolímero!puede!incrementarse!mediante!el!uso!de!bloques!más! hidrófobos! o! aumentando! el! número! de! unidades! hidrófobas! por! bloque.! Otro! factor! importante! a! tener! en! cuenta! es! la! temperatura:! por! ejemplo,! al! calentar! agua! ésta! se! vuelve! un! peor! disolvente! para! los! bloques! hidrófilos! de! óxido! de! etileno.! Además,! incrementando!la!temperatura!algunos!polímeros!modifican!también!su!solubilidad!debido! a!los!cambios!producidos!en!sus!interacciones!intra!e!interWmoleculares.! ! Una! característica! de! los! copolímeros! de! bloque! anfifílicos! es! su! capacidad! para! autoasociarse! en! disolución.! Este! proceso! de! agregación! para! los! copolímeros! lineales! depende!principalmente!del!disolvente!empleado!y!de!la!concentración.!Las!estructuras!más! simples! que! se! forman! por! autoasociación! son! las! micelas,! estructuras! en! las! que! las! cadenas! de! polímero! se! reconfiguran! espontáneamente! para! formar! estructuras! de! tipo! núcleoWcoraza.!Los!copolímeros!que!tienen!en!su!estructura!unidades!de!óxido!de!etileno!las! usan! para! formar! la! coraza! de! la! micela! (en! agua),! mientras! que! las! partes! hidrófobas! forman! el! núcleo.! Tanto! las! micelas! unimoleculares! como! las! polimoleculares! pueden! ser! empleadas!como!transportadores!de! fármaco.! El!fármaco!puede!incorporarse! tanto! en!la! coraza,!el!núcleo!o!la!interfase,!dependiendo!de!la!afinidad!de!éste!con!cada!parte.!Además,! los! copolímeros! con! bloques! de! óxido! de! etileno! dan! lugar! a! micelas! estéricamente! estabilizadas,!las!cuales!prolongan!su!circulación!sanguínea!al!evitar!ser!reconocidas!por!los! macrófagos!que!constituyen!el!sistema!retículoWendotelial!(SRE).!El!fármaco!cargado!en!el! núcleo! micelar! se! encuentra! asimismo! completamente! protegido! frente! a! la! dilución! y! a! otros! factores! externos.! Por! otra! parte,! el! tamaño! del! nanotranspotador! juega! un! papel! clave.!Así,!se!ha!probado!que!la!acumulación!de!nanopartículas!dentro!de!las!células!es!más! rápida! cuanto! más! pequeñas! son,! mientras! que! las! partículas! más! largas! son! retenidas! durante! un! tiempo! mayor.! Como! consecuencia,! se! ha! de! encontrar! un! equilibrio! entre! la! internalización,!la!acumulación!y!la!expulsión!de!la!célula!para!lograr!una!acción!terapéutica! efectiva.!! ! !Los! copolímeros! que! más! ampliamente! se! han! estudiado! para! aplicaciones! farmacéuticas! son! los! llamados! poloxámeros! (Pluronics®).! Los! Pluronics! son! copolímeros! cuya!estructura!está!formada!por!un!bloque!central!de!poli(óxido!de!propileno)!(PPO)!y!dos! bloques! laterales!de!poli(óxido!de!etileno),! PEO;! están! comercialmente!disponibles!en!un! amplio! rango! de! pesos! moleculares! y! longitudes,! su! capacidad! solubilizadora! está! sobradamente!demostrada!así!como!su!biocompatibilidad;!incluso,!algunos!están!ya!en!fase! de!ensayos!clínicos.!Sin!embargo,!los!Pluronic!muestran!también!diferentes!inconvenientes! como!nanosistemas!de!liberación!farmacológica,!como!son!una!micelización!incompleta!en! muchas!ocasiones!que,!por!lo!general,!conduce!al!autoWensamblaje!en!nanoestructuras!con! una!estabilidad!limitada!después!de!su!dilución!en!el!torrente!sanguíneo.! ! xx! ! El!objetivo!de!esta!tesis!doctoral!es!analizar!las!propiedades!y!capacidades!de!nuevos! copolímeros!de!bloque!como!sistemas!de!administración!de!fármacos!mediante!diferentes! soportes! micelares! formados! por! estos.! Debido! a! la! necesidad! de! nuevos! vehículos! de! administración!de!fármacos!que!permitan!un!aumento!de!la!solubilización!de!medicamentos! utilizando! la! concentración! de! polímero! más! baja! posible,! diferentes! copolímeros! fueron! sintetizados!y!caracterizados.!Para!alcanzar!este!objetivo,!la!presente!tesis!se!estructura!en! los!siguientes!capítulos:! ! En!el!capítulo'1!se!presentan!unas!nociones!básicas!en!relación!a!la!nanotecnología,! la!nanoterapia!y!a!los!sistemas!de!liberación!de!fármacos.!Con!la!finalidad!de!entender!las! necesidades!y!objetivos!de!los!sistemas!de!liberación,!se!ha!realizado!un!resumen!somero! acerca!de!algunos!de!los!distintos!tipos!de!sistemas!actualmente!usados!en!nanoterapia,!así! como!una! relación!de! los!requerimientos!y!ventajas!que!se!esperan!de!estos! sistemas.!El! capítulo! se! completa! con! un! apartado! referente! a! los! polímeros! y! sus! propiedades! de! agregación,!enfocado!principalmente!a!los!sistemas!micelares!y!las!ventajas!que!presentan! como!vehículos!de!liberación!de!fármacos.! ! En! el! capítulo' 2!se! presenta! un! análisis! de! los! copolímeros! EO33SO14EO33! y! EO38SO10EO38!(SO!=!óxido!de!estireno,!y!los!subíndices!indican!la!longitud!de!los!bloques).!Se! ha! evaluado! su! estructura! y! su! proceso! de! agregación! en! disolución! acuosa! como! paso! previo!y!necesario!para!su!empleo!como!agentes!transportadores!y!liberadores!de!fármacos.! Estos! copolímeros! se! han! elegido! como! alternativa! a! los! Pluronics! mencionados! anteriormente,!poseyendo!la!misma!estructura!molecular!pero!con!el!bloque!central!mucho! más! hidrófobo,! lo! que! debiera! conferirles! una! mayor! capacidad! de! solubilización! en! el! núcleo! micelar.! La! longitud! de! los! bloques! ha! sido! elegida! para! obtener! el! mejor! compromiso! entre! la! solubilidad! de! la! cadena! polimérica,! la! formación! de! micelas! y! el! tamaño! del! núcleo! micelar! en! base! a! estudios! previos.! Estos! copolímeros! se! sintetizaron! mediante!polimerización!secuencial!aniónica!y!se!caracterizó!su!composición!y!arquitectura! mediante!resonancia!magnética!nuclear!(RMN)!y!cromatografía!de!permeación!en!gel!(GPC).!! La! evaluación! de! las! micelas! como! nanovehículos! para! la! administración! de! fármacos! se! realizó! mediante! una! caracterización! física! para! conocer! los! detalles! de! su! estructura,! así! como!su! comportamiento!en!disolución!acuosa!en!un!amplio!rango!de!concentraciones!y! temperaturas,! seguida! por! estudios! in/ vitro! para! evaluar! la! capacidad! para! encapsular! fármacos! hidrófobos! (proporcionándoles! una! debida! estabilidad! y! protección),! su! biocompatibilidad!y!su!acción!citotóxica.!! ! El!proceso!de!autoasociación!de!los!copolímeros!y!consiguiente!formación!de!micelas! se! siguió! mediante! espectroscopía! de! fluorescencia,! empleando! el! método! del! pireno.! Se! verificó! que! el! proceso! de! agregación! se! inicia! a! concentraciones! inferiores! que! para! los! ! xxi! Pluronics!de!similar!longitud!de!bloque!al!ser!el!bloque!de!óxido!de!estireno!más!hidrófobo! que!el!de!óxido!de!propileno.! ! Empleando! las! técnicas! de! dispersión! estática! y! dinámica! de! luz! (SLS!y! DLS,! respectivamente)! y! analizando! un! amplio! rango! de! concentraciones! se! extrajo! la! información!necesaria!para!conocer!las! propiedades! de!las!micelas! poliméricas! formadas:! radio! hidrodinámico,! número! de! agregación! micelar,! peso! molecular! de! las! micelas,! y! el! volumen!de! la!corona!micelar!y!su!hidratación.!Se!encontró!que!en!disolución!acuosa!los! copolímeros! EOmSOnEOm! se! autoensamblan! a! concentraciones! muy! bajas! para! formar! micelas!de!tamaños!entorno!a!ca.!15!nm,!lo!que!los!hace!adecuados!para!la!administración! por!vía!parenteral.!Tales!micelas!son!esféricas!y!poseen!un!núcleo!hidrófobo!y!una!envoltura! hidrófila!de!PEO.!Para!corroborar!la!fiabilidad!de!los!análisis!de!los!datos!de!DLS!en!cuanto!a! los!tamaños!micelares,!las!muestras!se!visualizaron!también!por!microscopía!electrónica!de! transmisión! (TEM)! verificándose,! al! mismo! tiempo,! la! simetría! esférica! de! las! micelas.! Asimismo,! el! comportamiento! de! estos! copolímeros! de! bloque! en! un! rango! amplio! de! concentraciones!y!temperaturas!también!se!analizó!mediante!reometría,!de!modo!que!nos! permitió! conocer! en! detalle! el! comportamiento! fásico! y! las! propiedades! asociadas! a! las! distinta!fases!existentes!en!los!mismos.!! ! A! continuación,! la! capacidad! de! solubilización! de! fármacos! hidrófobos! de! los! copolímeros!EO33SO14EO33!y!EO38SO10EO38!se!probó!mediante!la!variación!de!la!relación!de! fármaco/copolímero,! siempre! a! concentraciones! de! polímero! por! encima! de! su! concentración!micelar!crítica.!Para!ello,!se!emplearon!dos!fármacos!poco!hidrosolubles,!el! antifúngico!griseofulvina!y!el!anticancerígeno!doxorubicina.!Se!observó!que!el!aumento!de! la!solubilidad!para!ambos!fármacos!fue!mayor!cuando!se!emplea!como!vehículo!el!polímero! EO33SO14EO33!debido!a!tener!un!núcleo!más!grande!y!más!hidrófobo!y,!por!consiguiente,!ser! más! afín! con! el! fármaco.! Las! pruebas! de! estabilidad! realizadas! muestran! que! estos! copolímeros! en! su! forma! micelar! mantienen! el! tamaño! inicial! después! de! un! ciclo! de! congelación!W!secado!W!reconstitución,!lo!que!los!hace!adecuados!para!ser!almacenados.!La! evolución!en!el!tiempo!bajo!condiciones!de!fuerte!dilución!tanto!de!las!micelas!vacías!como! para!las!micelas!cargadas!de!fármaco!muestran!que!estos!sistemas!son!físicamente!estables! hasta!12!días,!manteniendo!el!tamaño! prácticamente! invariable! y! la!carga!al!100%!de! su! valor!inicial!en!los!primeros!5!días.!! ! Otro! punto! importante! para! el! potencial! empleo! de! los! copolímeros! del! tipo! EOmSOnEOm!como!sistemas!de!liberación!es!la!velocidad!de!liberación!del!fármaco!una!vez! ha! llegado! a! su! destino.! Las! micelas! cargadas! con! el! fármaco! exhibieron! una! rápida! liberación!acumulada!in/vitro!a!tiempos!cortos!de!incubación!!para,!posteriormente!volverse! más!sostenida.!Se!ha!verificado!también!que!la!liberación!depende!del!pH!medio!en!el!que! se! encuentre! el! vehículo.! Para! comprobar! este! punto! se! emplearon! disoluciones! tamponadas! de! pH! 4.0,! 5.5! y! 7.4,! las! cuales! imitan! el! ambiente! ácido! en! las! células! ! xxix! 9.! Menzel,! C.;! Cambon,! A.;! Yeates,! S.! G.! Double! emulsion! template! suspension! polymerization:!towards!the!synthesis!of!polyelectrolyte!core!porous!hydrophobic! shell! particles! for! environmental! applications.! J.! Mat.! Chem.! A.,! 2013,! 1,! 125534 12559.! CHAPTER(1( ! INTRODUCTION( ! 2! 3! 1.1 NANOTECHNOLOGY! ! Nanotechnology! is! a! scientific! field! which! primarily! deals! with! the! synthesis,! characterization,!exploration!and!exploitation!of!nanostructured!materials.!The!National! Nanotechnology! Initiative! (NNI)! defines! nanotechnology! as! the! “understanding! and! control! of! matter! at! dimensions! of! roughly! 1! to! 100! nanometers,! where! unique! phenomena!enable!novel!applications,”!allowing!fabrication!of!devices!on!the!nanoscale.! Generally,! nanomaterials! are! characterized! by! having! at! least! one! dimension! in! the! nanometer! range! (1! nm! =! 10−9! m).! Nanostructured! materials! constitute! a! bridge! between!molecules!and!infinite!bulk!systems.!Individual!nanostructures!include!clusters,! quantum! dots,! nanocrystals,! nanowires! and! nanotubes,! amongst! others,! while! collections!of!nanostructures!involve!arrays,!assemblies!and!superlattices!of!individual! nanostructures!(1).!! ! Nanotechnological! devices! offer! a! broad! and! exciting! field! of! possibilities! in! a! wide! range! of! research! areas:! From! the! nanowires! used! in! electronic! devices! to! the! ferrofluids!used!as!contrast!agents!in!magnetic!resonance!imaging!(MRI),!their!potential! applications! are! growing! as! the! properties! of! each! nanomaterial! are! discovered! and! characterized,! and! new! combinations! of! different! nanosystems! emerge.! The! main! advantage! of! nanotechnology! is! neither! the! inherent! size! of! the! building! blocks! themselves!nor!another!step!in!miniaturization,!it!is!in!fact!the!generation!of!new!or!the! enhancement! of! previous! existing! properties! that! materials! exhibit! in! the! nanoscale! regime! (from! 1! to! 1000! nm,! the! soWcalled! mesoscale! too).! At! this! length! scale,! some! materials!properties!are!affected!by!the!laws!of!atomic!physics!rather!than!behaving!as! traditional!bulk!materials!do.!Hence,!the!nanoworld!lays!midway!between!the!scale!of! atomic!and!quantum!phenomena!and!the!scale!of!bulk!materials.! ! In! order! to! achieve! materials! in! the! mesoscale,! there! are! two! different! approaches:! topWdown! and! bottomWup! (see! Figure! 1).! The! topWdown! strategy! uses! physical! engineering! tools! for! carving! of! macroWsize! materials! to! obtain! a! mesoscale! smaller! material.! Along! this! process,! the! lateral! dimension! of! the! material! is! reduced! until! a! nanostructured! material! is! obtained! as! occurred,! for! example,! in! the! case! of! silicon! integrated! circuits! fabricated! by! selective! layer! deposition.! Conversely,! the! bottomWup! strategy! is! based! on! the! assembly! of! subWunits! in! a! controlled! and! reproducible!manner.!These!units!can!be!in!the!atomic,!molecular!or!colloidal!regime.! The!bottomWup!strategy!also!takes!advantage!of!the!selfWassociation!properties!of!!nanoW 4! sized!building!blocks!as,!for!example,!in!the!case!of!micelles!formed!by!surfactants!or! polymers!(2,3).!! ! ! ! Figure(1.!Schematic!representation!of!two!strategies!used!to!get!nanosized!materials:!a)! TopWdown!and!b)!BottomWup.! ! As! commented! previously,! the! physical! and! chemical! properties! of! nanostructures!are!distinctly!different!from!those!of!a!single!atom!(molecule)!and!bulk! matter! with! the! same! chemical! composition! (1).! These! differences! between! nanomaterials!and!the!molecular!and!condensedWphase!materials!pertain!to!the!spatial! structures! and! shapes,! phase! changes,! energetic,! electronic! structure,! chemical! reactivity,!and!catalytic!properties!of!large,!finite!systems,!and!their!assemblies.!Some!of! the!important!issues!in!nanoscience!are!related!to!size!and!shape!effects!which!affect,! for!example,!the!response!to!external!electric!and!optical!excitations!of!individual!and! coupled!finite!nanosystems!through!quantum!confinement.!! ! Surfaces!and!interfaces!are!also!important!in!explaining!nanomaterial!behaviour.! In!bulk!materials,!only!a!relatively!small!percentage!of!atoms!will!be!at!or!near!a!surface! or! interface! (like! a! crystal! grain! boundary).! In! nanomaterials,! the! small! feature! size! ensures! that! many! atoms,! more! than! a! half! in! some! cases,! will! be! near! interfaces.! Surface/Interfacial!properties!such!as!energy!levels,!electronic!structure,!and!reactivity! 5! can!be!quite!different!from!macrostates!and!may!give!rise!to!quite!different!material´s! properties!as,!for!example,!the!fluorescent!properties!of!quantum!dots!or!the!behaviour! of!superparamagnetic!iron!oxide!nanoparticles!(SPIONs)!(4,5).!! ! Nanomaterials! are! on! the! same! scale! as! the! critical! size! for! many! different! physical!phenomena!to!occur:!For!example,!the!tip!radius!of!a!crack!in!a!material!may! be!in!the!range!1W100!nm.!The!way!a!crack!grows!in!a!macroWsized!material!is!different! from!a!crack!propagation!in!a!nanomaterial,!where!crack!and!particle!are!comparable!in! size.! As! mentioned! before,! fundamental! electronic,! magnetic,! optical,! chemical,! and! biological!processes!are!also!different!at!this!level.!For!example,!proteins!are!10W1000! nm! in! size,! and! the! diameter! of! human! cells! spans! from! 10! to! 20! µm! being! the! organelles! diameter! ranging! from! a! few! nanometer! to! a! few! hundreds;! as! a! consequence,! the! behaviour! of! biological! entities! when! getting! into! contact! a! nanomaterial!may!be!quite!different!from!that!observed!when!contacting!to!largerWscale! materials!(6).!For!this!reason,!nanotechnological!devices!may!present!new!possibilities! for!drug!delivery,!gene!therapy!and!medical!diagnostics.!! ! ! 1.1.1##Nanopharmaceutics#and#nanomedicine# # In! relation! to! the! pharmaceutical/medical! aspects,! the! Federal! Drug! and! Administration!(FDA)!Office!of!USA!highlights!that!materials!in!the!nanoscale!often!have! different! physical,! chemical! and! biological! properties! than! their! bulk! counterparts! in! complex! biological! media! (7,8).! As! a! consequence,! their! biological! activities! can! also! exhibit! alterations.! A! very! good! example! is! silver:! Silver! atoms! do! not! exhibit! antibacterial! activity,! while! ionic! silver! ions! (Ag+)! are! really! effective! antibactericidal,! antimicrobial! and! antifungal! agents.! Because! of! their! water! solubility! and! ease! of! production,!Ag+!ions!are!a!potential!biocide,!but!their!high!toxicity!exclude!them!to!be! used! as! a! safe! product.! Other! silver! compounds! are! instead! employed! in! external! preparations!as!antiseptics,!for!example,!silver!nitrate.!In!addition,!silver!ions!cannot!be! directly! used! inside! the! human! body! because! they! quickly! combine! with! chloride! to! form! silver! chloride,! an! insoluble! compound! with! reduced! antimicrobial! activity.! To! avoid!this!issue,!colloidal!silver!nanoparticles!(Ag!NPs)!were!developed.!Solid!metallic!Ag! NPs!are!insoluble!in!water!and!can!release!ionic!silver!in!a!sustained!rate!once!into!the! body! but! with! suitable! functionalization! this! problem! can! be! overcome.! However,! concerns!still!remain!regarding!the!long!degradation!time!of!this!kind!of!particles!inside! living!systems!(9).!! ! 6! Nevertheless,!nanotechnology!offers!a!tremendous!potential!in!applications!such! as!biomedical!diagnosis!and! therapy!giving!rise! to!a!new!field! termed!Nanomedicine,! which! nowadays! constitutes! one! of! the! priority! areas! in! most! of! the! developed! countries.! For! example,! the! National! Institute! of! Health! of! USA! offered! a! budget! of! 1443WM$!for!the!period!2006W2011!to!encourage!and!develop!projects!in!this!research! field.!The!purpose!of!Nanomedicine!is!to!follow,!control,!construct,!repair,!defend!and! improve! biological! human! systems! for! different! applications! such! as! in! imaging! diagnosis!and!therapeutics.!To!do!that,!engineered!nanodevices!are!used,!these!possess! individual! functions! given! by! their! individual! components! integrated! in! an! single! architecture!and!can!even!meet!multiple!functions;!they!work!at!the!molecular!level!by! interacting! with! cellular! or! subWcellular! structures! and,! at! the! same! time,! to! look! for! producing!effective!responses!at!bigger!scales.!Hence,!the!final!goal!of!nanomedicine!is! to!overcome!some!or!all!of!the!drawbacks!of!current!clinical!practice!as!too!short!blood! circulating! times! of! active! chemicallyWactive! compounds,! lack! of! enough! image! resolution!for!an!early!detection!of!diseases!like!cancer,!unspecific!biodistribution,!nonW controlled!release,!unavailability!to!overcome!biological!barriers,!cytotoxicity...!! ! ! ! Figure(2.!Examples!of!nanomaterials!used!in!the!nanopharmaceutical/nanomedical!field:! a)! Fe3O4! SPION,! b)! gold! nanorods,! c)! silver! stars,! d)! metallic! fibres,! e)! micelles! and! f)! polymeric!nanocapsules!for!simultaneous!imaging!and!treatment.! 7! To! solve! some! of! these! problems,! nanotechnology! enabled! the! design! and! obtaining! of! the! soWcalled! drug! delivery! vehicles,! also! known! as! nanoplatforms! or! nanovectors–nanoparticles! capable! of! carrying! and! delivering! one! or! more! bioactive! molecules! (4,10,11),! giving! rise! to! a! field! known! as! pharmaceutical! nanotechnology.!! The!main!objective!of!these!nanovectors!is!to!transport!the!cargo!by!using!nanoparticles! composed! of! different! materials! (see! Figure! 2)! as! vehicles! offering! a! protective! environment!until!the!target!cell/tissue!is!reached!and!facilitating!its!controlled!release.! The!ultimate!goal!of!this!strategy!is!to!kill/cure!an!infected/tumour!cells/organ/tissue! without!affecting!healthy!ones,!avoiding!adverse!side!effects.!To!exert!the!therapeutic! function,! drug! carriers! must! be! accumulated! inside! cells! through! the! enhanced! permeation! and! retention! effect! (EPR)! and/or! by! specific! targeting,! should! enable! to! cross!the!cell!membrane!in!order!to!achieve!the!required!concentration!for!enough!time! to!carry!out!their!optimal!therapeutic!activity!levels.!! ! Once! reached! the! target,! the! release! must! be! controlled! to! avoid! underW! or! overdoses,!which!could!derive!in!an!ineffective!treatment!or!toxicity.!The!degradation! or!excretion!mechanism!of!the!nanocarriers!is!also!an!important!concern,!because!the! accumulation! in! some! organs! could! also! derive! in! toxicity.! To! solve! this! issue,! for! example! once! into! the! cell! the! nanocarrier! could! undergo! degradation! whilst,! at! the! same!time,!releases!its!cargo.!On!the!other!hand,!if!the!nanovehicle!remains!unaltered! after! releasing! the! cargo! it! could! be! captured! by! the! reticuloWendothelial! system! or! degraded!into!smaller!sizes,!which!would!allow!its!excretion!by!renal!clearance![8].!In! this!regard,!the!FDA!demands!an!exhaustive!control!over!the!specifications!and!quality! of!the!nanoparticles!designed!to!pharmaceutical/medical!applications,!being!the!main! requirements!the!control!over!(12):! ! W The!particle!size!and!their!distribution.! W The!surface!area,!the!chemical!properties!of!the!surfaces,!the!porosity!and!the! surface!coverage.! W The!hydrophobicity!and!the!charge!density!of!the!surface.! W Purity!and!sterility.! W Stability!(aggregation!or!protein!adsorption).! W Cell!internalization.! W Cytotoxicity.! W Drug!release!profile.! W Compelte!correlation!between!“in!vitro”!and!“in!vivo”!behaviour.! W Excretion!and!biodegradability.! W Evironmental!impact.! ! 8! Finally,! just! to! mention! that! amongst! the! most! wellWknown! representatives! of! nanocarriers!in! clinical! use,! for! example! for! cancer! treatment,! we! can! find! liposomes! (for!example,!DOXIL™,!liposome!loaded!with!the!anticancer!drug!doxorubicin!approved! in! 1995! for! the! treatment! of! Kaposi´s! sarcoma! (13);! albumin! nanoparticles! (as! ABRAXANE™,!approved!in!2005!for!the!treatment!of!methastasic!breast!cancer)!(14);!or! polymer! nanoparticles! (as! GENEXOLWPM,! a! formulation! of! polymeric! micelles! loaded! with!the!anticancer!drug!paclitaxel!and!free!of!CremophorWEl,!which!is!a!phase!II!trial!for! analyzing! its! efficacy! in! pancreatic! tumours! in! USA)! (15).! However,! the! generation! of! nanovectors! based! on! nanoparticles! in! the! market! nowadays,! around! a! couple! of! dozens,! are! nonWtargeting! passive! systems! whose! biodistribution! along! the! humans! body!cannot!be!traced!(8,11,16).!The!localization!of!these!systems!is!addressed!only!by! their!size!(in!particular,!by!the!enhanced!retention!and!permeation!effect,!EPR,!which! leads! to! the! nanovehicle! to! be! localized! in! areas! with! uncontrolled! increases! of! vascularity! as! occurred! in! solid! tumours! (17),! and! is! not! related! with! a! specific! recognition!by!the!targeted!cell!or!tissue.!Hence,!we!can!conclude!that!Nanomedicine!is! still!in!its!infancy!and!there!exist!a!huge!number!of!challenges!that!this!discipline!can! help!to!reach!in!next!years.!! ! ! 1.1.2#References# ! 1.!Gogocij,!J.G.!Nanomaterials,Handbook.!Boca!Raton:!Taylor!&!Francis,!2006.! 2.! Booth,! C.;! Price! C! Comprehensive, Polymer, Science.! Oxford:! Pergamon! Press,! 1989.! 3.! Holmberg,! K.;! Jönsson,! B.Kronberg,! B.! Surfactants, and, Polymers, in, Aqueous, Solution.!Nueva!York:!John!Wiley!&!Sons,!1989.! 4.!Kim,!K.Y.!Nanomedicine!Nanotechnol.!Biol.!Med.!2007,!3,!103.! 5.! Arya,!H.;!Kaul,!Z.;!Wadhwa,!R.;!Taira,!K.!;!Hirano,!T.!;!Kaul,!S.!C.!Biochem.!Biophys.! Res.!Commun.!2005,!329,!1173.! 6.! Yadav,! A.K.;! Mishra! P.;! Mishra! A.! K.;! Mishra! P.;! Jain! S.;! Agrawal! G.! P.! Nanomedicine!Nanotechnol.!Biol.!Med.!2007,!3,!246.! 7.!Preining,!O.!J.!Aerosol!Sci.,1998,!,29,(!481.! 8.! Hagens,!W.I.;!Oomen,!A.!G.;!de!Jong,!W.!H.;!Cassee,!F.!R.;!Sips,!A.!J.!A.!M.!Regul.! Toxicol.!Pharmacol.!2007,!49,!217.! 9.! Maillard,!J.WY.;!Hartemann,!P.!Crit.!Rev.!Microbiol.!2013,!39,!373.! 10.! Koo,! O.M.;! Rubinstein,! I.;! Onyuksel,! H.! Nanomedicine! Nanotechnol.! Biol.! Med.! 2005,!1,(193.! 11.! Mishra,!B.;!Patel,!B.B;!Tiwari,!S.!Nanomedicine!Nanotechnol.!Biol.!Med.!2010,!!6,! 9.! 12.! Tyner,!K.;!Sadrieh,!N.,Methods!Mol.!Biol.!2011,!697,!17.! 13.!Working,!P.K.;!Newman,!M.!S.;!Huang,!S.!K.;!Mayhew,!E.;!Vaage,!J.;!Lasic,!D.!!J.! Liposome!Res.!1994,!4,(!667.! 9! 14.! Ibrahim,!N.K.,!Samuels,!B.;!Page,!R.;!Doval,!D.;!Patel,!K.!M.;!Rao,!S.!C.;!Nair,!M.!K.;! Bhar,!Paul;!Desai,!N.;!Hortobagyi,!G.!N.,J.!Clin.!Oncol.!2005,!23,!6019.! 15.! !Werner,!M.E.,!Cummings,!N.!D.;!Sethi,!M.;!Wang,!E.!C.;!Sukumar,!R.;!Moore,!D.! T.;!Wang,!A.!Z.!Int.!J.!Radiation!Oncol.!Biol.!Phys.!2013,!86,!463.! 16.!Chouly,!C.;!Pouliquen,!D.;!Lucet,!I.;!Jeune,!J.!J.;!Jallet,!P.!J.!Microencapsulation! 1996,!13,!245.! 17.! Heidel,!J.;!Davis,!M.!Pharm.!Res.!2011,!!28,!187.! ! ! 16# 1.2.2$ $NPs$used$in$pharmaceutics$ # Different# nanoparticles# have# been# investigated# as# potential# drug# carriers# with# the#aim#of#increasing#the#efficacy#of#the#molecular#cargo#by#the#suitable#simultaneous# combination# of# the# drug# and# vehicle´s# properties.# As# mentioned# previously,# this# increase# in# effectiveness# might# be# originated# from# drug# protection,# enhancement# of# drug# solubility,# passive# or# active# targeting,# accumulation# in# the# targeted# area# and# avoidance#of#natural#excretion#mechanisms.#In#addition,#these#nanoparticles#must#meet# the#condition#of#aqueous#solubility#to#enable#their#transfer#to#biological#media#before# administration#to#living#systems.# # Inorganic!nanoparticles!are#those#composed#by#inorganic#materials,#as#silica#or# metals,#which#usually#are#presented#as#solid#spheres,#porous#structures#or#hollow#NPs# (53).# Their# main# advantages# are# related# to# their# great# aqueous# stability# and# the# possibility#of#choosing#an#appropriate#size#and#shape#on#demand.#Drugs#are#externally# attached#to#solid#structures#while#hollow#NPs#are#used#to#encapsulate#high#doses#into# their# inner# cavities.# Drugs# are# usually# loaded# onto# the# pores# surface# by# physical# adsorption# (in# porous# NPs)# because# of# their# high# surface# area:# the# subsequent# drug# loading#capacity#and#release#profile#results#very#different#depending#on#pore#diameter,# pore# topology,# surface# properties,# etc# (54).# Regarding# porous# carriers,# mesoporous## silica# NPs# are# the# most# extensively# studied# owing# to# the# great# amount# of# ordered# uniform# pores# on# their# surface# which# enables# a# precise# control# of# drug# loading# and# release#(55).#Hollow#nanocarriers#provide#an#excellent#isolated#cavity#for#drug#storage,# which# is# made# by# removing# the# template# used# to# create# the# NP.# Amongst# other# inorganic#materials#used#to# prepare#hollow#NPs# we#should#mention#silica#(56,57)# and# gold#(58,59)#as#the#most#common#found#in#literature.#As#examples,#we#could#mention# gold#nanoparticles#(Au#NPs),#which#has#been#deeply#studied#because#its#unique#physical# (localized# surface# plasmon# resonance,# catalytic# activity…)# and# chemical# properties# (chemical#stability,#ease#of#surface#functionalization…)#and#allow#controlled#drug#release# strategies# using# internal# or# external# stimuli,# such# as# glutathione,# pH,# heat# or# light,# amongst# others# (60L62);# or#iron# oxide# NPs# (SPION),# which# were# first# successfully# employed#as#contrast#agents#in#magnetic#resonance#imaging#(MRI)#and#now#are#being# used#to#magnetically#guide#and#deliver#drugs#taking#advantage#of#their#superb#magnetic# properties#and#biocompatibility#(63).# # On# the# other# hand,# organic! nanoparticles# are# those# composed# by# organic# materials# organized# in# supramolecular# architectures.# Amongst# the# most# common# employed# organic# materials# composing#drug# delivery# vehicles# we# can# mention# lipids,# proteins,#polymers#or#carbon#nanotubes.## 17# Some# amphiphilic# lipids# can# selfLassemble# to# form# vesicles,# spherical# bilayer# structures# whose# cores# comprise# the# same# solvent# as# their# surroundings.# Therefore,# these# vesicles# are# suitable# to# deliver# waterLsoluble# drugs# or# biomaterials,# including# enzymes,#antibodies#or#genes#(53).#Amongst#these,#liposomes#are#small#artificial#vesicles# of# spherical# shape# that# can# be# produced# from# natural# nontoxic# phospholipids# and# cholesterol# (64).# Liposomes# are# particularly# useful# as# drug# and# gene# therapy# devices# because# of# their# ability# to# pass# through# lipid# bilayers# and# cell# membranes.# As# an# example,#Doxil#is#a#formulation#carrying#the#chemotherapeutic#drug#doxorubicin#loaded# inside#pegylated#liposomes,#where#poly(ethylene#oxide)#chains#provides#the#system#with# hydrophilic#segments#for#aqueous# dispensability.#In#addition,#pegylated#shells#prevent# the#recognition#of#the#liposomes#by#the#reticuloLendothelial#system#(65),#increasing#the# nanocarrier#circulation#time#in#the#bloodstream#and#allowing#the#drug#to#be#released#for# longer#period#times.#Other#wellLstudied#lipid#structure#is#solid#lipid#nanoparticles#(SLN),# which#are#composed#by#solid#lipids#both#at#ambient#or#body#temperature#(66,67).#SLNs# are#composed#by#a#solid#lipid#core#matrix#that#can#solubilise#lipophilic#molecules,#while# the#used#emulsifier#prevent#agglomeration#between#NPs#and#improve#stability#(50).#SLN# are#produced#by#highLpressure#homogenization,#avoiding#the#use#of#organic#solvents#as# well# as# allowing# to# be# massively# produced,# a# required# condition# for# scalability# and# industrial#production.# # Proteins#are#large#biological#molecules#constituted#by#one#or#more#amino#acid# chains,#which#have#several#biological#functions#as#well#as#play#structural#and#mechanical# functions#in#cells.#As#a#result,#their#main#advantages#of#their#use#as#constituents#of#drug# delivery# systems# are# related# to# their# biocompatibility# into# the# human# body,# biodegradability,#and#their#nonLantigenic#and#metabolizable#character#(68).#In#addition,# they#can#also#be#easily#amenable#for#surface#modification#and#covalent#attachment#of# drugs# and# ligands.# A# relevant# example# of# protein# as# a# carrier# is# the# use# of# albumin# nanoparticles# to# transport# and# release,# for# example,# the# interferonLgamma# (IFNLγ)# (69,70),#keeping#active#the# bactericidal# properties# of# IFNLγ.#This#system#enhances#the# cellular# uptake# of# the# protein# inside# the# protein# nanocarrier,# and# even# it# exhibits# a# better# therapeutic# performance# than# liposomes# concerning# sustained# drug# release# profiles.## # Carbon#nanotubes#(CNTs)#are#a#distinct#molecular#form#of#carbon#atoms#that#was# discovered# in# the# late# 1980s.# Briefly,# CNTs# are# hexagonally# shaped# arrangements# of# carbon# atoms# that# have# been# rolled# into# tubes,# with# their# diameter# being# within# the# nanometer#scale.#CNTs#typically#have#diameters#ranging#from#<#1#nm#up#to#50#nm#while# their#lengths#are#typically#of#several#microns.#Their#long,#hollow#structure#with#the#walls# formed#by#oneLatomLthick#sheets#of#carbon#is#called#graphene.#These#sheets#are#rolled# at#specific#and#discrete#("chiral")#angles,#and#the#combination#of#the#rolling#angle#and# 18# particle#radius#control#the#nanotube´s#properties;#for#example,#whether#the#individual# nanotube# shell# is# a#metal#or#semiconductor.# There# has# been# tremendous# enthusiasm# over#carbon#nanotube#applications#in#many#industrial#sectors,#in#part#because#they#have# been#shown#to#be#100#times#stronger#than#steel#with#only#oneLsixth#of#its#weight,#and# with#unusual#heat#and#conductivity#properties#(71).#In#the#area#of#nanomedicine,#carbon# nanotubes#have#been#primarily#used#for#transporting#DNA#cargos#into#the#cell,#and#for# thermal#ablation#therapy#in#cancer#therapeutics.#For#example,#Kam#et#al.#have#shown# that#singleLwalled#carbon#nanotubes#of#1#to#2#nm#in#diameter#carrying#a#15Lmer#DNA# chain#adsorbed#onto#their#surfaces#as#a#cargo#molecule#can#be#internalized#by#cells#and# accumulate#in#their#cytoplasm#without#causing#cytotoxicity#(72).# # On# the# other# hand,# polymeric# nanoparticles# are# those# composed# of# polymers# (from#natural#to# synthetic#polymers#and# from#lineal#to#branched# or#star#copolymers),# offering# a# huge# field# of# possibilities.# The# most# common# structures# formed# are# nanospheres# and# nanocapsules.# Nanospheres# have# a# matrix# typeLstructure# allowing# drug#absorption#at#the#sphere#surface#or#encapsulated#within#the#particle.#Nanocapsules# are#vesicular#systems#in#which#the#drug#is#confined#inside#a#cavity#consisting#of#an#inner# liquid#core#surrounded#by#a#polymeric#membrane.#In#this#case,#the#active#substances#are# usually#dissolved#in#the#inner#core#but#may#also#be#adsorbed#onto#the#capsule#surface# (34).# # #Polymeric# particles# are# obtained# by# supramolecular# assembly# of# polymeric# chains.#There#exist#two#different#approaches#to#obtain#these#type#of#nanoparticles:#the# first# one# involves# the# in#situ' polymerization# of# monomers# and# their# subsequent# assembly;# the# second# one# is# based# on# the# dispersion# and# assembly# of# preformed# polymeric# chains# (34).# The# methodologies# to# obtain# NPs# by# monomer# polymerization# can# be# further# classified# into# emulsion# and# interfacial# polymerization# (34).# Besides,# emulsion# polymerization# can# be# either# organic# or# aqueous# depending# on# the# solvent# used;#the#polymerization#reaction#starts#when#the#monomer#is#dispersed#or#dissolved#in# the# chosen# solvent# in# the# presence# of# initiators.# Interfacial# polymerization# allows# to# obtain#polymeric#NPs#whose#surface#corresponds#to#the#contours#of#the#inner#phase#of# an# oil/water# or# water/oil# emulsion.# Another# type# of# interfacial# polymerization# is# interfacial# polycondensation,# that# relies# on# an# interfacial# reaction# between# two# monomers.# Nanoparticles# can# be# also# prepared# directly# from# preformed# synthetic# or# natural# polymers# and# by# desolvation# of# macromolecules.# Recently,# these# polymeric# systems#have#been#also#prepared#by#nebulization#techniques.## # Dispersion# of# preformed# polymers# can# also# be# classified# into# two# groups# regarding# the# polymer# nature,# i.e# synthetic# or# natural# polymers.# Synthetic# preformed# polymers# can# form# nanoparticles# by# different# techniques:# emulsification/solvent# 19# evaporation,# solvent# displacement,# interfacial# deposition,# emulsification/solvent# diffusion# or# salting# out.# Natural# preformed# polymers# can# be# assembled# by# emulsion# techniques#or#dropwise#extrusion.#Nevertheless,#the#main#synthetic#techniques#used#in# the#pharmaceutical#field#are#those#based#in#the#spontaneous#aggregation#properties#in# solution# (specially# in# aqueous# solution)# of# the# polymeric# monomer# chains# such# as# micellisation,#gelling#or#complex#formation.# # The# general# properties# of# polymeric# NPs# can# be# modulated# by# choosing# the# appropriate# polymer# monomeric# chain# and# the# chemical# surface# functionalization# (73,74).#Once#the#right#polymer#chain#has#been#found,#NP#size#can#be#tuned#choosing# between# high# or# low# molecular# weight# polymers# and/or# long# or# shorter# block# length# composition# (75).# The# employed# synthetic# technique# also# limits# the# NP# size# range,# structure# and# stability# (34).# As# an# example,# NPs# obtained# by# in# situ# bulk# monomer# polymerization#are#solid#NPs#which#cannot#be#degraded,#while#assembled#polymeric#NPs# as#micelles#are#reversibly#dynamic#systems.#In#the#same#way,#physical#gels#progressively# loss#their#structure#under#dilution#conditions#in#contrast#to#chemical#gels#(crosslinked)# which#shrink#while#keeping#intact#their#structure.## # Drug# encapsulation# in# polymeric# NPs# can# be# achieved# by# addition# of# the# compound#during#the#polymerization#process,#by#its#entrapment#during#NP#formation,# or# by# adsorption# after# the# formation# of# the# NP# upon# incubation# (76).# Entrapment# efficiency# depends# primarily# on# the# drugLreservoir# affinity,# but# also# on# the# encapsulation/incorporation#method#used.# # On# the# other# hand,# one# of# the# most# employed# strategies# to# enhance# the# circulation# time# of# polymeric# NPs# in# the# bloodstream# is# the# use#of# a# hydrophilic# coverage#or#shell#around#the#polymeric#NP#core.#The#surface#coverage#can#be#made#by# physical#adsorption#upon#NP#obtainment#or#by#covalent#bonding#to#the#polymer#chains# that# form# the# 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surrounding#medium,#that#is,#the#solvent#becomes#good#for#one#part#of#the#molecule# while#bad#for#the#other#(a#selective#solvent).#The#lyophobic#part#of#the#macromolecule# tends#to#segregate#and#autoJassociate#with#their#neighbouring#molecules#to#avoid#direct# contact# with# solvent# ones.# In# aqueous# media,# amphiphiles# usually# tend# to# form# supramolecular# spherical# colloidal# particles# termed# micelles# with# a# lyophobic# interior# (core)#and#a#lyophilic#exterior#(shell),#which#is#exposed#toward#the#bulk#solvent#phase.# Micelles#can#change#their#size#from#5#to#some#hundreds#of#nanometers#and#can#possess# different# geometries# (spheres,# wormJlike,# toroidal…)# (78).# Hydrophobic,# electrostatic,## and#Van#der#Waals#interactions#are#the#predominant#driving#forces#in#the#assembly#of# amphiphiles# in# aqueous# medium# (79).# Furthermore,# nanosized# micelles# have# polarity# gradients# from# the# highly# hydrated# corona# to# the# hydrophobic# core# (80)# and,# for# example,#can#be#used#for#solubilization#of#compounds#of#varying#polarities#by#physical# association#with#different#regions#within#the#micelles.## # One# of# the# most# important# types# of# nanosized# micellar# delivery# systems# are# made#of#amphiphilic#polymers#(81J85).#Polymers#are#macromolecules#composed#by#the# repetition# of# the# same# structural# unit,# known# as# monomer.# Basically,# they# are# hydrocarbon#chains#where#the#covalent#bonds#between#C,#H#and#O#atoms#constitute#the# main# polymeric# chain# structure# or# backbone.# The# backbone# can# be# flexible# and# may# adopt#different#spatial#configurations#provided#that#polymer#molecules#are#dynamic#in# nature,#may#continuously#deform,#and#are#able#to#return#their#original#shape#in#solution.# Other#bonds#present#in#polymeric#molecules#are#weak#bonds,#which#can#be#classified#as# nonJpolar# and# polar# (hydrophobic# and# hydrophilic# in# aqueous# solution).# Hydrophilic# bonds#are#formed,#for#example,#by#Van#der#Waals#interactions#and#hydrogen#bonding.## Hydrophobic# bonds# are# formed# by# repulsion# of# the# hydrophobic# counterparts# and# subsequent#mutual#interaction#regarding# the#polar#solvent#molecules.# Figure#1#shows# the# chemical# structure# of# some# monomers# commonly# found# in# many# wellJknown# polymers.# Glucose# is# a# monosaccharide# found# in# plants# along# with# fructose# and# galactose,#being#the#starting#monomer#for#the#polymerization#of#starch#and#cellulose.# Lysine#is#one#of#the#twenty#essential#amino#acids,#which#are#the#basic#structural#units#for# protein#formation.#Both#monomers#are#the#constitutive#parts#of#these#natural#polymers.# Conversely,#ethylene#is#a#natural#molecule#obtained#from#petrol#and#used#as#a#monomer# to# polymerize# polyethylene,# which# is# one# of# the# most# worldwide# extended# plastics.# 24# Although#ethylene#is#natural,#the#subsequent#manufacturing#and#processing#processes# make#the#produced#polymer#to#be#considered#as#synthetic.## # Name# Glucose# Lysine# Ethylene# Molecular# structure# # # # # # Chemical# formula# C6H12O6# C6H14N2O2# C3H4# Mw# (g/mol)# 180.16# 146.19# 28.05# # Figure- 3.# Chemical# structures# of# some# common# monomers# used# in# polymerization# processes#and#their#associated#molecular#weights#(Mw).# # # 1.3.1-----Block-copolymers- # #Amphiphilic( polymers# are# those# polymers# that# possess# lyophobic# and# lyophilic# monomers#or#chains#(hydrophobic#and#hydrophilic#in#water)#in#their#molecular#structure.# As# a# consequence,# they# can# aggregate# in# a# selective# solvent,# being# this# process# dependent#on#the#solvent#employed#as#well#as#the#polymer#concentration,#temperature,# the# presence# of# additives,# etc# (86).# Some# employed# monomers# can# posses# electrical# charge,#conferring# the#polymer# a#polar# character#that#makes#them#waterJsoluble,#but# this#also#enhances#their#dependence#with#the#medium#properties#(pH,#ionic#strength…)# as#in#the#case,#for#example,#of#DNA#and#proteins#(87).#Neutral#polymers#are#those#that# do# not# bear# electrical# charges# in# their# structure,# being# composed# of#nonJcharged# monomers# as,# for# example,# ethylene# oxide# (EO).# Aggregation# properties# for# neutral# polymers#depend#mainly#on#their#affinity#regarding#the#solvent#used#for#their#dissolution.# As#mentioned#previously,#amphiphilic#polymers#posses#one#soluble#part#in#the#chosen# solvent,#while#the#other#tend#to#evade#it;#in#aqueous#solution#this#behaviour#is#called#the# hydrophobic#effect.## # ( Block(copolymers#are#those#polymers#formed#by#two#or#more#blocks,#each#block# formed#by#the#covalent#bonding#of#the#same#repeating#monomer.#They#are#amphiphilic# in#nature#because#of#the#different#affinity#of#the#monomers#that#constitute#each#block# regarding# the# solvent# (88).# Block# copolymers# exhibit# those# aggregation# properties# 25# characteristics#of#amphiphilic#polymers#and#will#be#the#main#focus#of#the#present#PhD# work.## # There# exists# a# critical# concentration# range# where# all# amphiphilic# polymers,# and# hence,# amphiphilic# block# copolymers,# change# their# state# from# singly# dispersed# molecules# in# solution# to# micellar# aggregates# denoted# as# the# critical# micellar# concentration#(CMC).#Some#block#copolymers#have#also#the#ability#to#form#micelles#by# increasing#the#temperature#while#keeping#constant#the#copolymer#concentration;#this# temperature# is# known# as# the# critical# micellar# temperature# (CMT)# (see# Figure#3).# Both# CMC#and#CMT#depend#on#the#nature#and#length#of#the#copolymer#blocks,#solvent#nature,# presence# of# additives,# and# temperature# much# in# the# same# way# as# small# amphiphilic# molecules#do.#TemperatureJdependent#amphiphilic#polymers#also#exhibit#a#low#critical# solubility# temperature,# that# appears# when# hydrophilic# bonds# start# to# break# and# hydrophobic# ones# become# stronger# and,# as# a# consequence,# water# molecules# are# expulsed#from#the#micelle#interior.#Besides,#some#monomers#are#sensible#to#changes#in# the# surrounding# environment# (pH,# presence# of# additives,# temperature…)# modifying# their# solution# behaviour# as# a# consequence# of# variations# in# their# intraJ# and# intermolecular#interactions.## # # Ethylene# oxide# is# one# of# the# most,# if# not# the# most,# hydrophilic# unit# base# to# construct# amphiphilic# block# copolymers.# Common# hydrophobic# counterparts# are# ethylene,#styrene,#vinyl#chloride,#acrylonitrile,#methyl#methacrylate...#(89).#The#relative# hydrophobicity# ratio# of# some# different# hydrophobic# blocks# commonly# used# in# pharmaceutical#applications#has#been#studied#previously#in#terms#of#the#critical#micelle# concentration# value# of# the# obtained# block# copolymers# being#1:4:5:6:10:12:12:15# for# PO:L:C:BO:VL:CL:SO:G,#where#PO#denotes#propylene#oxide,#L#lactide,#C##methylene,#BO# butylene#oxide,#VL#valerolactone,#CL#caprolactone,#SO#styrene#oxide#and#G#glycidyl#ether,# respectively# (90,91).# Usually,# the# hydrophobic# character# of# the# polymer# can# be# increased# by# using# more# hydrophobic# monomers# or# increasing# the# hydrophobic/hydrophilic#block#ratio#(90,91).## # 1.3.2- -Block-copolymers-classification- # The#first# classification#for#polymers#which#can#be#established#is#based# on#their# origin:# natural# or# synthetic# ones.# Other# polymer# classifications# are# related# to# the# polymer# chemical# structure,# their# composition# or# spatial# order.# The# simplest# polymer# structure#is#the#homopolymer#formed#by#the#repetition#of#only#one#kind#of#monomer.# Copolymers# are# those# polymers# formed# by# two# or# more# different# monomers.# If# the# polymer#has#two#or#three#blocks#(each#block#formed#by#several#repetitive#monomers)#is# called#diblock#and#triblock#copolymer,#respectively.#Triblock#copolymers#can#be#formed# 32# 91.# Attwood,# D.;# # Booth,# C.;# Yeates,# S.G.;# Chaibundit,# C.;# Ricardo,# N.M.P.S.# Int.# J.# Pharm.#2007,#345,#35.# 92.# Hamley,# I.W.,# Block' Copolymers' in' Solution:' Fundamentals' and' Applications.# 2005,#Chichester,#England:#John#Wiley#&#Sons.# 93.# Yang,#Y.JW.;#Yang,#Z.;#Zhou,#Z.JK.;#Attwood,#D.;#Booth,#C.#Macromolecules,#1996,# 29,#670.# 94.# Nace,#V.M.,#Nonionic'Surfactants:'Polyoxyalkylene'Block'Copolymers.#Surfactant# Science#Series.#1996,#New#York:#Marcel#Dekker.# 95.# Booth,#C.;Attwood,#D.#Macromol.#Rapid#Commun.#2000,#21,#501.# 96.# Chiappetta,#D.A.;#Sosnik,#A.#Eur.#J.#Pharm.#Biopharm.#2007,#66,#303.# 97.# Croy,# S.R.;# Kwon,#G.S;#Polymeric' Micelles' for' Drug' Delivery.#Curr.# Pharm.# Des.# 2006,#12,#4669.# 98.# Hurter,#P.N.;#Hatton,#T.A.#Langmuir,#1992,#8,#1291.# 99.# Kabanov,#A.V.;#Alakhov,#V.Y.#Crit.#Rev.#Ther.#Drug#Carrier#Syst.#2002,#19,#1.# 100.# Tarcha#,#P.J.#Polymers'for'Controlled'Drug'Delivery.#1991,##CRC#Press,#Boca#Raton.# 101.# Hadjichristidis,#N.;#Pispas,#S.;#Floudas,#G.#Block'Copolymers'Synthetic'Strategies,' Physical'Properties'and'Applications.##2003,##John#Wiley#&#Sons.# 102.# Heidel,#J.;#Davis,#M.#Pharm.#Res.#2011,#28,#187.# 103.# Frisch,#H.L.''J.#Appl.#Polym.#Sci.#1970,#14,#1657.# 104.# De#Jong#W.H.;#Hagens,#W.I.;#Krystek,#P.;#Burger,#M.C.;#Sips,#A.#J.#A.#M.;#Geertsma,# R.#E.#Biomaterials#2008,##29,#1912.# 105.# Neuberger,# T.;# Schoepf,# B.;# Hofmann,# H.;# Hofmann,# M.;# Von# Rechenberg,# B.#J.# Magn.#Magn.#Mater.#2005,#293,#483.# 106.# Batrakova,#E.V.;#Li,#Shu;#A.;#Valery#Y.;#Miller,#D.W.;#Kabanov,#A.V.#J.#Pharmacol.# Exp.#Ther.#2003,#304,##845.# 107.# http://worldaccount.basf.com/wa/Startpage# 108.# Krupka,#T.M.;##Exner,#A.A.#Int.#J.#Hyperthermia,#2011,#27,#663.# 109.# Wei,#Z.;#Hao,#J.;#Yuan,#S.;#Li,#Y.;#Wu,#J.;#Sha,#X.;#Fang,#X.#Int.#J.#Pharm.#2009,#376,# 176.# 110.# http://www.supratek.com/rd/publications# 111.# Yu,#G.JE.;#Altinok,#H.;#Nixon,#S.#K.;#Booth,#C.;#Alexandridis,#P.;#Hatton,#T.#A.#Eur.# Polym.#J.#1997,#33,#673.# # # ! ! CHAPTER!2! ! EOmSOnEOm!COPOLYMERS! AS!NANOCARRIERS!! OF!HYDROPHOBIC!DRUGS! ! ! ! ! ! ! ! 34! 2.1!AIM!OF!THE!WORK!! ! ! Styrene! oxide! (PSO)?based! block! copolymers! are! of! particular! interest! as! a! consequence!of!their!wide!availability!of!architectures!and!molecular!weights!(1),!their! ability! to! self?assemble! at! very! low! concentrations! into! micelles! of! various! shapes! depending!on!relative!block!lengths!(2,3),!and!their!low!glass!transition!temperatures!(ca.! 40!ºC),!which!enables!the!incorporation!of!drugs!at!temperatures!that!are!compatible! with! thermolabile! agents! (4,5).! Despite! the! micellization! process! and! solubilisation! ability! of! some! PSO?based! block! copolymers! have! been! previously! studied! (5?11)! and! some!of!these!copolymers!are!already!commercially!available!from!Goldschmidt!AG,!as! far!as!we!know!only!one!study!about!diblock!copolymers!of!styrene!oxide!and!ethylene! oxide!(PSO?PEO)!copolymer!micelles!as!carriers!of!an!anticancer!drug!(docetaxel)!against! tumour!prostate!cancer!cells!has!been!reported!(12).!Moreover,!in!contrast!to!the!well? demonstrated! inhibitory! activity! of! several! Pluronics®! block! copolymers! against! drug! efflux! transporters! overexpressed! in! MDR! cells! and! the! subsequent! bioavailability! enhancement!of!their!substrates!in!different!tissues!and!organs!(13?15),!no!reports!are! available! about! the! potential! capabilities! of! PSO?based! copolymers! as! efflux?pump! inhibitors.!Therefore,!the!potential!capability!of!this!class!of!copolymers!as!potential!P? glycoprotein!efflux!pump!inhibitors!to!enhance!drug!accumulation!in!the!NCI?ADR?RES! which! overexpressed! P?glycoprotein! is! tested! in! this! work! for! the! first! time,! and! compared!to!that!observed!for!other!types!of!PEO?based!block!copolymers,!especially! Pluronics®.! ! 2.1.1!!Aim!of!the!work! ! ! In!the!present!work,!we!report!on!the!synthesis!and!characterization!of!the!self? assembly! properties! of! two! new! triblock! PEO?PSO! copolymers! (see! Figure! 2),! EO33SO14EO33!and!EO38SO10EO38,!where!the!subscripts!denotes!the!block!lengths.! ! 2.1.2!Methodology! ! !Physico?chemical! characterization! was! performed! by! means! of! fluorescence! spectroscopy,! light! scattering,! transmission! electron! microscopy! (TEM),! confocal! microscopy! and! rheometry.! The! triblock! structure! was! selected! for! comparison! with! those!data!previously!obtained!for!other!triblock!PEO?PPO?based!block!copolymers.! ! 35! ! ! Figure!1.!Constituent!monomers!of!PEO?PSO?PEO!block!copolymers." ! ! ! The! aim! of! this! work! is! to! assess! the! ability! of! copolymers! EO33SO14EO33! and! EO38SO10!EO38!to!dissolve!and!chemically!protect!different!hydrophobic!drugs,!evaluating:! a)!the!colloidal!stability!of!the!drug?loaded!polymer!micelles,!b)!the!drug!release!profile,! c)! the! safety! of! the! polymeric! nanocarrier,! and! d)! the! in" vitro! efficacy! as! an! antifungal/antitumor!formulation.!The!EO/SO!ratio!(∼1.5)!and!the!block!lengths!of!both! block!copolymers!were!selected!with!the!objective!of!attaining!an!optimal!compromise! between! chain! solubility,! micelle! formation! ability,! and! core! size! that! leads! to! an! enhanced!drug!solubility![16].!Previous!studies!have!shown!that!shorter!PEO!blocks,!as! those! of! EO10SO10EO10!copolymer,!lead! to! reduced! polymeric! chain! solubility! (4),! and! longer!hydrophobic!blocks!compromise!copolymer!solubility!(3,!17).!Conversely,!longer! PEO! blocks! and! shorter! PSO! blocks! may! lead! to! larger! cmc! with! i)! the! subsequent! increase!in!material!expense!to!form!micelles!able!to!solubilise!the!required!amount!of! drug,!and!ii)!the!existence!of!possible!adverse!side!effects!due!to!an!excess!of!polymer! accumulated! on! cells/tissues! (1).! Triblock! copolymers! EO33SO14EO33! and! EO38SO10EO38! may! also! enable! to! elucidate! the! possible! influence! of! copolymer! architecture! in! the! solubilization! and! controlled! release! abilities! by! comparison! with! experimental! data! previously!reported!by!Elsabathy!et"al.!(12)!for!PEO?PSO!diblock!copolymers.!! ! ! In! summary,! in! this! work! two! triblock! copolymers! have! been! designed! and! 36! synthesized! to! improve! their! drug! solubility! properties! in! the! micellar! range.! These! copolymers! were! physico?chemically! characterized! elucidating! their! structural! composition,! block! length! and! purity! (by! means! of! the! polydispersity! index).! Their! behaviour!in!aqueous!solution!was!tested!in!a!broad!range!of!concentrations!in!order!to! clearly!define!the!micellar/gel!regions!and!the!properties!that!exhibit!each!copolymer!in! each!state.!In!this!regard,!micellar!parameters!revealed!the!shape,!size!and!aggregation! number!of!the!formed!polymeric!micelles,!while!the!analysis!of!physical!gels!reported! valuable!information!on!their!rheological!properties!and!enable!the!construction!of!their! corresponding! phase! diagrams.! Once! the! physico?chemical! behaviour! of! the! block! copolymers! polymers! was! determined,! their! capability! as! drug! reservoirs! for! two! different!drugs!were!studied,!the!antifungal!compound!griseofulvin!and!the!anticancer! drug!doxorubicin.!Spherical!micelles!were!obtained!in!aqueous!solution,!having!a!highly! hydrophobic!core!and!a!PEO!shell.!The!PEO!shell!is!expected!to!minimise!the!recognition! by! the! RES! in! the! blood! stream,! while! the! PSO! core! is! expected! to! increase! the! drug! entrapment!respect!Pluronic!micelles!owned!to!the!higher!affinity!to!hydrophobic!drugs.! The!entrapment!efficiency!for!these!drugs!was!tested!for!both!copolymers,!as!well!as! the!drug!loading!capacity.!Colloidal!stability!and!release!rates!of!the!cargo!from!micelles! into! different! buffer! media! were! also! performed! in! vitro,! to! mimic! the! different! pH! medium!the!system!should!go!through.!Stability!test!should!permit!to!elucidate!the!drug! release! mechanism.! Cellular! toxicity! and! bioavailability! were! studied! for! empty! and! drug?loaded! micelles! in! cell! culture.! Finally,! the! capability! of! both! copolymers! as! potential! P?glycoprotein! efflux! pump! inhibitors! to! enhance! drug! accumulation! in! an! ovarian!MDR!NCI?ADR/RES!cell!line!was!tested!and!compared!to!that!observed!for!other! different!block!copolymers.!! ! 2.1.3!References! ! ! 1.! Booth,!C.;!Attwood,!D.;!Price,!C.!Phys.!Chem.!Chem.!Phys.!2006,!!8,!3612.! 2.!Castro,! E.;! Barbosa! S.;! Juarez! J.;! Taboada! P.;! Katime! I.! A;! Mosquera! V.! J.! Phys.! Chem.!B,!2008,!112,!5296.! 3.!Juárez,(J.;!Taboada,!P.;!Valdez,!M.A.;!Mosquera,!V.!Langmuir,!2008,!24,!!7107.! 4.! Crothers,!M.;!!Zhou,!Z.;!Ricardo,!N.M.P.S.;!Yang,!Z.;!Taboada,!P.;!Chaibundit,!C.;! Attwood,!D.;!Booth,!C.!Int.!J.!Pharm.!2005,!293,!91.! 5.!Zhou,!N.;!Lodge,!T.P.;!Bates,!F.S.!J.!Phys.!Chem.!B,!2006,!110,!3979.! 6.! Kabanov,!A.V.;!Alakhov,!V.Y.!Crit.!Rev.!Ther.!Drug!Carrier!Syst.!2002,!19,!1.! 7.! Ribeiro,!M.E.N.P.;!Vieira,!I.G.P.;!Cavalcante,!I.M.;!Ricardo,!N.M.P.S.;!Attwood,!D.;! Yeates,!S.G.;!Booth,!C.!Int.!J.!Pharm.!2009,!378,!!211.! 8.! Yang,!Z.;!Crothers,!M.;!Ricardo,!N.M.P.S.;!Chaibundit,!C.;!Taboada,!P.;!Mosquera,! V.;!Kelarakis,!A.s;!Havredaki,!V.;!Martini,!L.;!Valder,!C.!Langmuir!2003,!19,!!943.! 9.! Yang,! Z.;! Crothers,! M.;! Attwood,! D.;! Collett,! J.H.;! Ricardo,! N.M.P.S.;! Martini,! L.! G.A.;!Booth,!C.!J.!Colloid!Interface!Sci.!2003,!263,!!312! 37! 10.! Chaibundit,! C.;! Ricardo,! N.M.P.S.;! Crothers,! M.;! Booth,! C.! Langmuir! 2002,! 18,! 4277.! 11.! Crothers,!M.;!Ricardo,!N.M.P.S.;!Heatley,!F.;!Nixon,!S.K.;!Attwood,!D.;!Booth,!C.! Int.!J.!Pharm.!2008,!358,!!303.! 12.! Elsabahy,!M.;!Perron,!M.?E.;!Bertrand,!N.;!Yu,!G.;!Leroux,!J.?C.!Biomacromolecules,! 2007,!!8,!2250.! 13.! Batrakova,!E.V.;!Kabanov,!A.V.!J.!Controlled!Release,!2008,!130,!98.! 14.! Kabanov,!A.V.;!Batrakova,!E.V;!Alakhov,!V.Y.!Adv.!Drug!Deliv.!Rev.!2002,!54,!759.! 15.! Alvarez?Lorenzo,!C.;!!Rey?Rico,!A.;!Brea,!J.;!Loza,!M.!I.;!Concheiro,!A.;!Sosnik,!A.! Nanomedicine,!2010,!5,!1371.! 16.! Yamamoto,! Y.;! Nagasaki,! Y.;! Kato,! Y.;! Sugiyama,! Y.;! Kataoka,! K.! J.! Controlled! Release,!2001,!77,!!27.! 17.! Taboada,! P.;! Velasquez,! G.;! Barbosa,! S.;! Castelletto,! V.;! Nixon,! S.! K.;! Yang,! Z.;! Heatley,!F.;!Hamley,!I.W.;!Ashford,!M.;!Mosquera,!V.!Langmuir!2005,!!21,!5263.! ! ! ! 39 2.2#POLY# (ETHYLENE# OXIDE)# 1# POLY# (STYRENE# OXIDE)#–#POLY(ETHYLENE#OXIDE)#COPOLYMERS:# MICELLIZATION,# DRUG# SOLUBILISATION# AND# GELLING#FEATURES# # ! # 2.2.1 Abstract! ! Two# new# poly(ethylene# oxide)1poly(styrene# oxide)# triblock# copolymers# (PEO1 PSO1PEO)#with#optimised#block#lengths#selected#on#the#basis#of#previous#studies#were# synthesized# with# the# aim# of# achieving# a# maximal# solubilisation# ability# and# a# suitable# sustained# release,# while# keeping# very# low# material# expense# and# excellent# aqueous# copolymer# solubility.# The# self1assembling# and# gelling# properties# of# these# copolymers# were# characterized# by# means# of# light# scattering,# fluorescence# spectroscopy,# transmission# electron# microscopy# and# rheometry.# Both# copolymers# formed# spherical# micelles# (12114# nm)# at# very# low# concentrations.# At# larger# concentration# (># 25# wt%),# copolymer#solutions#showed#a#rich#phase#behavior,#with#the#appearance#of#two#types#of# rheologically# active# (more# viscous)# fluids# and# of# physical# gels# depending# on# solution# temperature#and#concentration.#The#copolymer#behaved#notably#different#despite#their# relatively# similar# block# lengths.# The# ability# of# the# polymeric# micellar# solutions# to# solubilize#the#antifungal#drug#griseofulvin#was#evaluated#and#compared#to#that#reported# for#other#structurally1related#block#copolymers.#Drug#solubilization#values#up#to#55#mg#g1 1# were# achieved,# which# are# greater# than# those# obtained# by# previously# analyzed# poly(ethylene# oxide)1poly(styrene# oxide),# poly(ethylene# oxide)1poly(butylene# oxide),# and#poly(ethylene#oxide)1poly(propylene#oxide)#block#copolymers.#The#results#indicate# that# the# selected# SO/EO# ratio# and# copolymer# block# lengths# were# optimal# for# simultaneously#achieving#low#critical#micelle#concentrations#(cmc)#values#and#large#drug# encapsulation# ability.# The# amount# of# drug# released# from# the# polymeric# micelles# was# larger#at#pH#7.4#than#at#acidic#conditions,#although#still#sustained#over#1#day.# # 2.2.2 Introduction! ! Advances# in# materials# science# and# nanotechnology# offer# novel# approaches# to# address# formulation# issues# and# to# regulate# drug# biodistribution# and# release# patterns# (1,2).#Block#or#graft1copolymers#consisting#of#hydrophilic#and#lipophilic#domains#are#able# to#form#polymeric#micelles#and#nanocompartmentalized#particles,#via#self1assembly#in# an#aqueous#environment,#that#exhibit#a#long#circulation#half1life#due#to#the#stabilization# 40 provided#by#the#hydrophilic#shell.#These#core1shell1type#nanostructures#are#particularly# suitable#to#host#poorly1soluble#drugs#and#to#target#them#to#the#required#tissue#or#cells# (319).#As#a#consequence,#the#local#drug#bioavailability#and#the#safety#of#the#treatment# are#improved#(10112).# # Probably,# the# most# widely# studied# amphiphilic# triblock# copolymers# are# those# composed# of# hydrophilic# PEO# blocks# and# hydrophobic# propylene# oxide# (PPO)# blocks,# which#can# be#classified#in#two#families:#the#linear#poloxamers#(Pluronics®),#and#the#X1 shaped# poloxamines# (Tetronic®)# (13116).# The# reasons# for# their# popularity# can# be# summarized#in:#i)#commercial#availability#in#a#very#broad#range#of#compositions#(i.e.,#a# wide# variety# of# molecular# weights,# block# lengths# and# PEO/PPO# ratios);# ii)# proven# solubilization# capacity# and# sustained# drug# release;# iii)# high# biocompatibility# of# most# varieties;#iv)#enhancement#of#drug#transport#across#cellular#barriers;#and#v)#approval#of# some# varieties# by# US# FDA# and# EMA# to# be# used# in# pharmaceutical# formulations# and# medical# devices# (13116).# Nevertheless,# PEO–PPO# block# copolymers# still# present# a# number#of#limitations#that#could#curtail#their#application,#such#as#i)#limited#stability#of# the# self1assembled# nanostructures# upon# dilution# in# the# bloodstream,# particularly# for# derivatives#with#high#EO/PO#ratios,#ii)#incomplete#micellization#of#the#unimers,#and#iii)# variability# from# batch# to# batch# in# micellar# sizes,# drug# delivery# capacities# and# release# profiles.## To# overcome# some# of# these# limitations,# more# hydrophobic# block# copolymer# counterparts#with#similar#architecture,#but#with#the#PPO#segment#replaced#by#a#more# hydrophobic# one,# such# as# poly(butylene# oxide)# (PBO),# poly(styrene# oxide)# (PSO)# or# phenylglycidyl#ether#(PG),#have#been#developed#by#the#Attwood#and#Booth´s#group#in# collaboration# with# us# during# last# years# (17121).# Polystyrene# oxide1based# block# copolymers#are#of#particular#interest#due#to#i)#their#ability#to#self1assemble#at#very#low# concentrations# into# micelles# with# improved# solubilization# ability# and# stability# (22123)# and#ii)#the#low#glass#transition#temperatures#(ca.#40#ºC)#of#the#core1forming#block,#which# enables# the# incorporation# of# drugs# at# temperatures# that# are# compatible# with# termolabile# agents# (22,24125).#In# general,# triblock# PSO1based# block# copolymers# show# larger# solubilization# capacity# of# hydrophobic# drugs#if# compared# to# commercially# available# Pluronics®# or# Tetronic®# copolymers# thanks# to# their# more# hydrophobic# cores,# although# such# an# enhancement# depends# on# copolymer# structure,# block# length# ratios,# micellar#shape#and#drug#affinity#for#the#block1forming#micellar#core#(18,22123).#Some#of# these#factors#are#also#key#in#providing#suitable#polymeric#chain#solubility#and#stability;#in# fact,# when# designing# styrene1oxide# copolymers# for# enhancing#drug# solubility# by# increasing/decreasing#the#length#of#the#hydrophobic/hydrophilic#block,#the#copolymer# chain#solubility,#the#micelle#stability#and/or#the#drug#solubilization#capacity#have#been# found#to#be#compromised#(18,24).#Shorter#EO#and#longer#SO#block#lengths#typically#have# led# to# reduced# polymeric# chain# solubility,# whereas# copolymers# with# longer# EO# blocks# 41 and/or#very#short#SO#block#self1assemble#at#high#concentrations#and#form#micelles#with# lower#drug#entrapment#abilities#(20).# # In#the#present#work#we#report#on#the#synthesis,#the#characterization#of#the#self1 assembling# properties,# and# the# drug# solubilization# and# release# profiles# of# two# new# triblock#PEO1PSO#copolymers,#EO33SO14EO33#and#EO38SO10EO38#(the#subscripts#denoting# the# block# lengths)# using# fluorescence# spectroscopy,# light# scattering,# transmission# electron#microscopy#(TEM)#and#rheometry.#The#main#goals#of#the#present#work#were#to# target# optimized# block# lengths# and# hydrophilic/hydrophobic# block# molar# ratios# of# the# copolymers#on#the# basis#of#previous#studies# to#simultaneously#achieve# a#compromise# between# polymer# chain# solubility# and# micelle# formation# at# very# low# copolymer# concentrations;# and# to# study# the# effect# of# subtle# differences# on# the# copolymer# block# lengths# to# obtain# a# micellar# core# with# a# suitable# size# for# hosting# great# amounts# of# a# poorly1soluble#drug#such#as#the#antifungal#griseofulvin,#used#as#a#model#for#comparison# purposes# with# other# block# copolymer# structures.# The# micellar# systems# based# on# EO33SO14EO33#and#EO38SO10EO38#block#copolymers#largely#reach#these#goals,#improving# griseofulvin#encapsulation#and#release.#Hence,#these#results#prove#the#potential#benefits# of# this# class# of# copolymers# as# components# of# drug# delivery# systems# improving# the# performance# of# Pluronic# and# Tetronic# block# copolymers,# while# exhibiting# the# biocompatibility,#cytocompatibility#and#capacity#of#inhibiting#efflux#pumps#of#the#latter# (26).## # 2.2.3 Experimental!section! ! 2.2.3.1 !!!Materials! ! #EO33SO14EO33# and# EO38SO10EO38# copolymers# were# synthesized# as# previously# described#(27,28).#Briefly,#high#vacuum#and#ampule#techniques#were#used#to#eliminate# unwanted# moisture.# Initiation# of# the# bifunctional# precursor# was# potassium# hydroxide# and#1,21butanediol#partly#in#the#form#of#its#potassium#salt.#The#mole#ratio#OH/OK#was# ∼9,# this# being# chosen# to# achieve# a# suitable# polymerization# rate.# The# monomers# were# distilled#and#dried#immediately#before#use.#Styrene#oxide#was#added#to#the#ampule#by# syringe,# and# for# the# second# stage# of# polymerization,# ethylene# oxide# was# distilled# through#the#vacuum#line.#The#polymerization#of#styrene#oxide#at#85#°C#was#slow,#taking# as#long#as#8#weeks.#Weight1averaged#(Mw)#to#number1averaged#(Mn)#molecular#weight# ratios#were#determined#at#25ºC#using#a#Waters#gel#permeation#chromatography#(GPC)# system# equipped# with# a# 1515# isocratic# pump# and# a# 2410# refractive# index# detector# (Waters,#Milford,#MA).#Chloroform#was#used#as#the#eluent,#and#monodisperse#PEO#was# employed#as#standard.#Mn$values#were#estimated#from$1H#NMR#spectra#recorded#on#a# Bruker# ARX400# spectrometer# (Bruker,# Milton,# ON,# Canada)# in# deuterated# chloroform.# Table# 1# summarises# the# molecular# characteristics# of# both# copolymers.# Water# was# 42 double# distilled# and# degassed# before# use.# Pyrene# and# griseofulvin# were# from# Sigma1 Aldrich.## # Table!1.!Molecular#characteristics#of#the#copolymers.$ Polymer# Mn#/g#mol11# (NMR)# wt#%#SO# (NMR)# Mw/Mn# (GPC)# Mw#/g#mol11# EO33SO14EO33# 4790# 40.0# 1.01# 4850# EO38SO10EO38# 5055# 34.1# 1.02# 5130# # Estimated#uncertainty:#Mn#to#±3#%;#wt%#S#to#±1#%,#Mw/Mn#to#±0.01.#Mw#calculated#from#Mn#and# Mw/Mn.# # 2.2.3.2 Methods! ! a. Characterization$of$block$copolymer$micelles$ $ a1.$ Fluorescence$ measurements:$Values# of# cmc# were# obtained# from# pyrene# fluorescence# measurements# at# 37# ±# 0.1# ºC# (Cary# Eclipse# fluorescence# spectrophotometer,#Agilent.,#Germany)#as#described#by#Lee#et$al.$(29).#Stock#solutions# were#prepared#by#dissolving#the#copolymers#in#water#for#24#h#before#being#diluted#to# the#desired#concentrations#within#the#range#1150#g# dm13.#Pyrene#dissolved#in#acetone# was#added#to#the#copolymer#solution#and,#after#acetone#evaporation,#was#allowed#for# equilibration#during#24#h.#The#final#copolymer#solution#contained#3#x#1017#M#pyrene.#The# fluorescence# spectrum# (λexc# =# 335# nm)# was# the# average# of# three# scans# and# was# corrected#for#scattering#using#an#equivalent#blank#solution#before#determining#the#ratio$ I1/I3#of#the#first#and#third#vibronic#peaks.#Reproducibility#was#better#than#2#%.#$ a2.$Dynamic$and$static$light$scattering$measurements:#DLS#and#SLS#intensities#were# measured#at#37#°C#by#means#of#an#ALV15000F#(ALV1GmbH,#Germany)#instrument#with# vertically# polarized# incident# light# (λ# =# 488# nm)# supplied# by# a# diode1# pumped# Nd:YAG# solid1state#laser#(Coherent#Inc.,#CA,#USA)#and#operated#at#2#W,#and#combined#with#an# ALV# SP186# digital# correlator# with# a# sampling# time# of# 25# ns# to# 100# ms# (for# DLS).# The# intensity# scale# was# calibrated# against# scattering# from# toluene.# Measurements# were# made#at#a# scattering#angle# θ #=#90°# to#the#incident#beam,# as#appropriate#for# particles# smaller# than# the# light# wavelength.# Solutions# were# filtered# through# Millipore# Millex# filters# (Triton# free,# 0.22# µm# porosity)# directly# into# cleaned# scattering# cells# and# let# to# equilibrate# at# 37ºC# for# 30# min# before# measurement.# Experiment# duration# was# in# the# range#5110#min,#and#each#experiment#was#repeated#at#least#two#times.#The#correlation# functions#from#DLS#runs#were#analyzed#by#the#CONTIN#method#to#obtain#the#intensity# distributions# of# decay# rates# ( Γ )# (30).# From# the# decay# rate# distributions# the# apparent# diffusion# coefficients# (Dapp# =# Γ/q2,# q$ =# (4πns/λ)sin(θ/2))# were# derived,# being# ns# the# refractive#index#of#solvent.#Values#of#the#apparent#hydrodynamic#radius#(rh,app,#radius#of# 49 010 20 30 40 50 0 20 40 60 80 100 SOFT-GEL Temperature (ºC) concentration (wt.%) HARD-GEL SOL b) 010 20 30 40 50 0 20 40 60 80 100 SOFT-GEL Temperature (ºC) concentration (wt.%) SOL HARD-GEL a) 010 20 30 40 50 0 20 40 60 80 100 SOFT-GEL Temperature (ºC) C (wt.%) SOL HARD-GEL a) 010 20 30 40 50 0 20 40 60 80 100 SOFT-GEL Temperature (ºC) C (wt.%) SOL HARD-GEL a) # Figure! 3:! Gel# boundaries# of# aqueous# micellar# solutions# of# a)# EO33SO14EO33# and# b)# EO38SO10EO38.# # As#observed#in#Figure#3a,#copolymer#EO33SO14EO33#at#concentrations#lower#than# 25#wt.%#leads#to#a#transparent#and#isotropic#sol#phase#in#the#0#to#ca.#80#ºC#temperature# range.# At# larger# concentrations,# the# gel1like# phase# is# formed,# with# an# upper# limit# temperature#of#ca.#60#ºC#for#concentrations#lower#than#40#wt.%#which#increases#up#to# ca.#90#ºC#for#a#concentration#of#50#wt.%.#There#was#no#low1temperature#boundary#for# copolymer#gels#as#a#consequence#of#the#stability#of#the#polymeric#micelles#in#water#at# low#temperatures#due#to#the#water#insolubility#of#PSO#blocks,#in#agreement#with#their# very# low# standard# micellization# enthalpy# values# (28).# Also,# the# gel1viscous# fluid# spans# from# ca.# 20# wt.%# up# to# ca.# 50# wt.%,# with# their# lower# temperature# boundary# concentration1dependent#and#the#upper#temperature# limit# close#to#90# ºC.# This#upper# limit#of#the# viscous#fluid#region# reached#within#the# temperature#range#investigated# is# consistent# with# a# decrease# in# the# stability# of# the# hard# gels# of# EO33SO14EO33# as# temperature#increases,#in#contrast#to#other#block#sequences#(21,24).#At#temperatures# larger# than# 90# ºC,# the# appearance# of# a# new# sol# phase# agrees# with# the# melting# of# copolymer# chains# at# such# high# temperature.# The# phase# behavior# of# copolymer# EO38SO10EO38#is#similar#to#that#found#for#EO33SO14EO33#but#two#main#differences#were# detected:#the#viscous#fluid1gel#boundary#was#shifted#to#a#lower#concentration#and#the# viscous#fluid#region#became#very#narrow.### # #In# order# to# get# deeper# insight# on# the# rheological# behavior# of# the# present# copolymers,#temperature#scans#of#storage#and#loss#moduli#at#concentrations#below#and# above# their# critical# gel# concentration,# cgc,# (Figures# 4# and# 5)# were# used# to# verify# and# complete#the#phase#diagrams.#A#cgc$of#19#wt%#was#estimated#for#both#copolymers#by# means# of# the# expression# cgc# =# 102ρaφc/δt,# where# φc# =# 0.68# is# the# volume# fraction# of# spherical# micelles# packed# in# a# body1centered# structure.# The# rheological# behavior# of# EO33SO14EO33#and#EO38SO10EO38#copolymers#at#concentrations#below#20#wt%#exhibits#a# 50 predominant# viscous# behavior# (G´´># G´)# that# is# a# characteristic# feature# of# a# sol# phase# (Figure#4a#and#Figure#S3).#At#a#temperature#of#ca.#70#°C,#both#G’#and#G”#increased#in# about#two#decades.#This#effect#may#be#due#to#an#increment#in#the#number#of#micelles#in# solution# due#the#insolubility#of#the# PSO# chains.# The# sample# at#20#wt.%#concentration# exhibited# a# similar# trend# than# that# depicted# for# the# 10# wt.%# solution# at# a# lower# temperature.#At#higher#temperatures#two#transitions#were#observed:#the#first#at#78#ºC,# corresponding#to#the#appearance#of#a#sol1viscous#fluid#transition,#and#the#second,#at#ca.# 90#ºC,#due#to#the#melting#of#the#viscous#fluid#(Figure#4b).#This#area#of#the#phase#diagram# is#rather#narrower#for#EO38SO10EO38#(Figure#S3).#A#more#viscous#fluid#developed#from#a# sol# solution# should# originate# from# weak# attractions# of# spherical# micelles# in# water# at# elevated#temperatures,#where#the#solvent#is#poorer#for#the#micelles.#The#transition#from# sol# to# viscous# fluid# may# well# occur# when# aggregates# of# spherical# micelles# well# would# reach# a# percolation# threshold# yielding# sufficient# structure# to# cause# a# characteristic# rheological#effect#(46147).#This#additional#structuration#is#more#important#in#the#case#of# EO38SO10EO38,#which#even#formed# a# gel#within#this#temperature# range#(Figure#S3# and# text#in#Supporting#Information#for#further#details).#At#25#wt%#(Figure#4c),#the#copolymer# solution#behaved#as#a#gel#below#10#ºC#due#to#the#formation#of#cubic#liquid#crystals#in#a# body#centered#structure#(bcc);#at#higher#temperatures#two#transitions#were#observed:# the#first#one#corresponding#to#a#gel1viscous#fluid#at#10#°C,#and#a#second#one#at#17#ºC#due# to# a# viscous# fluid1sol# transition.# The# former# transition# can# be# assigned# to# a# defective# version# of# the# cubic1packed# gel# as# the# temperature# increases,# i.e.,# small# structured# domains#in#an#overall#fluid#matrix#(gel1defective#viscous#fluids).#This#viscous#region#can# be#identified#in#Figure#4c#as#a#narrow#low1T#shoulder#on#the#G(T)#curve#of#the#25#wt%# solution#(or#as#a#distinct#high1T#shoulder#on#the#G(T)#curve#of#the#30#wt%#in#Figure#S3d).# Viscous#fluids#of#the#present#type#have#been#previously#identified#in#aqueous#micellar# solutions#of#a#wide#range#of#block#copolymers,#including#PEO1PPO,#(18,#47150)#and#other# PEO1PSO#block#copolymers#(21,24,51).##In#addition,#G´and#G”#increased#in#about#three# magnitude#orders#at#ca.#70#ºC#to#give#a#new#viscous#fluid#region#between#76#and#90#ºC# due# to# further# copolymer# micelle# structuration.# This# additional# structuration# is# more# important#in#the#case#of#EO38SO10EO38#(Figure#S3#and#text#in#SI#for#further#details).# # On#the#other#hand,#an#increase#in#polymer#concentration#(up#to#30#wt%.)#led#to# both#a#widening#of#the#low1temperature#gel#region#at#the#expense#of#the#gel1defective# viscous# fluid# region# as# a# consequence# of# enhanced# intermicellar# interactions;# also,# a# certain# narrowing# of# the# mild1temperature# sol# region# took# place# until# completely# disappearance# (Figure# 4d# for# a# 27# wt.%# solution# as# an# example),# whilst# the# high1T# viscous#fluid#region#emerged#at#relatively#lower#temperatures#(ca.#70#ºC,#see#Figure#4d)# and#remained#almost#invariable#in#shape.#This#is#in#contrast#to#the#behavior#observed#for# EO38SO10EO38,# for# which# the# high1T# viscous# fluid# region# was# very# narrow# within# this# copolymer# concentration# range# (Figure# S3).# At# larger# copolymer# concentrations# (># 30# wt.%),# the# attractive# intermicellar# interactions# become# much# stronger.# This# involves# 51 firstly#the#avoidance#of#the#gel#phase#disruption#in#the#temperature#range#60170ºC#(see# Figure#4d)#and,#then,#the#widening#of#the#gel#phase#at#the#expense#of#the#gel1defective# viscous# fluid# region# (see# Figure# 4e)# until# it# completely# disappears# at# 50# wt.%# for# copolymer#EO33SO14EO33#(Figure#4f#and#Figure#S3d).## # 010 20 30 40 50 60 70 80 90 100 100 101 102 103 104 105 Temperature (°C) G´, G´´ (Pa) HARD GEL SOFT-GEL SOL e) 010 20 30 40 50 60 70 80 90 100 100 101 102 103 104 SOL SOFT GEL HARD GEL G´, G´´ (Pa) Temperature (°C) f) 010 20 30 40 50 60 70 80 90 100 10-2 100 102 104 106 Temperature (°C) G´, G´´ (Pa) d) HARD GEL SOFT GEL SOL 0 10 20 30 40 50 60 70 80 90 100 10-4 10-2 100 102 104 106 SOL SOFT-GEL HARD GEL Temperature (°C) G´, G´´ (Pa) c) ! 0 10 20 30 40 50 60 70 80 90 100 10-3 10-2 10-1 100 101 SOFT-GEL SOL Temperature (°C) G´, G´´ (Pa) b) 0 10 20 30 40 50 60 70 80 0.01 0.1 1 G´, G´´ (Pa) Temperature (°C) a) SOL # ! Figure!4:#Temperature#scans#of#(#)#storage,#G´,#and#(!)#loss#moduli,#G´´,##for#a)#10#wt# %.;#b)#20#wt#%.;#c)#25#wt#%.;#d)#27#wt#%.;#e)#40#wt#%.;#and#f)#50#wt#%.#of#EO33SO14EO33.# # # # To#get#a#more#detailed#picture#about#the#rheological#behavior#of#the#observed# viscous#fluids,#frequency#sweeps#within#the#linear#viscoelastic#region#of#block#copolymer# 52 solutions#were#also#performed.#Frequency#scans#obtained#for#20#and#30#wt%.#solutions# of# copolymer# EO33SO14EO33# at# different# temperatures# are# shown# in# Figure# 5.# Similar# plots#were#obtained#for#copolymer#EO38SO10EO38#(not#shown).#The#20#wt.%#copolymer# solution#at#60#and#90#ºC#is#a#sol#(Figure#3a);#at#these#temperatures,#the#system#exhibits#a# predominant# viscous# behavior# (G’<# G”)# and# only# the# terminal# zone# is# observed,# with# increasing#values#of#G´$and#G´´#with#frequency#(see#Figure#5a,c).#In#contrast,#at#80#ºC#the# solution#behaves#as#a#viscous#fluid#(or#“soft#gel”)#and#shows#a#viscoelastic#behavior,#with# low#G´#values.#G’#and#G”#exhibit#a#crossover#at#a#characteristic#frequency#(φc)#of#2#rad/s,# which# would# correspond# to# a# Maxwell# fluid,# at# most,# showing# localized# cubic# order# (Figure# 5b);# the# reciprocal# of# the# frequency# crossover# corresponds# to# the# main# relaxation# time#of#the#system,# φc# =# 0.5s.# At# frequencies#lower#than#φc#the# rheological# behavior# is# predominantly# viscous,# and# at# higher# frequencies# the# system# exhibits# a# predominant#elastic#behavior#(G’#>#G”).#Both#moduli#increased#with#frequency#but#the# plateau#modulus#(G0)#was#not#detected#in#the#frequencies#range#studied.#This#behavior# is#a#consequence#of#the#weak#attraction#of#spherical#micelles#in#water#at#temperatures# at#which#this#is#a#poor#solvent#for#micelles,#as#commented#previously.#Furthermore,#the# 30#wt.%#solution#at#20#ºC#is#within#the#gel#region,#the#G’,#G”#crossover#shifts#to#lower# frequencies#(φc# =#0.2#rad/s)#increasing#the#predominant#elastic#behavior#in#almost#the# whole#interval#of#frequencies#studied.#G´#exhibits#a#plateau#(G0)#with#a#value#around#of# 4·∙104#Pa,#and#G´#decreases#in#around#one#magnitude#order#indicating#an#increasing#in# the# hardness# of# the# sample# with# frequency# (Figure# 5d).#The# insensitivity# of# storage# moduli# to# frequency# justifies# our# use# of# a# single# frequency# (6.28# rad/s)# in# the# temperature# scans# done# to# confirm# the# gel# boundary.# As# temperature# rises,# the# copolymer#solution#becomes#more#fluid#and#transforms#into#a#viscous#fluid,#as#shown#in# Figure#5e.#This#plot#shows#again#the#characteristic#moduli#crossover#and#a#predominant# elastic#behavior#is#observed#in# the#interval#of#frequencies#studied.# As#a#consequence,# temperatures#at#the#soft1gel/sol#boundary#are#dependent#on#the#frequency#used,#and# those#drawn#in#Figure#3#give#only#an#indication#of#the#viscoelasticity#of#the#systems.## # From# a# pharmaceutical# point# of# view,# it# is# clear# that# at# 37ºC# the# transition# between#the#sol#and#the#gel#state#can#be#achieved#through#a#tiny#change#in#copolymer# concentration# around# ca.# 25%# (see# Figure# 3).# The# present# PEO1PSO1PEO# copolymers# could#be#suitable#for#preparing#syringeable#drug#depots,#which#can#easily#flow#from#the# syringe#as#a#solution#at#a#temperature#some#degrees#above#37ºC,#but#transform#at#the# body# temperature# (for# example,# once# injected# in# the# subcutaneous# tissue)# in# a# viscoelastic#gel#that#can#sustain#drug#release.#On#the#other#hand,#one#can#envision#that#if# a#polymeric#physical#gel# is# formed# in# the#implantation#site#in# the# body,# it# can#rapidly# become# a# sol# if# the# temperature# raises# some# degrees# above# 37ºC,# as# occurs# when# a# pathological# process# is# on1going# or# if# an# external# source# of# heat# is# applied.# Such# a# behaviour# may# enable# to# trigger# drug# release# by# a# systemic# or# local# increase# in# temperature.#It#should#be#noticed#that#although#the#micelles#of#both#block#copolymers# 53 tested#are#too#large#to#be#directly#cleared#by#renal#filtration,#the#molecular#weight#of#the# unimers# is# much# below# the# urinary# threshold,# so# after# drug# release# and# micelle# destabilization#copolymer#chains#might#be#excreted#through#this#route.# # 100101102 101 102 G', G" (Pa) ω (rad/s) e) 10-2 10-1 100101102 103 104 105 ω (rad/s) G', G" (Pa) d) 100101102 10-2 10-1 100 G', G" (Pa) ω (rad/s) c) 10-1 100101102 100 101 102 ω (rad/s) G', G" (Pa) b) 10 20 30 40 10-2 10-1 100 ω (rad/s) G', G" (Pa) a) # # Figure! 5:! Frequency# scans# of# storage# (#)# and# loss# (!)# moduli# obtained# for# 20# wt.%# solutions#of#EO33SO14EO33#at#a)#60#ºC;#b)#80#ºC;#c)#90#ºC;#and#30#wt.%#at#d)#20#ºC;#and#e)# 90ºC.# # 2.2.4.3!Solubilization!capacity!studies! # In#order#to#establish# the# solubilization# capability# of#the#present#copolymers# as# drug# carriers,# encapsulation# experiments# were# carried# out# employing# the# antifungal# drug#griseofulvin.#This#drug#is#commonly#used#as#a#model#for#solubilization#assays#and,# thus,#it#was#chosen#in#order#to#compare#the#solubilization#ability#of#EO33SO14EO33#and# EO38SO10EO38#copolymers#(at#0.2#wt%,#far#above#the#cmc)#with#that#previously#reported# for#other#structure1related#block#copolymers.#To#investigate#the#impact#of#the#amount# 54 of# feeding# drug# used# to# prepare# the# drug1loaded# micelles,# the# entrapment# efficiency# and# drug1loaded# amount# were# determined# in# loaded# micelles# with# varying# drug/copolymer# weight# ratio.# In# general,# the# higher# the# feeding,# the# lower# the# entrapment# efficiency# was# (Table# 3)# as# a# consequence# of# the# saturation# of# the# inner# micellar#core.#Drug#precipitation#was#also#observed#when#very#large#griseofulvin#feeding# concentrations#were#used,#which#confirms#that#the#micelles#can#enhance#drug#solubility# but# up# to# a# maximum# beyond# which# further# addition# of# drug# leads# to# precipitation.# Comparing# both# copolymers,# EO33S14EO33#exhibited# a# slightly# larger# solubilization# capacity,#which#can#be#attributed#to#its#longer#hydrophobic#block#and#the#consequent# higher# affinity# of# the# hydrophobic# drugs# for# the# micelle# core.# Also,# the# solubility# per# gram#of#copolymer#(SCP,#Table#3,#maximum#uncertainty#of#±#1#mg#g11)#was#concentration1 dependent,#reaching#values#of#up#to#55#mg#g11.#These#solubility#values#are#larger#than# those# previously# reported# for# other#triblock# PSO1PEO# block# copolymer# counterparts# (22,23,39)#and#similar#to#related#PSO1PEO#diblocks#despite#the#effective#shorter#block# length#of#the#present#copolymers#due#to#looping#of#the#PSO#blocks#in#the#micellar#core# (22).#This#fact#confirms#that#the#selected#EO:SO#ratio#and#copolymer#block#lengths#were# optimal#for#efficient#drug#solubilization.#In#addition,#EO33SO14EO33#and#EO38SO10EO38#also# display#a#notably#much#larger#solubility#than#other#types#of#block#copolymers#such#as# PEO1PPO,# PEO1PBO# and# PEO1PG# ones# (18,19,23,52,53),# or# different# surfactant# and# organic# solutions# (53).$For# example,# we# noted# solubilization# increases# 101fold# larger# than# for# Pluronic# F127# [55]# and# Poloxamine# T904# (38);# these# data# highlight# the# importance# of# a# judicious# choice# of# both# block# composition# and# length# to# simultaneously# minimize# copolymer# concentration# (i.e.# material# expense)# while# maximizing#solubilization#ability#without#compromising#polymeric#micelles#stability.## # Table! 3:! Griseofulvin# loaded# amount# (D.L.),# entrapment# efficiency# (E.E.)# and# solubilisation#capacity#(SCP)#of#the#copolymers.# Feeding$ Drug/Polymer$ %$(w/w)$ EO33SO14EO33$ EO38SO10EO38$ D.L.$ %$ E.E.$ %$ Scp$ /mg$g]1$ D.L.$ %$ E.E.$ %$ Scp$ /mg$g]1$ 4# 3.8# 98.0# 39.6# 3.5# 91.4# 36.6# 10# 2.4# 24.4# 34.7# 2.2# 22.2# 32.4# 15# 2.2# 4.3# 43.1# 2.8# 5.5# 55.6# 25# 1.4# 8.6# 17.2# 1.2# 6.9# 13.8# 50# 1.2# 3.7# 18.4# 1.3# 4.0# 19.9# 100# 1.2# 2.4# 23.5# 1.1# 2.3# 22.8# # ! ! ! ! 55 2.2.4.4!In!vitro!release!of!griseofulvin! ! The#in$vitro#release#profiles#of#griseofulvin#encapsulated#inside#EO33SO14EO33#and# EO38SO10EO38#block#copolymer#micelles#were#monitored#by#dialysis#at#37#ºC#in#pH#4.0,# 5.5#and#7.4#buffers#(Figure#6).#An#initial#burst#release#was#observed#in#all#cases,#followed# by# a# more# gradual# phase# until# equilibration# was# attained# over# 1# day.# Based# on# this# observation,#it#can#be#concluded#that#an#important#fraction#of#the#drug#probably#existed# at#the#interstices#of#the#self1assembled#micelles#causing#the#burst#release,#while#those# drug# molecules# located# at# the# micellar# interior# followed# a# slow# and# stepwise# release# kinetics#(56).#Furthermore,#while#roughly#35%#and#45%#of#the#loaded#griseofulvin#was# released# in# 5# h# at# pH# 4.0# and# 5.5# respectively,# the# release# at# pH# 7.4# was# faster,# and# almost#75%#of#the#drug#diffused#out#the#micelles.## 010 20 30 40 50 60 0 20 40 60 80 100 % cumulative release time (h) b) 010 20 30 40 50 60 0 20 40 60 80 100 % cumulative release time (h) a) % (1) # Figure!6.!In$vitro#release#kinetics#of#griseofulvin#encapsulated#inside#a)#EO33SO14EO33#and# b)#EO38SO10EO38#block#copolymer#micelles,#under#dialysis#at#37#ºC#and#pH#4.0#(!),#5.5# (¢)#and#7.4#(∆).# # # Drug#release#profiles#from#the#micellar#systems#were#fitted#to#the#typical#square1 root#kinetics#(57)## Mt/Mα=$k·∙t0.5######(9)## # and#to#the#Fickian#diffusion#model#considering#the#micelles#as#perfect#spheres#(58)# # Mt/Mα=$k1+$k2·∙t0.5$]$k3·∙t#####(10)# # Only# the# Fickian# diffusion# model# fitted# well# the# whole# release# profile# (Table# 4,# correlation# coeffient# R2# ># 0.90).# This# model# has# been# previously# applied# to# swellable# matrices#to#explain#the#coupling#of#the#diffusion#and#relaxation#events#(57)#and#more# recently# to# micellar# systems# to# describe# the# radial# diffusion# of# the# drug# through# the# 56 core1shell# phases# (58).# Both# conformational# changes# in# the# micellar# structure# during# drug#release#and#partial#transfer#of#drug#from#one#micelle#to#another#may#play#a#role#in# the#release#rate#of#the#drug.#Moreover,#since#the#release#data#were#obtained#from#the# decay# of# the# drug# concentration# in# the# micellar# solution# inside# the# dialysis# bag,# drug# released#from#one#micelle#can#enter#in#an#already#empty#micelle#nearby.#That#situation# may#mimic#the#drug#release#in#a#cellular#environment,#where#the#drug1loaded#micelles# would# be# at# a# short# distance# of# the# cellular# acceptors# (which# can# be# simulated# with# empty#micelles)#(58).#Thus,#drug#release#from#micelles#to#the#aqueous#buffer#and#from# one#micelle#to#another#may#likely#occur.#It#should#be#noticed#that#the#release#tests#were# carried#out#using#dialysis#bags#of#MWCO#3500Da,#which#is#below#the#molecular#weight# of#the#copolymer.#Therefore,#the#copolymer#concentration#inside#the#dialysis#bag#should# remain#almost#constant#along#the#test#and,#consequently,#the#micelles#breakdown#may# occur#in#a#timescale#considerably#larger#than#that#of#the#diffusion#of#the#drug.### # Table!4:!Results#of#the#fitting#to#equation#10#of#the#griseofulvin#release#profiles#from#the# micellar# solutions# in# aqueous# buffer# of# different# pH.# The# release# rate# constants# are# given#as#mean#values,#with#standard#deviations#in#parenthesis.# Formulation# Release# medium# k1$ $ k2$ k3$ F# P1value# R2# E33SO14EO33# pH#4# 30.60# (2.12)# 9.19# (1.28)# 0.81# (0.16)# 65.67# 0.001# 0.9563# pH#5.5# 12.39# (6.13)# 19.22# (3.71)# 1.84# (0.46)# 26.35# 0.002# 0.8978# pH#7.4# 118.07# (10.50)# 37.88# (6.36)# 3.53# (0.78)# 38.19# 0.001# 0.9272# E38SO10EO38# pH#4# 15.57# (5.28)# 16.98# (3.22)# 1.64# (0.40)# 27.17# 0.001# 0.9005# pH#5.5# 25.18# (3.84)# 15.25# (2.34)# 1.34# (0.29)# 58.16# 0.001# 0.9509# pH#7.4# 19.33# (3.76)# 19.22# (2.28)# 1.58# (0.28)# 120.66# 0.001# 0.9757# # The# constant# associated# to# drug# diffusion# (k2)# became# larger# as# the# pH# raised# from#4.0#to#7.4,#particularly#in#the#case#E33SO14EO33.#The#overall#amount#released#in#the# first# 24# h# was# larger# at# pH# 7.4# (78# %# for# EO33SO14EO33# and# 71%# for# EO38SO10EO38,# respectively)# than# under# acidic# conditions# (56# %# at# pH# 4.0# for# both# copolymers).# The# reasons# for# this# effect# are# unclear.# One# hypothesis# is# that# at# acid# pH# there# is# an# strengthening# of# the# hydrogen# bonds# between# the# PEO# blocks# and# water# molecules,# resulting# in# the# stretching# of# PEO# chains# and# enlargement# of# the# micellar# shell#(59),# making#the#diffusion# path#longer.#An#effective# increase#in#the# SO/EO#ratio#might# also# 57 occur#as#the#pH#decreases,#due#to#a#partial#hydrolysis#of#the#EO#chains#(60),#which#may# result#in#an#enhanced#drug/hydrophobic#block#affinity,#hence,#slowing#drug#release.#The# amount#of#drug#released#from#EO38SO10EO38#micelles#was#slightly#larger#than#from#the# EO33SO14EO33# ones,# probably# as# a# consequence# of# a# slightly# lower# micellar# stability# (previously#discussed)#due#to#a#less#compact#hydrophobic#core#of#the#former#polymer,# which#may#favour#the#formation#of#hydrophilic#channels#(61,62).## # 2.2.5 Conclusions! # In#aqueous#solution,#poly(ethylene#oxide)1poly(styrene#oxide)#block#copolymers# EO33SO14EO33# and# EO38SO10EO38#self1assembled# at# very# low# concentrations# to# form# spherical# micelles# of# sizes# ca.# 13114# nm.# Also,# these# copolymers# present# a# rich# phase# behavior,#with#the#formation#of#soft#and#hard#gels.#In#this#regard,#two#different#types#of# soft#gels#were#observed,#one#usually#presented#at#high#temperatures#in#the#copolymer# concentration# range# 20130# wt%# as# a# consequence# of# percolation# between# copolymer# micelles#acting#as#hard#spheres,#and#other#after#hard#gel#regions#originated#by#defective# cubic# structures.# On# the# other# hand,# both# copolymers# display# an# important# ability# to# solubilise# hydrophobic# drugs# as# observed# by# comparing# the# solubilisation# extent# of# griseofulvin.#Solubility#factors#up#to#ca.#55#mg#g11,#obtained#by#optimization#of#the#SO/EO# ratio# and# the# copolymer# block# lengths,# were# not# previously# achieved# in# any# other# micellar#system.#Drug#release#profiles#show#an#initial#burst#followed#by#a#more#sustained# pH1dependent#delivery.#The#slower#release#rate#observed#at#acid#pH#may#be#the#result# of#i)#conformational#changes#in#the#EO#blocks#that#lead#to#large#shells#and#thus#longer# diffusional# paths# and# ii)# a# more# hydrophobic# microenvironment# inside# the# micelle,# increasing#the#drug1hydrophobic#copolymer#chains#affinity,#due#to#partial#hydrolysis#of# EO# chains# which# leads# to# an# effective# increase# in# SO/EO# ratio.# Overall# the# results# obtained#indicate#that#EO33SO14EO33#and#EO38SO10EO38#can#act,#at#low#concentrations,#as# suitable#components#of#micellar#carriers#for#drug#administration#by#either#parenteral#or# oral#route,#subsequent#drug#transport#in#the#body#and#sustained#release,#and#at#higher# concentrations,#as#components#of#gel#systems#that#can#undergo#gel1to1sol#transitions#as# a#function#of#tiny#increases#in#temperature#above#37ºC.## # 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Taboada,# P.;# Velasquez,# G.;# Barbosa,# S.;# Yang,# Z.;# Nixon,#S.K.;#Zhou,# K.;Heatley,##F.;#Ashford,#M.;#Mosquera,#V.;#Attwood,#D.;#Booth,#C.#Langmuir,# 2006,#22,#7465.# # 20. Booth,#C.;#Attwood,#D.;#Price,#C.#Phys.#Chem.#Chem.#Phys.,#2006,#8,#3612.# # 21. Barbosa,# S.;# Cheema,# M.A.;# Taboada,# P.;# Mosquera,# V.# #J.# Phys.# Chem.# B,# 2007,#11,#10920.# # 22. Crothers,#M.;#Zhou,#Z.;#Ricardo,#N.#M.#P.#S.;#Yang,#Z.;#Taboada,#P.;#Chaibundit,# C.;#Attwood,#D.;#Booth,#C.#Int.#J.#Pharm.,#2005,#293,#91.# # 23. Ribeiro,#M.#E.# N.# P.;#Vieira,#I.# G.#P.;#Cavalcante,#I.M.;# #Ricardo,#N.# M.# P.#S.;# Attwood,#D.;#Yeates,#S.G.;#Booth,#C.#Int.#J.#Pharm.,#2009,#378,#211.# # 24. Juarez,# J.;# Taboada,# P.;# # Valdez,# M.A.;# Mosquera,# V.# Langmuir,# 2008,# 24,# 7107.# # 25. #Zhou,#N.;#Lodge,#T.P.;#Bates,#F.S.#J.#Phys.#Chem.#B,#2006,#110,#3979.# # 26. Cambón,#A.;#Rey1Rico,#A.;#Barbosa,#S.;#Soltero,#J.F.A.;# Yeates,#S.G.;#Brea,#J.;# 65 2.4$POLY$(STYRENE$ OXIDE)$3$POLY(ETHYLENE$ OXIDE)$ BLOCK$ COPOLYMERS:$ FROM$ “CLASSICAL”$ CHEMOTHERAPEUTIC$ NANOCARRIERS$ TO$ ACTIVE$ CELL3RESPONSE$ INDUCERS$ $ ! 2.4.1 Abstract! ! Two$ poly(styrene$ oxide)3poly(ethylene$ oxide)$ (PSO3PEO)$ triblock$ copolymers$ with$different$chain$lengths$were$analyzed$as$potential$chemotherapeutic$nanocarriers,$ and$their$ability$to$inhibit$the$P3glycoprotein$(P3gp)$efflux$pump$in$a$multidrug$resistant$ (MDR)$cell$line$were$measured$in$order$to$establish$possible$cell3responses$induced$by$ the$ presence$ of$ the$ copolymer$ molecules.$ Thus,$ EO33SO14EO33$ and$ EO38SO10EO38! polymeric!micelles$ were$ tested$ regarding$ doxorubicin$ (DOXO)$ entrapment$ efficiency$ (solubilisation$ test),$ physical$ stability$ (DLS),$ cytocompatibility$ (fibroblasts),$ release$ profiles$ at$ various$ pHs$ (in# vitro$ tests),$ as$ well$ as$ P3gp$ inhibition$ and$ evasion$ and$ cytotoxicity$of$the$DOXO3loaded$micelles$in$an$ovarian$MDR$NCI3ADR/RES$cell$line$and$ in$ DOXO3sensitive$ MCF37$ cells.$ EO33SO14EO33$ and$ EO38SO10EO38!formed$ spherical$ micelles$ (∼13$ nm)$ at$ lower$ concentration$ than$ other$ copolymers$ under$ clinical$ evaluation$(e.g.$Pluronic®),$exhibited$0.2$to$1.8%$loading$capacity,$enhancing$more$than$ 60$times$drug$apparent$solubility,$and$retained$the$cargo$for$long$time.$The$copolymer$ unimers$inhibited$P3gp$ATPase$activity$in$a$similar$way$as$Pluronic$P85,$favoring$DOXO$ accumulation$in$the$resistant$cell$line,$but$not$in$the$sensitive$cell$line.$DOXO$loaded$in$ the$micelles$accumulated$more$slowly$inside$the$cells,$but$caused$greater$cytotoxicity$ than$free$drug$solutions$in$the$NCI3ADR3RES$cell$line,$which$overexpressed$P3gp.$Hence,$ PSO3PEO$ block$ copolymers$ offer$ interesting$ features$ as$ new$ biological$ response$ modifiers$to$be$used$in$the$design$of$efficient$nanocarriers$for$cancer$chemotherapy.$$ $ 2.4.2 Introduction! ! Advances$in$materials$science$offer$tremendous$opportunities$to$develop$novel$ nanocarriers$ able$ to$ improve$ the$ pharmacokinetics$ and$ the$ local$ bioavailability$ of$ a$ variety$of$drugs,$apart$from$providing$additional$functionalities$(134).$Among$the$diverse$ nanoparticulate$ systems$ suitable$ for$ encapsulating$ and$ delivering$ drugs,$ micelles$ formed$ by$ amphiphilic$ polymers$ occupy$ a$ relevant$ position$ (5,6).$ Self3assembly$ of$ 66 biocompatible$ copolymers$ consisting$ of$ two$ or$ more$ blocks$ with$ different$ hydrophobicity$may$result$in$the$formation$of$micelles$with$a$hydrophobic$core$and$a$ hydrophilic$shell.$Copolymers$bearing$hydrophilic$poly(ethylene$oxide)$(PEO)$blocks$lead$ to$ sterically$ stabilized$ micelles$ that$ show$ prolonged$ blood$ circulation,$ and$ passively$ accumulate$ in$ solid$ tumors$ (4).$ The$ most$ widely$ studied$ amphiphilic$ copolymers$ are$ those$composed$of$PEO$and$poly(propylene$oxide)$(PPO)$blocks,$particularly$the$linear$ and$ bifunctional$ poloxamers$ (Pluronics®)$ and$ the$ X3shaped$ poloxamines$ (Tetronic®).$ PEO–PPO$block$copolymers$have$gained$popularity$over$the$last$decades$due$to:$i)$their$ commercial$ availability;$ ii)$ proven$ fair$ solubilization$ capacity$ and$ sustained$ drug$ delivery;$ iii)$ high$ biocompatibility$ of$ most$ varieties;$ iv)$ inhibition$ of$ different$ efflux$ transporters$overexpressed$in$multidrug$resistant$(MDR)$cells;$v)$ability$to$enhance$drug$ transport$ across$ cellular$ barriers;$ and$ vi)$ regulatory$ status,$ i.e.,$ approval$ of$ some$ varieties$by$US$FDA$and$EMA$to$be$used$in$pharmaceutical$formulations$and$medical$ devices$(739).$Nevertheless,$PEO–PPO$block$copolymers$display$several$drawbacks,$such$ as$uncomplete$micellization$of$unimers$and$limited$drug$solubility$and$colloidal$stability$ upon$dilution$in$the$bloodstream$particularly$when$the$EO/PO$ratio$is$high.$$ $ $ To$ achieve$ a$ more$ efficient$ aggregation$ and$ micelle$ stability,$ a$ series$ of$ other$ block$ copolymer$ counterparts$ of$ similar$ architecture$ but$ with$ the$ PPO$ segment$ replaced$by$a$more$hydrophobic$one$such$as$poly(butylene$oxide)$(PBO),$poly(styrene$ oxide)$ (PSO)$ or$ phenylglycidyl$ ether$ (PG)$ has$ been$ developed$ by$ the$ Attwood$ and$ Booth´s$group$in$collaboration$with$us$during$last$years$(10313).$The$micelles$of$these$ copolymers$ showed$ improved$ solubilization$ capacity$ and$ stability$ (11,14315).$ In$ particular,$PSO3based$block$copolymers$are$of$interest$due$to$the$wide$availability$of$ architectures$and$molecular$weights$(12),$their$ability$to$self3assemble$into$micelles$of$ different$shapes$at$very$low$concentrations$depending$on$their$relative$block$lengths$ (16),$ and$ their$ low$ glass$ transition$ temperature$ (ca.$ 40$ ºC),$ which$ enables$ the$ incorporation$of$termolabile$drugs$(11,17).$Despite$the$micellization$and$solubilisation$ ability$of$some$PSO3based$copolymers$have$been$previously$studied$(10,12,13,16),$as$ far$as$we$know$only$one$study$has$analysed$the$role$of$PSO3PEO$copolymer$micelles$as$ carriers$ of$ an$ anticancer$ drug$ (docetaxel)$ (18).$ Moreover,$ in$ contrast$ to$ the$ well3 demonstrated$ inhibitory$ activity$ of$ several$ PEO3PPO$ Pluronic®$ and$ Tetronic®$block$ copolymers$against$drug$efflux$transporters$overexpressed$in$MDR$cells$(8,19320),$no$ reports$are$available$about$the$potential$capabilities$of$PSO3based$copolymers$as$efflux3 pump$inhibitors.$$ $ $ Hence,$we$evaluate$the$ability$of$copolymers$EO33SO14EO33$and$EO38SO10EO38$to$ dissolve$and$chemically$protect$doxorubicin$(DOXO),$analyzing$the$colloidal$stability,$the$ drug$release$profiles,$the$safety,$and$the$in#vitro$efficacy$of$the$drug3loaded$polymeric$ micelles$as$an$antitumoral$formulation.$The$EO/SO$ratio$and$the$block$lengths$of$both$ copolymers$were$selected$to$attain$an$optimal$compromise$between$chain$solubility,$ 67 micelle$ formation$ ability,$ and$ core$ size$ that$ lead$ to$ enhanced$ drug$ solubility,$ while$ ensuring$renal$clearance$of$unimers$as$required$for$non3biodegradable$polymers$(21).$ Shorter$PEO$blocks,$as$those$of$EO10SO10EO10$(16),$and$longer$PSO$blocks$compromise$ copolymer$ solubility.$ By$ contrast,$ longer$ PEO$ and$ shorter$ PSO$ blocks$ may$ lead$ to$ greater$ cmcs$ with$ the$ subsequent$ increase$ in$ material$ expense$ to$ solubilise$ the$ required$ amount$ of$ drug$ (12).$ EO33SO14EO33$ and$ EO38SO10EO38$ may$ also$ help$ to$ elucidate$the$effect$of$copolymer$architecture$in$solubilisation$and$controlled$release$ performance$by$comparison$with$the$data$reported$for$PSO$diblock$copolymers$(18).$On$ the$other$hand,$the$ability$of$the$present$copolymers$to$inhibit$the$P3glycoprotein$(P3gp)$ efflux$pump$was$investigated$for$the$first$time.$DOXO$accumulation$in$an$in#vitro$model$ of$MDR$cell$line$with$high$expression$of$P3gp$(ovarian$tumor$cell$line$NCI3ADR3RES)$was$ evaluated$and$compared$with$that$achieved$in$a$non3resistant$cell$line$(breast$cancer$ cell$line$MCF37).$Moreover,$the$effect$of$the$copolymers$on$P3gp$ATPase$activity$was$ analyzed$ and$ compared$ to$ that$ caused$ by$ Pluronic®$P85,$ the$ most$ efficient$ cell$ sensitizing$block$copolymer$so$far$described$(8,19).$Overall,$the$results$indicate$that$the$ micellar$ systems$ based$ on$ PSO3PEO$block$ copolymers$ improves$ DOXO$ encapsulation$ and$ its$ systemic$ delivery,$ resulting$ in$ lower$ cytotoxicity$ and$ enhanced$ chemotherapeutic$activity$by$ the$combined$effect$ of$the$ controlled$drug$release$and$ the$inhibition$of$the$P3gp$efflux$pump.$ $ 2.4.3!Experimental!section! ! 2.4.3.1!Materials! $ EO33SO14EO33$ and$ EO38SO10EO38$ (Table$ 1)$ were$ synthesized$ as$ previously$ described$(10).$Weight3averaged$(Mw)$to$number3averaged$(Mn)$molecular$weight$ratios$ were$determined$at$25ºC$using$a$Waters$gel$permeation$chromatography$(GPC)$system$ (Waters,$Milford,$MA).$Mn#values$were$estimated$from#1H$NMR$spectra$recorded$on$a$ Bruker$ARX400$spectrometer$(Bruker,$Milton,$ON,$Canada).$Pluronic®$P85$was$supplied$ by$ BASF$ (New$ Milford,$ CT,$ USA).$ Verapamil$ (VER),$ calcein$ AM,$ and$ doxorubicin$ hydrochloride$(DOXO·∙HCl)$were$from$Sigma3Aldrich.$DOXO$base$for$solubilisation$inside$ copolymer$ micelles$ was$ obtained$ by$ means$ of$ aqueous$ precipitation$ of$ DOXO·∙HCl$ (1$ mg/ml)$ adding$ triethylamine$ (three$ moles$ per$ drug$ mol)$ and$ methylene$ chloride.$ Hereinafter,$ DOXO$ refers$ to$ DOXO$ base.$ Water$ was$ double$ distilled$ and$ degassed$ before$use.$All$other$reagents$were$analytical$grade.$ $ $ $ $ $ $ 68 Table!1.!Molecular$characteristics$of$the$copolymers.# $ Mn*$(g$mol31)$ SO$content*$$ (wt.%)$ Mw/Mn**$ $ Mw$$(g$mol31)$ EO33SO14EO33$ 4790$ 40.0$ 1.01$ 4850$ EO38SO10EO38$ 5055$ 34.1$ 1.02$ 5130$ *Estimated$ from$ NMR$ data;$ **Determined$ by$ GPC;$ Mw$ was$ calculated$ from$ Mn$ and$ Mw/Mn$ratio.$Uncertainty:$Mn$to$±3$%;$wt%$SO$to$±1$%,$Mw/Mn$to$±0.01.$ $ $ !2.4.3.2! Methods! ! a. ##Drug#solubilisation# # #Solubilization$ of$ DOXO$ (intrinsic$ solubility$ in$ water$ 0.130.5$mg$ dm33)$ (22)$ in$ micellar$ copolymer$ solutions$ (0.2$ wt%)$ was$ tested$ in$ triplicate$ following$ a$ methodology$ previously$reported$(15)$(see$Supplementary$Material).$$ $ b. Physical#stability#of#the#drug:loaded#micelles#upon#dilution# # #DOXO$micellar$solutions$were$diluted$(1/50)$with$either$0.01$M$phosphate$buffer$pH$ 7.4$ or$ cell$ culture$ medium$ with$ 10%$ FBS$ and$ incubated$ at$ 37$ oC,$ and$ the$ drug$ concentration$was$monitored$over$time$by$UV$spectrophotometry.$In$parallel,$changes$ in$the$size$of$drug3loaded$micelles$were$monitored$by$dynamic$light$scattering$at$37$°C$ using$an$ALV35000F$(ALV3GmbH,$Germany)$instrument$with$vertically$polarized$incident$ light$( λ $=$488$nm)$supplied$by$a$diode3pumped$Nd:YAG$solid3state$laser$(Coherent$Inc.,$ CA,$USA)$operated$at$2$W,$and$combined$with$an$ALV$SP386$digital$correlator$(sampling$ time$25$ns$to$100$ms)$as$previously$reported$(10)$(see$Supplementary$Material).$$ $ c. In#vitro#DOXO#release# # #Aliquots$ (4$ mL)$ of$ DOXO3loaded$ micellar$ systems$ (0.2$ wt.$ %$ copolymer)$ in$ 0.01$ M$ phosphate$buffer$pH$7.4,$0.01$M$sodium$citrate$buffer$pH$5.5,$or$cell$culture$media$at$ pH$ 7.4$ or$ 5.5$ were$ placed$ into$ dialysis$ tubes$ (SpectraPore®,$ MWCO$ 3500),$ and$ immersed$into$the$same$medium$(500$mL)$used$to$prepare$the$micellar$solutions.$The$ medium$was$kept$at$37ºC$and$replaced$every$6$hours$to$maintain$sink#conditions.$The$ released$ drug$ concentration$ was$ spectrophotometrically$ monitored$ at$ 480$ nm,$ by$ removing$ a$ small$ volume$ (20$ µL)$ that$ was$ diluted$ in$ methanol$ in$ order$ to$ fit$ the$ calibration$curve$range.$Assays$were$carried$out$in$triplicate.$ $ $ # 69 d. #Copolymer#cytocompatibility#evaluation# # #The$cytocompatibility$of$the$bare$copolymer$micelles$was$first$assessed$using$BALB/3T3$ clone$ A31$ mouse$ embryonic$ fibroblast$ cells$ (CCL$ 163,$ ATCC),$ following$ a$ previously$ reported$procedure$(23)$(see$Supplementary$Material$for$further$details).$$ $ e. Cellular# uptake# of# DOXO# after# incubation# with# unimers# and# empty# polymeric# micelles#(P:gp#inhibition)# # #MDR$ NCI3ADR/RES$ and$ drug3sensitive$ MCF37$ cells$ (American$ Type$ Culture$ Collection,$MD,$USA)$were$separately$seeded$in$a$243wells$plate$(1.5x105$cells/well,$ 1000$μL/well)$in$supplemented$RPMI$1640$and$EMEM$medium,$respectively,$for$48$ h.$ The$ medium$ was$ replaced$ by$ serum3free$ one$ containing$ 43(23hydroxyethyl)313 piperazineethanesulfonic$ acid$ (HEPES,$ 25$ mM,$ pH$ 7.4).$ Polymer$ samples$ were$ added$(20$µL;$final$concentrations$in$the$medium$0.001%,$0.01%$and$0.2%)$and$cells$ incubated$ at$ 37ºC$ for$ 30$ min.$ Polymer3free$ medium$ and$ VER$ solution$ (100$ µM)$ were$ used$ as$ blank$ and$ positive$ control,$ respectively.$ Immediately$ after$ 30$ min$ incubation,$50$µl$of$a$DOXO$solution$(100$mM$in$water)$was$added$and$the$samples$ incubated$for$60$additional$min.$The$medium$ was$ removed$and$the$cells$washed$ (PBS,$3$x$500$µL)$to$remove$DOXO$and$copolymer$residues.$Quantification$of$DOXO$ inside$ the$ cells$ was$ carried$ out$ as$ previously$ reported$ (20)$ (see$ Supplementary$ Material$ for$ details).$ Confocal$ microscopy$ analysis$ (Leica$ TCS3SP2,$ LEICA$ Microsystems$Heidelberg$GmbH,$Germany)$was$carried$out$upon$cell$staining$with$ Bodipy®$phalloidin$(30µl/ml)$in$0.2%$Triton$X3100$(permeabilizer),$and$subsequent$ washing$and$mounting$on$glass$slides$using$anti3fading$solution.$Visualization$was$ made$at$20X$and$63X$using$green$channel$for$doxorrubicin$(λexc.$561nm)$and$red$ channel$ for$ Bodipy®$ Phalloidin$ (λexc.$633$ nm,$ see$ Supplementary$ Material$ for$ details).$ $ f. Cellular#uptake#of#calcein#AM#after#incubation#with#the#polymers## # The$calcein$AM$assay$was$performed$following$the$method$described$by$Dong#et#al.$ (24)$(see$Supplementary$Material$for$details).$$ $ g. P:gp#ATPase#assay$$ $ The$effect$of$EO33SO14EO33,$EO38SO10EO38$and$Pluronic$P85$at$0.001%,$0.01%$and$0.2$ wt%$on$the$ATPase$activity$of$Pgp$was$measured$using$Pgp3Glo™$Assay$System$with$ P3glycoprotein$ (V3601,$ Promega$ Biotech$ Ibérica,$ SL,$ Madrid,$ Spain)$ following$ the$ manufacturer´s$protocol.$Na3VO4$and$verapamil$(12$mM)$were$used$as$controls$of$ inhibition$and$stimulation,$respectively.$The$luminescence$of$the$samples$detected$ 70 using$ a$ Tecan$ Ultra$ Evolution$ (Tecan,$ Switzerland)$ reflected$ the$ ATP$ level,$ which$ negatively$correlated$with$the$activity$of$P3gp$ATPase.$ $ h. Cellular#uptake#of#DOXO:loaded#polymeric#micelles#(P:gp#evasion)# # #NCI3ADR/RES$seeded$in$243wells$plates$(1x105$cells/well)$in$RPMI$1640$medium$with$2$ mM$L3glutamine,$10%$FBS$and$1%$penicillin/streptomycin$over$sterile$glass$covers.$After$ 48$h,$culture$medium$was$replaced$with$RPMI$1640$medium$with$HEPES$25$mM$(pH$ 7.4).$Cells$were$incubated$with$formulations$containing$DOXO$for$1$and$24$h$at$37ºC.$ Then,$ DOXO$ formulations$ were$ removed$ and$ the$ cells$ were$ washed$ and$ stained$ as$ explained$above.$As$a$control,$the$cells$were$incubated$with$DOXO$solution$(50$µM)$in$ PBS$at$pH$7.4.$ $ i. #In#vitro#cytotoxicity#of#drug#loaded:polymeric#micelles$ $ #Human$NCI3ADR/RES$and$MCF37$cells$were$seeded$in$963wells$plates$(15.000$cells/well)$ as$described$above.$Then,$DOXO3loaded$micellar$systems$or$DOXO·∙HCl$solutions$(100$ µM$and$50$µM$final$concentration)$in$PBS$pH$7.4$were$added.$As$controls,$copolymers$ at$0.01$and$0.2%$(final$concentrations)$were$used.$Cytotoxicity$was$evaluated$at$24$and$ 48$h$applying$the$crystal$violet$method$(see$Supplementary$Material$for$details).$ $ 2.4.4 Results!and!discussion! ! The$molecular$characteristics$of$copolymers$EO33SO14EO33$and$EO38SO10EO38$and$ the$ physico3chemical$ properties$ of$ their$ micelles$ in$ diluted$ and$ concentrated$ regime$ were$ previously$ characterized$ in$ detail$ (25).$ Briefly,$ EO33SO14EO33$ and$ EO38SO10EO38$ displayed$very$low$cmc$in$aqueous$medium$(2.5·∙1033$and$3.7·∙1033$wt.$%,$respectively),$ and$formed$monodisperse$spherical$micelles$of$ca.$13$nm$in$diameter$and$association$ numbers$of$37$and$14,$respectively$(Table$S1$and$Figure$S1$in$Supplementary$Material).$ $ 2.4.4.1 Solubilization!capacity!! ! $ Some$ PSO3PEO$block$ copolymers$ have$ shown$ superior$ solubilisation$ ability$ compared$to$Pluronic®$and$Tetronic®$ones$(14,15,17).$Optimization$of$the$SO/EO$ratio$ and$the$blocks$length$can$enhance$their$perfomance.$Apparent$solubility$of$DOXO$was$ tested$in$0.2$wt.%$copolymer$solutions$(above$the$cmc)$by$adding$different$amounts$of$ drug$in$ order$ to$ evaluate$ the$ impact$ of$ the$ feeding$ amount$ on$ the$ entrapment$ efficiency$and$the$total$drug$loaded.$In$general,$the$higher$the$drug/copolymer$weight$ ratio,$the$lower$the$entrapment$efficiency$was$(Table$2)$due$to$drug$saturation$of$the$ micelles.$EO33S14EO33$copolymer$exhibited$a$slightly$larger$solubilisation$capacity,$which$ can$be$attributed$to$its$longer$hydrophobic$block$and$subsequent$higher$affinity$of$the$ 71 micelle$core$for$hydrophobic$drugs.$Nevertheless,$both$copolymers$encapsulated$DOXO$ very$ efficiently$ with$ a$ hydrosolubility$ excess$ of$ 30$ mg/l;$ i.e,$ more$ than$ 60$ times$ the$ aqueous$ solubility$ of$ free$ DOXO.$ The$ maximum$ loading$ capacity$ was$ ca.$ 1.8%,$ with$ entrapment$ efficiencies$ ranging$ from$ 20%$ to$ 50%$ depending$ on$ the$ drug$ feeding$ concentration.$ These$ values$ are$ also$ slightly$ larger$ than$ those$ reported$ for$ DOXO$ in$ previous$solubilisation$studies$with$other$block$copolymers,$such$as$PEO3based$poly(DL3 lactic3co3glycolic$ acid),$ PEG3PLGA,$ poly(caprolactone),$ PEO3PCL,$ or$ poly[N3(23 hydroxypropyl)$ methacrylamide3lactate),$ PEG3p(HPMAm3Lac),$ that$ had$ entrapment$ efficiencies$of$ca.$23%,$48%$or$5%,$respectively$(26330).## # Table! 2:! Doxorubicin$ loaded$ amount$ (D.L.),$ entrapment$ efficiency$ (E.E.)$ and$ solubilisation$capacity$(SCP)$of$the$copolymers$at$0.2$wt.%.$ ! Feeding# drug/polymer# %#(w/w)# EO33SO14EO33! EO38SO10EO38! D.L.# %# E.E.# %# Scp# mg#g:1# D.L.# %# E.E.# %# Scp# mg#g:1# 0.1$ 0.05$ 46.7$ 0.4$ 0.05$ 51.8$ 0.5$ 0.5$ 0.2$ 52.3$ 2.4$ 0.2$ 46.0$ 2.2$ 1$ 0.3$ 36.8$ 3.4$ 0.4$ 44.7$ 4.2$ 2.75$ 1.0$ 37.6$ 10.2$ 1.0$ 38.6$ 10.5$ 4.25$ 1.4$ 34.5$ 14.5$ 0.9$ 22.1$ 9.3$ 6$ 1.8$ 30.8$ 16.1$ 1.3$ 21.2$ 11.6$ $ ! 2.4.4.2 !Size! distribution! and! physical! stability! of! DOXOIloaded! polymeric! micelles! $ $ Size$is$critical$for$the$biodistribution$profile$and$the$interactions$of$micelles$with$ cells.$Drug$incorporation$could$increase$the$micellar$size$due$to$either$the$enlargement$ of$ the$ core$ (31)$ and/or$ the$ fusion$ of$ drug3containing$ micelles$ into$ larger$ ones$ (32).$ However,$ both$ non3loaded$ and$ DOXO$ loaded3micelles$ showed$ similar$ narrow$ and$ monodisperse$ intensity$ distribution$ functions$ by$ DLS.$ The$ loaded$ micelles$ could$ be$ readily$freeze3dried$and$their$initial$size$distribution$was$recovered$upon$reconstitution$ in$aqueous$solution$(Figure$1A).$The$micellar$sizes$also$remained$stable$upon$extensive$ incubation,$which$points$to$a$great$micelle$stability$and$the$capability$of$the$PEO$stealth$ layer$ to$ avoid$ protein$ binding$ and$ subsequent$ micellar$ aggregation$ (Figure$1B$ and$ Supplementary$Material,$Figure$S2A).$$ $ DOXO3loaded3copolymer$ micellar$ solutions$ were$ strongly$ diluted$ (1/50)$ in$ medium$with$or$without$10%$FBS$to$mimic$the$events$after$body$administration,$and$ the$drug$concentration$was$monitored$over$time.$In$any$tested$medium,$the$loaded3 72 polymeric$ micelles$ were$ physically$ stable$ until$ 10312$ days.$ DOXO$ solubility$ remained$ above$ 86%$ of$ the$ initial$ value$ for$ EO33S14EO33$ and$ ca.$ 75%$ for$ EO38SO10EO38$when$ incubating$ in$ the$ cell$ culture$ medium$ (Figure$ 1C).$ In$ the$ absence$ of$ proteins,$ DOXO$ solubility$remained$slightly$larger:$ca.$92%$and$85%$for$EO33S14EO33$and$EO38SO10EO38,$ respectively,$ at$ 20$ days$ of$ incubation$ (Supplementary$ Material$ Figure.$ S2B).$ The$ observed$ slightly$ lower$ stability$ of$ EO38SO10EO38$ micelles$ possibly$ arises$ from$ a$ less$ compact/smaller$hydrophobic$core$and$higher$cmc.$In$addition,$the$colloidal$stability$of$ EO33SO14EO33$ and$ EO38SO10EO38$ micelles$ is$ apparently$ larger$ than$ that$ previously$ reported$ for$ EO45SO15$ and$ EO45SO26$diblock$copolymers$ (18);$ nevertheless,$ it$ is$ necessary$ to$ bear$ in$ mind$ that$ the$ latter$ copolymers$ were$ subjected$ to$ stronger$ destabilizing$ conditions$ which$ might$ accelerate$ their$ disintegration.$ Also,$ micellar$ stability$ of$ EO33SO14EO33$ and$ EO38SO10EO38$ is$ greater$ than$ that$ of$ some$ structurally$ related$ PBO3PEO$ and$ PPO3PEO$ block$ copolymers,$ for$ which$ the$ drug$ solubilized$ decreased$more$than$60%$upon$extended$incubation$(18,23,32).$$ $ ! Figure!1:!Intensity$fraction$size$distribution$of$(−)$non3loaded,$(·∙·∙·∙)$DOXO3loaded$and$(33)$ reconstituted$freeze3dried$DOXO3loaded$EO33SO14EO33$micelles$(A);$temporal$evolution$ of$ the$ size$ of$ DOXO3loaded$ EO33SO14EO33$ (¢)$ and$ EO38SO10EO38$ (")$ micelles$ under$ strong$dilution$in$cell$culture$medium$(B);$and$%DOXO$that$remained$solubilised$in$the$ polymeric$micelles$over$time$when$diluted$with$cell$culture$medium$at$37$ºC$(C).$$ $ 2.4.4.3!!In!vitro!release! # ## DOXO3loaded$micellar$solutions$(0.2$wt.%$copolymer)$were$dialysed$against$pH$ 7.4$ and$ 5.5$ buffer$ and$ serum3containing$ (10$ %$ FBS)$media$ using$ dialysis$ tubing$ that$ 1 10 100 0.0 0.2 0.4 0.6 0.8 1.0 Intensity (a.u.) rH,app A) 0 5 10 15 20 0 2 4 6 8 10 rh (nm) Incubation time (days) B) 0 5 10 15 20 60 75 90 105 120 % (DOXO) Incubation time (days) C) 73 ensured$ that$ no$ micellar$ diffusion$ occurred.$ In$ general,$ in# vitro$ cumulative$ DOXO$ release$profiles$at$both$neutral$and$acidic$conditions$in$the$presence$of$FBS$showed$a$ burst$followed$by$a$sustained$release$pattern$(Figure$2).$At$pH$7.4,$ca.$25%$DOXO$was$ released$ from$ both$ micellar$ systems$ in$ the$ first$ 5$ h$ of$ incubation,$ and$ then$ a$ more$ sustained$release$was$observed$with$ca.$35$%$released$at$60$h.$In$general,$the$amount$ of$ drug$ released$ from$ EO38SO10EO38$ micelles$ was$ slightly$ larger$ than$ from$ the$ EO33SO14EO33$ ones,$probably$as$a$consequence$of$the$observed$slightly$lower$micelle$ stability$ due$ to$ a$ less$ compact$ hydrophobic$ core$ of$ the$ former$ polymer,$ which$ may$ favor$the$formation$of$hydrophilic$channels$(32).$The$drug$release$rate$was$higher$at$pH$ 5.5,$which$is$consistent$with$previous$reports$(27,33334)$(for$modellization$of$release$ profiles,$ see$ Supplementary$ Material).$ At$ pH$ 5.5,$ EO38SO10EO38$ and$ EO33SO14EO33$ micelles$released,$respectively,$ca.$39%$and$47%$of$the$initially$loaded$DOXO$during$the$ first$5$h$and$ca.$76%$and$63$%$at$60$h.$As$occurred$for$the$stability$micellar$tests,$no$ significant$ differences$ in$ the$ release$ profiles$ were$ observed$ when$ serum$ was$ not$ present$in$the$medium$(Supplementary$Material,$Figure$S3).$The$faster$release$under$ acidic$ conditions$ is$ originated$ from$ the$ reprotonation$ of$ the$ amine$ group$ of$ DOXO,$ which$ involves$ an$ increase$ in$ its$ hydrophilicity$ and$ a$ decrease$ in$ the$ affinity$ for$ the$ hydrophobic$ blocks.$ This$ in$ turn$ favors$ its$ escape$ from$ the$ micellar$ core$ by$ an$ out3 diffusion$ process$ through$ the$ core3shell$ structure$ whose$ diffusion$ rate$ depends$ on$ factors$ such$ as$ copolymer$ crystallinity,$ viscosity,$ and$ drug$ association$ state$ (35).$ Reprotonation$would$enable$DOXO$to$be$preferentially$released$in$acidic$tumor$sites,$ compared$to$healthy$tissues.$In$this$regard,$it$is$plausible$that$copolymer$micelles$are$ passively$ targeted$ to$ the$ tumor$ tissue$ through$ the$ EPR$ effect$ with$ minimised$ DOXO$ release$along$circulation$in$the$bloodstream.$After$accumulation$in$the$vicinity$of$the$ tumor$ cells,$ DOXO$ could$ be$ selectively$ released$ from$ the$ micelles$ in$ the$ acidic$ solid$ tumor$ microenvironment$ for$ passive$ cellular$ uptake$ (33).$ More$ importantly,$ intact$ copolymer$ micelles$ might$ be$ also$ taken$ up$ by$ tumor$ cells$ through$ nonspecific$ endocytosis$and$located$preferentially$at$the$acidic$endosome$compartments,$in$which$ the$ decreasing$ pH$ values$ might$ induce$ a$ faster$ DOXO$ release$ and$ a$ subsequent$ diffusion$in$the$cytosol.$This$cellular$uptake$mechanism$could$bypass,$to$certain$extent,$ the$ multidrug$ resistance$ (MDR)$ effect,$ which$ is$ often$ observed$ when$ free$ DOXO$ penetrates$in$the$cell$by$passive$diffusion.$$ 74 $ Figure! 2:! In$ vitro$ drug$ release$ from$ DOXO3loaded$ EO33SO14EO33$ (filled$ symbols)$ and$ EO38SO10EO38$(open$ symbols)$ micelles$ in$ cell$ culture$ medium$ (10$ %$ FBS)$ at$ pH$ 7.4$ (squares)$and$5.5$(circles).$$ $ 2.4.4.4 Cytocompatibility!of!EO33SO14EO33!and!EO38SO10EO38$ $ Cytocompatibility$tests$were$carried$out$against$the$BALB/3T3$fibroblast$cell$line$ because$ of$ its$ high$ sensitiveness$ to$ the$ presence$ of$ toxic$ species.$ The$ LDH$ assay$ enabled$to$measure$if$this$cytosolic$enzyme$was$released$to$the$culture$medium$due$to$ increased$membrane$permeability,$indicating$cell$damage$or$lysis$(36).$For$the$lowest$ concentration$tested$(0.1$wt.%)$viability$extents$of$ca.$100$%$were$observed$for$both$ copolymers,$decreasing$up$ to$ca.$92%$(for$EO38SO10EO38)$and$ 84%$(for$EO33SO14EO33)$ when$copolymer$concentration$increased$up$to$1.66$wt.%$(Figure$3).$In$order$to$prevent$ false$positives$caused$by$possible$delayed$LDH$release$from$cells$after$induction$of$cell$ apoptosis$ or$ necrosis,$ the$ activity$ of$ the$ mitochondrial$ dehydrogenase$ enzyme$ was$ studied$by$means$of$the$MTT$assay.$Whilst$cell$viability$for$EO33SO14EO33$was$ca.$100%$ in$ the$ whole$ range$ of$ concentrations$ analyzed,$ proliferation$ in$ the$ presence$ of$ EO38SO10EO38$ was$ lower$ and$ slightly$ decreased$ as$ the$ copolymer$ concentration$ increased:$95%$at$0.1$wt.%,$82.5%$at$1.0$wt.%,$to$ca.$60%$at$a$concentration$of$1.66$ wt.%,$ respectively.$ In$ any$ case,$ none$ of$ the$ copolymer$ concentrations$ led$ to$ cell$ viabilities$below$50%$(37).$Therefore,$both$copolymers$can$be$considered$as$safe$and$ non3toxic,$ being$ even$ more$ cytocompatible$ than$ most$ of$ the$ commercially$ available$ and$FDA3approved$Pluronic®$and$Tetronic®$block$copolymers$(20,32).#$ 0 10 20 30 40 50 60 0 25 50 75 100 % cumulative DOXO release time (h) 81 cells,$ favoring$ DOXO3induced$ apoptotic$ cell$ death,$ as$ reported$ for$ Pluronic®$ and$ its$ derivatives$(12,20,21,41,46).$$ $ 2.4.5!Conclusions! The$results$obtained$here$with$the$present$PEO3PSO$block$copolymers$highlight$the$ role$ of$ a$ judicious$ choice$ of$ the$ hydrophobic$ block$ and$ the$ relative$ block$ lengths$ to$ achieve$ a$ successful$ cytotoxic$ effect$ of$ the$ drug3loaded$ polymeric$ nanocarriers$ on$ cancer$ cells.$ Aqueous$ solutions$ of$ block$ copolymers$ EO33SO14EO33$ and$ EO38SO10EO38$ self3assemble$ at$ very$ low$ concentrations$ to$ form$ cytocompatible$ spherical$ micelles$ suitable$for$sterilizing$filtration$and$administration$by$parenteral$route.$These$polymeric$ micelles$efficiently$entrap$DOXO$and$display$a$release$profile$with$an$initial$burst$phase$ at$ very$ short$ times$ followed$ by$ a$ more$ sustained$ rate.$ The$observed$ pH3dependent$ release$ may$ prompt$ drug$ accumulation$ in$ tumoral$ tissues$ due$ to$ the$ acidic$ pH$ conditions$ in$ both$ cell$ membrane$ surroundings$ (caused$ by$ hypoxia)$ and$ in$ some$ intracellular$compartments$(i.e.,$lysosomes).$The$cytotoxic$activity$of$the$DOXO3loaded$ polymeric$ micelles$ is$ exclusively$ ascribed$ to$ the$ DOXO$ therapeutic$ action.$ Confocal$ microscopy$images$showed$that$DOXO$progressively$accumulates$inside$the$MDR$cells$ (evading$efflux$pumps),$prolonging$their$residence$inside$and,$consequently,$enhancing$ cytotoxicity$over$that$observed$for$free$DOXO$solutions.$Moreover,$EO33SO14EO33$and$ EO38SO10EO38$unimers$ have$ certain$ ability$ to$ inhibit$ P3gp$ efflux$ pump,$ as$ occurs$ for$ some$ Pluronic®$ and$ Tetronic®$block$ copolymers.$ Hence,$ the$ role$ of$ the$ present$ copolymers$evolve$from$plain$“inert$drug$nanocarriers”$to$relevant$“biological$response$ inducers”$as$a$consequence$of$complementation$of$the$drug3cytotoxic$activity$with$a$ moderate$ inhibition$ of$ the$ P3gp$ activity,$ which$ highlights$ the$ complexity$ of$ the$ cell$ response$to$the$presence$of$the$block$copolymer$micelles.$$ $ 2.4.6!!References! ! 1. 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Exner,$A.A.;$Krupka,$T.M.;$Scherrer,$K.;$Teets,$J.M.$J.$Controlled$Release,$2005,$ 106,$188.$ ! ! ! ! ! ! ! ! ! ! ! ! ! 84 2.5$SUPPORTING$INFORMATION$FOR$POLY$ (STYRENE$ OXIDE)$ 3$ POLY(ETHYLENE$ OXIDE)$ BLOCK$ COPOLYMERS:$ FROM$ “CLASSICAL”$ CHEMOTHERAPEUTIC$ NANOCARRIERS$ TO$ ACTIVE$CELL3RESPONSE$INDUCERS$ ! ! 2.5.1!!Materials!and!methods! ! Drug# solubilisation:$ DOXO$ was$ added$ in$ excess$ to$ 0.2$ wt%$ copolymer$ solutions.$ The$ systems$were$kept$under$magnetic$stirring$at$37$ºC$for$3–5$days$and$then$were$filtered$ (Millipore,$ 0.45$ µm).$ The$ filtered$ solutions$ were$ diluted$ (1/1000)$ with$ methanol$ to$ disrupt$ the$ self3assembled$ structures,$ the$ amount$ of$ water$ after$ dilution$ being$ low$ enough$to$allow$direct$use$of$a$calibration$plot$for$methanol$medium.$The$amounts$of$ DOXO$solubilised$were$determined$spectrophotometrically$at$480$nm$(Cary$50$UV3Vis$ spectrophotometer,$ Agilent,$ Germany).$ Solutions$ of$ each$ copolymer$ at$ the$ same$ dilution$were$used$as$a$blank.$In$order$to$confirm$that$solubilisation$was$predominantly$ in$the$core$rather$than$in$the$EO3block$corona,$the$apparent$drug$solubility$in$5–30$wt%$ aqueous$ solutions$ of$ polyethylene$ glycol$ (Mn$6000$ g$ mol31)$ was$ determined$ as$ previously$ reported$ (S1).$ The$ minimum$ solubilization$ observed$ indicate$ that$ only$ residual$solubilisation$occurs$by$incorporation$in$the$corona.$$$ # $ Drug$ loaded,$ D.L.,$ and$ entrapment$ efficiency,$ E.E.,$ in$ the$ micelles$ were$ calculated$as$follows:$ $ $ $ $ $ (1)$ $ $ $ $ (2)$ $ The$solubilisation$capacity$(SCP)$was$estimated$as$the$ratio$between$the$amount$ of$drug$dissolved$at$37$ºC$in$100$ml$of$copolymer$solution$and$the$amount$dissolved$in$ the$same$volume$of$water.$ $ Physical# stability# of# the# drug:loaded# micelles# upon# dilution:# DLS#measurements$ were$ made$at$a$scattering$angle$ θ $=$90°$to$the$incident$beam.$Experiment$duration$was$in$the$ range$ 5310$ min,$ and$ each$ experiment$ was$ repeated$ at$ least$ twice.$ The$ correlation$ %100 drug +polymer ofweight solutionmicellar in drug theofweight .%.×=LD %100 drug feeding ofweight solutionmicellar in drug theofweight .%.×=EE 85 functions$from$DLS$runs$were$analyzed$by$the$CONTIN$method$to$obtain$the$intensity$ distributions$of$decay$rates$( Γ ),$from$which$the$apparent$diffusion$coefficients$(Dapp$=$ Γ # /q2,$q#=$(4 π ns/ λ )sin( θ /2))$were$derived$(being$ns$the$refractive$index$of$solvent).$Values$ of$the$apparent$hydrodynamic$radius$(rh,app)$were$calculated$from$the$Stokes3Einstein$ equation# $$$$rh,app#=$kT/(6 πη Dapp)$$$$$(3)$ where$k$is$the$Boltzmann$constant$and$ η $is$the$viscosity$of$water$at$temperature$T.$ $ Copolymer# cytocompatibility# evaluation.# The$ cytocompatibility$ of$ the$ bare$ copolymer$ micelles$was$first$assessed$using$BALB/3T3$clone$A31$mouse$embryonic$fibroblast$cells$ (CCL$163,$ATCC),$following$a$previously$reported$procedure$(S2).$Cells$were$trypsinated$ and$ cultured$ in$ 963well$ plates$ (2·∙104$ cells/well).$ Autoclaved$ copolymer$ solutions$ in$ phosphate$ buffer$ pH$ 7.4$ (final$ copolymer$ concentration$ 0.1,$ 0.5,$ 1.0$ or$ 1.7%)$ were$ added$and$the$cells$incubated$for$24$h.$The$medium$was$replaced$by$fresh$one$(200$µL)$ containing$MTT$solution$(20$µL,$5$mg/mL)$and$the$well$plates$were$incubated$for$4$h$ (37ºC,$5%$CO2).$Immediately$after$incubation,$the$supernatant$was$removed,$formazan$ crystals$ were$ dissolved$ (0.1N$ HCl$ in$ anhydrous$ isopropanol)$ and$ the$ absorbance$ measured$within$1$h$using$a$microplate$reader$(BIORAD$Model$680,$USA)$at$570$nm.$ Cells$exposed$to$copolymer3free$culture$medium$were$used$as$negative$control$(100%$ viability).$Cell$viability$was$quantified$as:$ $ $$%$viability$=$(Abssample$/Abscontrol)$x$100$$$(4)$ $ where$Abssample$and$Abscontrol$represent$the$absorbances$of$the$sample$of$cell$culture$in$ the$presence$and$in$the$absence$of$copolymer,$respectively.$The$assay$was$carried$out$ in$ triplicate.$ Cell$ survival$ was$ also$ evaluated$ monitoring$ the$ release$ of$ lactate$ dehydrogenase$(LDH)$using$the$cytotoxity$detection$KitPLUS$(Roche,$Spain).$Triton$X3100$ (0.1%)$ and$ copolymer3free$ culture$ medium$ were$ used$ as$ positive$ control$ (total$ cell$ death)$ and$ blank,$ respectively.$ The$ viability$ (%)$ was$ determined$ from$ absorbance$ measurements$at$490$nm$according$to$the$kit$instructions.$$ $ Cellular# uptake# of# DOXO# after# incubation# with# empty# polymeric# micelles# (P:gp# inhibition).#DOXO$accumulation$in$the$absence$and$the$presence$of$copolymer$unimers$ and$micelles$was$tested$by$using$MDR$NCI3ADR/RES$cells$and$MCF37$(American$Type$ Culture$ Collection,$ MD,$ USA)$ seeded$ in$ a$ 243wells$ plate$ (1.5x105$cells/well,$ 1000$ μL/well)$in$supplemented$medium$for$48$h$following$a$previously$reported$method$(S3).$ The$ medium$ was$ replaced$ by$ serum3free$ DMEM$ containing$ 43(23hydroxyethyl)313 piperazineethanesulfonic$acid$(HEPES,$25$mM,$pH=7.4).$Polymer$samples$were$added$ (20$µL)$and$cells$incubated$at$37ºC$for$30$min.$Polymer3free$medium$and$VER$solution$ (100$µM)$were$used$as$blank$and$positive$control,$respectively.$Then,$50$µl$of$a$DOXO$ 86 solution$(100$µM$in$water)$was$added$and$the$samples$incubated$for$60$additional$min.$ The$medium$was$removed$and$the$cells$washed$(PBS,$3$x$500$µL)$to$eliminate$DOXO$ and$copolymer$residues.$Cells$were$lysed$(1%$Triton$X3100,$300$µL,$20$min),$supernatant$ aliquots$(200$µL)$transferred$to$opaque$963well$plates,$and$the$fluorescence$measured$ in$ a$ plate$ reader$ (λexc$ =$ 485$ nm;$ λem$ =$ 580$ nm;$ Tecan$ Ultra$ Evolution,$ Männedorf,$ Switzerland).$The$remaining$100$µl$were$103fold$diluted$with$PBS$and$protein$content$ was$measured$using$Bradford$method.$DOXO3free$medium$was$used$as$blank.$DOXO$ concentrations$ were$ calculated$ using$ a$ calibration$ curve$ (0.2$ pmol30.2$ nmol,$ R2$ =$ 0.997).$Determinations$were$carried$out$three$separate$times,$each$in$triplicate.$Data$of$ DOXO$ concentration$ were$ normalized$ to$ the$ protein$ content$ in$ each$ well.$ DOXO$ accumulation$factors$were$calculated$as$follows:$ $ fDOXO$=$ADs/AD0$$$$$$(5)$ $ ADs$and$AD0$being$the$accumulated$DOXO$for$the$sample$and$the$basal$AD$obtained$ with$a$DOXO$solution$in$absence$of$polymer$or$VER.$Statistical$significance$was$analyzed$ applying$ANOVA$(post$hoc$Dunnet´s$T3)$with$SPSS$15.1$software.$ Additionally,$confocal$microscopy$analysis$was$carried$out$by$seeding$the$NCI3 ADR/RES$cells$on$coverslips$in$a$243wells$plate$(1.5x105$cells/well,$1000$μL/well)$in$RPMI$ 1640$ medium$ with$ 2$ mM$ L3glutamine,$ 10%$ FBS$ and$ 1%$ penicillin/streptomycin$ over$ sterile$glass$covers$(from$Invitrogen).$After$48$hours$the$culture$medium$was$replaced$ with$RPMI$1640$containing$HEPES$25$mM$(pH$7.4).$The$cells$were$incubated$at$37ºC$for$ 30$minutes$with$50$μl$of$VER$100$μM$or$0.2$wt.%$polymeric$dispersions.$Then,$DOXO$ (50$μM,$50$μl)$was$added$and$the$cells$incubated$for$another$60$minutes$at$37ºC.$The$ formulations$ were$ removed$ and$ the$ cells$ were$ washed$ three$ times$ with$ phosphate$ saline$buffer$pH$7.4$(PBS,$Sigma)$and$then$fixed$with$paraformaldehyde$4%$for$10$min,$ washed$ and$ stained$ with$ Bodipy®$ phalloidin$ (30µl/ml)$ in$ 0.2%$ Triton$ X3100$ (permeabilizer).$The$cells$were$washed$again$with$PBS$pH$7.4$(3x10$min),$mounted$on$ glass$ slides$ using$ anti3fading$ solution$ (Bio3Rad$ laboratories,$ Hercules,$ CA,$ USA),$ and$ visualized$at$20X$and$63X$using$a$Confocal!Espectral!Microscope!Leica!TCSISP2$(LEICA# Microsystems#Heidelberg#GmbH,#Mannheim,#Germany);$green$channel$for$doxorrubicin$ (λexc.$561nm)$and$red$channel$for$Bodipy®$Phalloidin$(λexc.$633$nm).$ $ Cellular#uptake#of#calcein#AM#after#incubation#with#the#copolymers:$NCI3ADR$RES$cells$ were$ seeded$ in$ black$ 963well$ pretreated$ plates$ (1$ ×$105$ cells/well)$ and$ cultured$ in$ supplemented$medium$for$48$h.$The$medium$was$replaced$by$serum3free$RPMI$1640$ containing$43(23hydroxyethyl)313piperazineethanesulfonic$acid$(HEPES,$25$mM,$pH=7.4).$ Aliquouts$ of$ copolymer$ solutions$ were$ added$ (20$ µL,$ 0.01$ and$ 0.2$ wt.%$ final$ concentrations)$and$the$cells$incubated$at$37°C$for$30$min.$Then,$0.25$µM$calcein3AM$ was$added$and$the$cells$incubated$for$other$30$min$at$37ºC.$The$medium$was$removed,$ the$cells$washed$(PBS,$3$x$100$µL)$and$the$intracellular$fluorescence$of$calcein$measured$ 87 in$a$plate$reader$(λexc$=$485$nm;$λem=$535$nm)$at$5$minutes$intervals$during$1$h$in$order$ to$attain$stable$fluorescence$lectures.$Cells$were$lysed$(1%$Triton$X3100,$100$µL)$and$the$ protein$ content$ was$ measured$ according$ to$ the$ Bradford$ method.$ The$ experiments$ were$ repeated$ four$ times.$ The$ calcein$ accumulation$ in$ the$ cells$ was$ expressed$ as$ fluorescence$relative$units$(RFU)$of$calcein$/mg$of$protein,$as$follows:$ # CA$=Ct/C0$$$$$(6)$ $ Ct$and$C0$being$the$calcein$accumulation$in$the$presence$and$absence$of$the$copolymer,$ respectively.$Statistical$significance$was$analyzed$applying$ANOVA$(post$hoc$Dunnet´s$ T3;$SPSS$15.1$software).$ $ In# vitro# cytotoxicity# of# drug# loaded:polymeric# micelles.# Crystal# violet# assay:# At$ the$ selected$ time$ points,$ the$ culture$ medium$ was$ removed$ and$ cells$ were$ fixed$ with$ glutaraldehyde$(10$μl,$11%)$for$15$min$and,$then,$washed$to$eliminate$glutaraldehyde$ residues.$Cells$were$stained$with$100$μl$of$crystal$violet$solution$0.1%$in$pH$6$buffer$ (orthophosphoric$ acid$ 200$ mM,$ formic$ acid$ 200$ mM,$ and$ 23N3morpholine3 ethanesulfonic$acid$200$mM)$at$room$temperature$for$15$min,$and$then$washed$with$ distilled$water$and$dried.$Finally,$cells$were$treated$with$100$μl$of$10%$acetic$acid$at$ room$temperature$for$15$min$under$gentle$stirring$and$the$absorbance$was$measured$ in$a$plate$reader$(λ$=$595$nm;$Tecan$Ultra$Evolution).$Copolymer3free$PBS$was$used$as$a$ control$and$showed$234%$inhibition$with$respect$to$cells$incubated$in$serum3containing$ medium.$ Experiments$ were$ carried$ out$ in$ triplicate.$ The$ growth$ inhibition$ was$ quantified$as:$# $$$$(7)$ $ AO$and$AT$being$the$absorbances$of$the$sample$and$of$the$PBS$control,$respectively.$$ $ 2.5.2! !Modellization!of!release!profiles$$ $ Drug$ release$ profiles$ from$ the$ micellar$ systems$ were$ fitted$ to$ the$ following$ Fickian$diffusion$model$considering$the$micelles$as$perfect$spheres$(S4):$ $ ####Mt/M∞=#k1+#k2·∙t0.5#:#k3·∙t$$$$(8)$ $ where$ Mt#and$ M∞$ represent$ the$ drug$ amount$ released$ at$ time$ t$ and$ that$ initially$ contained$in$the$formulation,$respectively,$and$k1,$k2$and$k3$are$release$rate$coefficients.$ This$model$ takes$ into$account$ drug$ diffusion,$ conformational$ changes$in$ the$ micellar$ structure$during$release$and$partial$transfer$of$drug$from$one$micelle$to$another.$The$ model$fitted$well$the$release$profiles$(Table$4,$R2>0.90),$unlike$the$simpler$square3root$ model$which$could$ not$explain$ the$whole$profiles.$The$coefficient$associated$to$ drug$ )/100(100%ATAOinhibition ⋅−= 88 diffusion,$k2,$became$larger$as$the$pH$decreased$from$7.4$to$5.5$for$both$copolymers,$as$ expected$for$an$enhanced$out3diffusion$process$of$the$reprotonated$DOXO$under$acidic$ conditions$(Supplementary$Material,$Table$S2).$ $ 2.5.3!Calcein!accummulation!studies! ! Calcein3AM$ tests$ were$ carried$ out$ to$ gain$ an$ insight$ into$ the$ mechanism$ of$ inhibition$of$P3gp$pump$(S5).$Calcein3AM$is$a$lipid$soluble$dye$recognized$as$a$substrate$ for$ both$ P3gp$ and$ MRP$ transporters$ (S6).$ Upon$ entering$ cells,$ endogenous$ esterases$ cleave$calcein3AM$to$form$the$hydrophobic$fluorescent$calcein.$ABC3transporters$cause$ rapid$efflux$of$calcein3AM,$but$they$cannot$expel$calcein$once$formed$inside$the$cells$ (S7).$ Although$ EO33SO14EO33$ and$ EO38SO10EO38$ enhanced$ DOXO$ accumulation$ in$ NCI3 ADR$ RES$ cells,$ they$ did$ not$ increase$ the$ accumulation$ of$ calcein.$ At$ copolymer$ concentration$ of$ 0.01$ wt.%,$ calcein$ accumulation$ was,$ respectively,$ 0.80$ ±$ 0.16$ and$ 0.92$ ±$ 0.15,$ and$ at$ 0.2$ wt.%$ it$ was$ 0.89$ ±$ 0.20$ and$ 0.73$ ±$0.103fold$ the$ basal$ level$ obtained$for$non3pretreated$cells.$This$finding$may$be$the$result$of$several$concomitant$ facts.$One$possibility$is$that,$although$MRP1$is$scarcely$found$in$NCI/ADR3RES$cells$(S8),$ the$efficient$inhibition$of$P3gp$pump$exerted$by$the$copolymers$may$favor$calcein$efflux$ by$ MRP1$ and$ MRP2,$ as$ observed$ by$ Evers$ and$ coworkers$ for$ Pluronic$ L61$ (S9).$ Batrakova$ and$ Kabanov$ (S10)$ found$ that$ Pluronic$ P85$ can$ inhibit$ P3gp$ drug$ efflux$ system,$but$cause$an$only$partial$inhibition$on$MRP$activity.$Tetronics$have$shown$no$ inhibition$of$MRP1$in$hepatocarcinoma$cell$lines$(S11).$Furthermore,$it$might$occur$that$ since$ the$ P3gp$ pump$ has$ several$ binding$ domains$ [S12],$ different$ substrates$ may$ interact$with$different$sites$of$the$protein$and$the$same$may$happen$with$the$inhibitors.$ For$example,$Pluronic$F127$cannot$block$the$binding$of$nelfinavir$to$the$pump,$while$it$ effectively$inhibits$verapamil$association$(S13).$This$phenomenon$makes$the$prediction$ of$ the$ inhibitory$ activity$ of$ a$ pump$ with$ respect$ to$ a$ specific$ substrate$ by$ a$ certain$ polymer$ very$ complex.$ On$ the$ other$ hand,$ the$ micellar$ encapsulation$ of$ calcein3AM$ could$contribute$to$a$lower$accumulation$(S14).$The$inherent$property$of$the$unimers$to$ self3assemble$as$micelles$could$lead$to$a$strong$trapping$of$calcein3AM$outside$the$cells.$ The$ high$ affinity$ of$ the$ calcein3AM$ for$ the$ lipophilic$ core$ provided$ by$ styrene$ oxide$ block$ could$ even$ prompt$ the$ self3assembling$ of$ more$ unimers$ into$ micelles,$ as$ previously$observed$for$other$copolymers$and$hydrophobic$drugs$(S15).$Nevertheless,$ these$aspects$would$require$further$elucidation.$ $ $ $ $ ! 89 Table! S1:! Critical$micelle$ concentration,$ cmc,$ expansion$ factor,$ δt;# micellar$ molecular$ mass,$ Mw;$ hydrodynamic$ radius,$ rh;$ association$ number,$ Nw;$ and$ number$ of$ water$ molecules$per$EO$group,$nwater,$of$EO33SO14EO33$and$EO38SO10EO38$at$37$ºC.$! Copolymer$ cmc# (wt%)$ # δ t# #Mw$ (mol$g31)$ #rh# (nm)$ #Nw# !nwater# EO33SO14EO33$ 2.5·∙1033$ 4.7$ 17.8·∙104$$$ 6.2$$ 37$$ 14$ EO38SO10EO38$ 3.7·∙1033$ 3.5$ 6.9·∙104$$$ 6.2$ 14$$ 9$ ! Table! S2:! Results$ of$ the$ fitting$ to$ equation$ 8$ of$ the$ DOXO$ release$ profiles$ from$ EO33SO14EO33$and$EO38SO10EO38$ micellar$solutions$in$pH$5.5$and$7.4$media$without$or$ with$ 10%$ FBS.$ The$ release$ rate$ coefficients$ are$ given$ as$ mean$ values,$ with$ standard$ deviations$in$parenthesis.$ Copolymer$ pH$ FBS$ (%)$ k1$ k2$ k3$ *F2,8d.f$ *P3 value$ R2$ E33SO14EO33$ $ 5.5$ 0$ 5.28$ (10.56)$ 27.48$ (8.22)$ 33.22$ (1.38)$ 16.77$ 0.0114$ 0.8934$ 7.4$ 0$ 0.06$ (2.71)$ 11.24$ (2.11)$ 31.20$ (0.35)$ 56.85$ 0.0012$ 0.9660$ E38SO10EO38$ $ 5.5$ 0$ 30.32$ (3.68)$ 13.76$ (2.86)$ 31.28$ (0.48)$ 65.29$ 0.0009$ 0.9703$ 7.4$ 0$ 5.99$ (3.38)$ 11.68$ (2.63)$ 31.39$ (0.44)$ 28.59$ 0.0043$ 0.9346$ E33SO14EO33$ $ 5.5$ 10$ 11.08$ (4.67)$ 17.28$ (2.81)$ 31.32$ (0.31)$ 44.16$ 0.0000$ 0.9169$ 7.4$ 10$ 2.10$ (3.00)$ 12.46$ (2.03)$ 31.16$ (0.25)$ 34.23$ 0.0001$ 0.8954$ E38SO10EO38$ $ 5.5$ 10$ 31.67$ (1.85)$ 21.22$ (1.11)$ 31.37$ (0.12)$ 649.67$ 0.0000$ 0.9939$ 7.4$ 10$ 30.14$ (2.00)$ 13.25$ (1.35)$ 31.13$ (0.17)$ 112.34$ 0.0000$ 0.9656$ *$From$ANOVA$of$the$regression.$ ! ! ! ! ! ! ! 90 Table! S3:! DOXO$ accumulation$ factors$ (fDOXO)$ attained$ after$ preincubation$ of$ drug3 sensitive$and$drug3resistant$cells$for$30$min$with$the$copolymers$before$adding$DOXO$to$ the$medium$(final$drug$concentration$50$μM),$and$P3gp$ATPase$activity$recorded$using$ the$Pgp3Glo$assay$system.$Mean$values$and,$in$parenthesis,$standard$deviations$of$three$ independent$experiments.$Cell$preincubation$with$VER$100$mM$led$to$1.043fold$(1.05$ nmolDOXO/mg$ protein)$ and$ 2.123fold$ (6.8$ nmolDOXO/mg$ protein)$ increase$ in$ DOXO$ accumulation$ in$ MCF37$ and$ NCI3ADR3RES$ cells,$ respectively.$ VER$ 12$ mM$ resulted$ in$ 2.683fold$increase$in$ATPase$activity.$*P<0.05;$**P<0.01.$ $ Parameter$ EO33SO14EO33$ EO38SO10EO38$ Pluronic®$P85$ 0.001%$ 0.01%$ 0.20%$ 0.001%$ 0.01%$ 0.20%$ 0.001%$ 0.01%$ 0.20%$ fDOXO$in$MCF3 7$cells$ 1.52$ (0.18)$ 1.18$ (0.28)$ 1.09$ (0.22)$ 1.13$ (0.22)$ 1.06$ (0.22)$ 1.02$ (0.19)$ 1.16$ (0.28)$ 1.07$ (0.26)$ 1.03$ (0.19)$ fDOXO$in$NCI3 ADR3RES$cells$ 1.56**$ (0.37)$ 1.44**$ (0.24)$$ 1.58**$ (0.18)$$ 1.54**$ (0.34)$ 1.47**$ (0.26)$ 1.34**$ (0.12)$ 1.96**$ (0.36)$ 2.18**$ (0.36)$ 2.23**$ (0.56)$ P3gp$ATPase$ activity$ 0.15**$ (0.20)$ 0.30**$ (0.13)$ 0.55$ (0.20)$ 1.17$ (0.26)$ 0.84$ (0.25)$ 0.86$ (0.19)$ 0.42*$ (0.28)$ 0.66$ (0.22)$ 1.37$ (0.22)$ $ Table! S4:$IC50$(µM)$of$DOXO.$Mean$values$and,$in$parenthesis,$standard$error$of$the$ mean$(n=3).$ $ Cell$line$ 24$h$ 48$h$ MCF37$cells$ 7.61$(0.70)$ 0.97$(0.13)$ NCI3ADR3RES$cells$ 20$(2)$ 31$(3)$ $ $ 0.1 110 100 1000 0.0 0.5 1.0 Intensity (a.u.) rh, app (nm) a) $ Figure! S1:! a)$ Intensity$ fraction$ size$ distributions$ of$ EO33SO14EO33$ (−)$ and$ EO38SO10EO38(·∙·∙·∙·∙)$ at$ a$ concentration$ of$ 90$ g$ dm33$ $ and$ 37$ ºC;$ b)$ TEM$ image$ of$ EO38SO10EO38$micelles$.! $ b) 97! ! 3.1!AIM!OF!THE!WORK! ! ! In!the!previous!chapter!two!triblock!copolymers!were!synthesized!and!tested!as! drug!nanocarriers!because!their!similar!structure!to!Pluronics,!one!of!the!most!deeply! studied!copolymers!family!up!to!day!in!nanopharmaceutics.!ESE!copolymers!have!a!very! hydrophobic!central!block!that!implies!lower!cmc!values!and,!as!a!consequence,!lower! polymer!concentrations!needed!to!form!micelles!and!en!capsulate!and!release!similar! cargo!amounts.!In!addition,!the!block!lengths!and!E/S!ratio!of!these!copolymers!were! chosen! to! attain! an! optimal! compromise! between! chain! solubility,! micelle! formation! ability,! and! core! size! that! lead! to! an! enhanced! drug! solubility,! while! ensuring! renal! clearance!of!unimers!as!required!for!nonObiodegradable!polymers!(1O4).! ! In! order! to! improve! the! properties! related! to! micellar! nanocarriers,! specially! micelle! formation! ability,! we! have! analyzed! the! potential! use! of! telechelic! block! copolymers!generally!used!use!as!associative!thickeners!as!potential!drug!nanocarriers,! new! reverse! triblock! copolymers! formed! by! an! inner! very! long! poly(ethylene! oxide)! (PEO)!hydrophilic!and!two!side!poly(butylene!oxide)!(PBO)!blocks!were!synthesized!and!! tested,!in!which!the!poly(butylene!oxide)!(PBO)!is!less!hydrophobic!than!PSO!ones!(in!a! ratio!of!1:1.5!based!on!cmc!value!of!structurally!related!linear!block!coolymers!of!the! two! families! (see! Figure! 1)! (5,! 6).! Nevertheless,! by! using! reverse! triblock! copolymers! having!one!central!very!long!hydrophilic!block!(PEO)!and!two!side!hydrophobic!blocks,! an!increase!in!hydrophobicity!needed!to!improve!the!drug!solubility!is!expected.!Owing! to!have!longer!and!less! hydrophobic! blocks,! bigger! nuclei! are!expected,!which!would! also!be!related!to!an!increase!in!drug!solubility!ability.! ! Five! PBOOPEOOPBO! copolymers! were! synthesised! by! sequential! oxyanionic! polymerization.!To!ensure! aqueous!solubility,!they!were! provided!with!very! long!PEO! central! block.! These! polymers! were! designed! with! different! PBO! block! lengths! and! PEO/PBO! ratios! with! the! aim! to! correlate! the! block! composition! and! length! with! the! observed!physicoOchemical!properties!in!solution!(7).!Aggregation!properties!in!aqueous! solutions!at!low!concentrations!are!the!first!focus!point,!provided!that!these!copolymers! could!form!unimolecular!micelles!as!diblock!EOOBO!copolymers!do!(5).!As!well!as!EOOSOO EO! copolymers,! BOOEOOBO! ones! are! expected! to! form! spherical! micelles! in! aqueous! solution! but! with! a! very! loosely! corona! (the! soOcalled! flowerOlike! micelles)! with! an! optimal! size! for! intravenous! injection! of! 10O30! nm! (8).! Micelles! have! a! core–shell! structure! as! expected,! where! the! hydrophobic! core! might! act! as! a! drug! reservoir! for! hydrophobic! drugs! and! the! PEO! shell! should! contribute! to! extend! blood! circulation! 98! ! times.![9,!10].!As!a!final!goal,!reverse!triblock!copolymers!are!expected!to!gel!under!low! concentrations! and/or! temperature! in! order! to! create! drug! depots! and! achieve! a! prolonged!sustained!release,!maintaining!the!therapeutic!effect!for!weeks!or!months.! ! ! ! Figure!1.!Constituent!monomers!of!PBOOPEOOPBO!block!copolymers.! ! ! 3.1.1!Aim!of!the!work! ! ! In!the!present!work,!we!report!on!the!synthesis!and!characterization!of!the!selfO assembly! properties! of! five! new! triblock! PBOOPEOOPEO! copolymers! with! lengthy! PEO! blocks! (see! Figure! 1):! BO8EO90BO8,! BO12EO227O12,! BO14EO378BO14,! BO20EO411O20! and! BO21EO385O21! (where! the! subscripts! denote! the! block! lengths).! To! reveal! the! selfO aggregation!properties!for!these!polymers!in!aqueous!solution!is!the!previous!step!to! test! the! ability! of! this! class! of! copolymers! to! encapsulate,! protect,! and! ensure! a! sustained!release!of!the!loaded!drug!(doxorubicin),!which!are!the!main!goals!to!achieve! in!this!part!of!the!work.!Also,!the!cytotoxicity!of!empty!and!drugOloaded!micelles!was! tested!in!order!to!verify!the!biocompatibility!of!the!different!types!of!micelles!and!to! ensure!their!therapeutic!activity.! ! 3.1.2!Methodology! ! !PhysicoOchemical!characterization!was!performed!by!means!of!isothermal!titration! 99! ! calorimetry,! UVOvis! and! fluorescence! spectroscopy,! light! scattering,! transmission! electron!microscopy!(TEM),!confocal!microscopy!and!rheometry.!! ! ! !Briefly,! five! PBOOPEOOPBO! triblock! copolymers! with! lengthy! inner!hydrophilic! blocks!and!relatively!longer!side!hydrophobic!blocks!than!those!previously!reported!(11O 13),! have! been! designed! and! synthesized! in! order! to! obtain! polymeric! micelles! at! relatively!low!concentrations.!The!present!block!copolymers!have!been!characterized!to! exactly! determine! their! structural! conformation,! composition! block! length! and! purity! (polydispersity! index).! A! deep! physicoOchemical! study! on! their! aqueous! solution! properties! in! a! broad! copolymer! concentration! range! was! performed,! from! dilute! solutions!to!gel!structures.!The!micellization!process!was!carefully!analyzed!in!order!to! verify!or!neglect!the!existence!of!unimolecular!micelles!as!an!intermediate!aggregation! step! between! unimers! and! polymolecular! micelles,! previously! observed! in! PEOOPBO! diblock!copolymers!(14,!15).!In!addition,!the!spherical!shape!of!polymolecular!micelles! was!verified,!as!well!as!other!micellar!properties!as!the!hydrophobic!radius,!molecular! micellar!weight!and!aggregation!number,!amongst!others.!Phase!diagrams!showing!the! clouding!point!and!the!gelation!temperatures!were!performed!in!order!to!complete!the! polymer!aggregation!map!from!dilute!aqueous!solutions!to!physical!gels!(11O13,!16).!To! fulfill!the!study!about!the!aggregation!properties!of!PBOOPEOOPBO!triblock!copolymers,! the! gel! region! was! deeply! analyzed! (10,17).! Storage! and! loss! modulus! (G´! and! G´´,! respectively),! which! can! be! ascribed! to! different! internal! forces! of! the! polymeric! network,! were! analysed! under! varying! temperature! and! frequency! conditions.! To! complete!the!rheological!study,!master!curves!were!constructed!to!extend!their!profile! to!frequencies!that!are!not!achievable!by!the!rheometer.!These!curves!permit!to!extract! a! common! rheological! profile! independent! on! the! structure/composition! of! the! corresponding!polymer.!! ! ! Efficiency! entrapment! tests! were! performed! with! the! different! copolymers! in! order!to!correlate!the!copolymer!composition!with!their!capabilities!as!drug!reservoirs,! in! particular,! of! the! chemotherapeutic! drug! doxorubicin.! Colloidal! stability! and! drug! release! experiments! with! the! three! most! optimal! drug/copolymer! formulations! into! different! buffer! media! were! also! performed.! Despite! some! additional! studies! were! previously! performed! in! order! to! reveal! the! drug! solubilisation! ability! of! these! copolymers! (18,19),! there! are! not! studies! regarding! cytotoxicity,! cytocompatibility,! cellular!uptake!mechanisms.!Here,!the!cytocompatibility!of!these!block!copolymers!was! tested!against!BALB/3T3!clone!A31!mouse!embryonic!fibroblast!cells!(CCL!163,!ATCC),! which!is!highly!sensitive!to!the!presence!of!toxic!species.!Citotoxicity!and!bioavailability! of!empty!and!drugOloaded!micelles!were!also!analyzed!in!an!ovarian!MDR!NCIOADR/RES! cell! line! in! order! to! achieve! an! optimal! chemocytotoxic! effect! and! to! confirm! their! 100! ! potential!use!as!effective!and!safe!drug!delivery!systems.!Finally,!the!capability!of!two!of! the! present! PBOOPEOOPBO! copolymers! as! potential! POglycoprotein! efflux! pump! inhibitors!to!enhance!doxorubicin!accumulation!in!the!same!ovarian!MDR!NCIOADR/RES! cell!line!was!confirmed!and!compared!to!that!observed!for!other!block!copolymers.! ! 3.1.3!References! ! 1.! Yamamoto,! Y.;! Nagasaki,! Y.;! Kato,! Y.;! Sugiyama,! Y.;! Kataoka,! K.! J.! Controlled! Release,!2001,!77,!27.! 2.! Crothers,!M.;!Zhou,!Z.;!Ricardo,!Nagila!M.P.S.;!Yang,!Z.;!Taboada,!P.;!Chaibundit,! C.;!Attwood,!D.;!Booth,!C.!Int.!J.!Pharm.!2005,!293,!91.! 3.! Taboada,! P.;! Velasquez,! G.;! Barbosa,! S.;! Castelletto,! V.;! Nixon,! S.! K.;! Yang,! Z.;! Heatley,!F.;!Hamley,!I.W.;!Ashford,!M.;!Mosquera,!V.!Langmuir!2005,!!21,!5263.! 4.!Juárez,(J.;(Taboada,!P.;!Valdez,!M.A.;!Mosquera,!V.!Langmuir!2008,!24,!7107.! 5.! Booth,!C.;!Attwood,D.;!Price,!C.!Phys.!Chem.!Chem.!Phys.!2006,!8,!3612.! 6.! Attwood,! D.;! Booth,! C.;! Yeates,! S.G.;! Chaibundit,! C.;! Ricardo,! N.M.P.S.! Int.! J.! Pharm.!2007,!345,!!35.! 7.! Booth,!C.;!!Attwood,!D.!Macromol.!Rapid!Commun.!2000,!!21,!!501.! 8.! Heidel,!J.;!Davis,!M.!Pharm.!Res.!2011.,!28,!187.! 9.! Branco,!M.C.;!Schneider,!J.P.!Acta!Biomaterialia,!2009,!5,!817.! 10.! Alexandridis,! P.;! Lindman,! B.!Amphiphilic! Block! Copolymers.! Self4Assembly! and! Applications.!2000,!Amsterdam:!Elsevier.! 11.! Mistry,!D.;!Annable,!T.;!Yuan,!X.OF.;!Booth,!C.!Langmuir!2006,!22,!2986.! 12.! Kelarakis,!A.;!Yuan,!X.OF.;!Mai,!S.OM.;!Yang,!Y.OW.;!Booth,!C.!Phys.!Chem.!Chem.! Phys,!2003,!5,!2628.! 13.!Zhou,!Z.;!Yang,!Y.OW.;!Booth,!C.;!Chu,!B.!Macromolecules!1996,!29,!8357.! 14.! Ribeiro,!M.E.N.P.;!de!Oliveira,!S.!A.;!Ricardo,!N.M.P.S.;!Mai,!S.OM.;!Attwood,!D.;! Yeates,!S.G.;!Booth,!C.!Int.!J.!Pharm.!2008,!362,!193.! 15.! Kelarakis,!A.!!Havredaki,!V.;!Yu,!G.OE.;!Derici,!L.;!Booth,!C.!Macromolecules!1998,! 31,!944.! 16.!Liu,!T.;!Nace,!V.M.;!Chu,!B.!J.!Phys.!Chem.!B!1997,!101,!8074.! 17.! Booth,!C.;!Price,!C.!Polymer!Characterization.!Comprehensive! Polymer!Science.! Vol.!1.!1989,!Exeter:!Pergamon!Press.! 18.! Ribeiro,! M.E.;! Cavalcante,! I.! M.;! Ricardo,! N.M.P.S.;! Mai,! S.OM.;! Attwood,! D.;! Yeates,!S.G.;!Booth,!C.!Int.!J.!Pharm.!2009,!369,!196.! 19.! Elsabahy,!M.;!Perron,!M.OE.!Bertrand,!N.;!Yu,!G.;!Leroux,!J.OC.!Biomacromolecules! 2007,!8,!2250.! ! 101 3.2$ MICELLISATION$OF$TRIBLOCK$COPOLYMERS$ OF$ETHYLENE$OXIDE$AND$1,2:BUTYLENE$OXIDE:$ EFFECT$OF$BO:BLOCK$LENGTH$ $$ $ 3.2.1%Abstract% % $ We$ have$ used$ pyrene$ fluorescence$ spectroscopy$ and$ isothermal$ titration$ calorimetry$(ITC)$to$investigate$the$effect$of$hydrophobic:block$length$on$values$of$the$ critical$ micelle$ concentration$ (cmc)$ for$ aqueous$ solutions$ of$ triblock$ poly(butylene$ oxide):poly(ethylene$ oxide):poly(butylene$ oxide)$ block$ copolymers$ (BOnEOmBOn,$ where!m$and$n$denote$the$respective$block$lengths)$with$hydrophobic$block$lengths$in$ the$ range!n$=$ 12:21.$ Combined$ with$ results$ from$ previous$ work$ on$ BOnEOmBOn$copolymers$with$shorter$BO$blocks,$plots$of$log10(cmc)$(cmc$in$molar$units$ and$reduced$to$a$common$EO:block$length)$against$total$number$of$BO$units$(nt$=$n!for$ diblock$or$nt$=$2n$for$triblock$copolymers)$display$transitions$in$the$slopes$of$the$two$ plots,$which$indicate$changes$in$the$micellisation$equilibrium.$These$occur$at$values$of$ nt$which$can$be$assigned$to$the$onset$and$completion$of$collapse$of$the$hydrophobic$BO$ blocks,$an$effect$not$previously$observed$for$reverse$triblock$ copolymers.$ The$results$ are$compared$with$related$data$for$diblock$EOmBOn$copolymers.$ $$ 3.2.2.%%Introduction% $ $ The$ properties$of$ polymer$ surfactants$ combining$ hydrophilic$ poly(ethylene$ oxide)$ with$ various$ hydrophobic$ components$ have$ been$ reviewed$ in$ compilations$ edited$ by$ Nace$ (1)$ and$ by$ Alexandridis$ and$ Lindman$ (2),$ more$ recently$ by$ Booth$et! al.$(3)$ Triblock$ copolymers$ which$ combine$ poly(oxyethylene)$ with$ poly(oxypropylene)$ are$ much$ in$ use,$with$ important$ applications,$ for$ example,$ as$ emulsifiers$ (4),$ drug$ delivery$ systems$ (5),$ efflux$ pump$ inhibitors$ (6)$and$coating$ materials$ (7).$ To$ describe$ their$ repeat$ units$ we$ use$ the$ notation$ EO$=$oxyethylene,$ OCH2CH2$and$ PO$ =$ oxypropylene,$ OCH2CH(CH3),$ with$ block$ copolymers$ of$ the$ two$ triblock$ architectures$ denoted$ EOmPOnEOm!and$ POmEOnPOm,$ where$ the$ subscripts$m!and$n$denote$ number: average$ block$ lengths$ in$ repeat$ units.$ However,$ we$ note$ that$ the$ oxyanionic$ 102 polymerisation$ of$ propylene$ oxide$ is$ not$ straightforward,$ the$ problem$ being$ the$ transfer$ reaction$ originating$ from$ hydrogen$ abstraction$ rather$ than$ addition$ (8).$$For$ example,$ EOmPOnEOm$copolymers$ often$ have$ a$ diblock$ component,$ detected$ as$ a$ pronounced$ shoulder$ on$ the$ high:elution:volume$ side$ of$ their$ gel$ permeation$ chromatography$ curves,$ and$ leading$ to$ variation$ in$ micellisation$ (9)$ and$ gelation$(10)$ behaviour$from$batch$to$batch.$$As$a$consequence,$effects$of$EO$and$PO$block$length$on$ the$association$properties$of$these$copolymers$in$aqueous$solution$are$well$understood$ qualitatively$but$less$so$quantitatively.$ $ During$ the$ last$ few$ years,$ a$ series$ of$ more$hydrophobic$ block$ copolymer$ counterparts$ with$ similar$ architecture$but$ free$ from$ this$ complication$ has$ been$ examined.$$ In$ particular,$ we$ have$ studied$ copolymers$ in$ which$the$ PPO$ segment$ was$ replaced$by$a$more$hydrophobic$one$(e.g.$poly(butylene$oxide),$poly(styrene$oxide)$or$ poly(phenylglycidyl$ ether))$$ in$ order$ to$better$ elucidate$ the$ effects$ of$ block$ hydrophobicity$on$ micellisation$ and$micellar$ properties$ (3,11),$with$ the$ aim$ of$ improving$ the$ rheological$ properties$ of$ micellar$ solutions$ and$ the$ solubilisation$ capacities$of$the$micelles$for$poorly:water$soluble$drugs$(12).$Special$attention$has$been$ paid$to$copolymers$with$1,2:butylene$oxide$as$the$hydrophobic$component.$Transfer$is$ not$ a$ problem$ in$ the$ laboratory$ polymerisation$ of$ 1,2:butylene$ oxide$ (13),$but$ this$ monomer$ (as$ does$ propylene$ oxide)$ adds$ to$ the$ growing$ chain$ to$ give$ a$ secondary$ oxyanion,$and$slow$initiation$of$EO$chains$at$the$secondary$termination$may$lead$to$a$ broadened$ E:block:length$ distribution$ (14).$However,$ this$ effect$is$ eliminated$ if$ 1,2: butylene$oxide$blocks$ are$ polymerised$last$when$ forming$EOBO$diblock$and$BOEOBO$ triblock$copolymers.$Reverse$BOEOBO$triblock$copolymers$have$potential$for$the$control$ of$ rheological$ properties$ in$ aqueous$ systems,$ particularly$ associative$ thickeners$ (15).$The$effect$originates$from$molecular$association$of$the$hydrophobic$ends$of$the$ chains$ in$ dilute$ solution$ and,$ above$ the$ critical$ micelle$ concentration$ (cmc),$ from$ association$ of$ unimers$ into$ micelles$ in$ which$ the$ chains$ can$ either$ loop$ or$ extend.$ Bridging$ of$ extended$ chains$ between$ micelles,$ a$ dynamic$ process,$ leads$ to$ the$ formation$ of$ transient$ micelle$ clusters,$ and$ at$ high$ enough$ concentrations$ to$the$formation$of$transiently:linked$networks.$ $ The$rheological$effect$depends$on$a$balance$between$EO:$and$BO:block$lengths.$$ A$ lengthy$ EO:block$ length$ promotes$ bridging$ but$ at$ the$ expense$ of$ micellisation.$$ Lengthening$ the$ B:block$ length$ restores$ micellisation,$ but$ a$ limit$ will$ be$ reached$ at$ which$the$longest$hydrophobic$blocks$in$the$individual$molecules$will$collapse$to$form$ a$globule$ (16),$ i.e.$ the$ coiled$ unimers$ will$ form$ so:called$ unimolecular$ micelles.$$ The$ 103 collapsed$state$involves$a$reduction$in$the$number$of$contacts$of$the$chain$units$of$the$ core:forming$block$with$solvent$and,$hence,$a$reduction$in$the$hydrophobic$effect$which$ drives$micellisation,$the$physics$of$which$has$been$described$in$detail$elsewhere$(17).$$ An$accurate$knowledge$of$the$conformational$state$of$the$dispersed$copolymer$ in$solution$is$desirable$if$the$complexities$of$micellisation$and$micelle$bridging$in$these$ associative$systems$is$to$be$understood.$$The$effect$of$hydrophobic:block$collapse$on$ the$ block:length$ dependence$ of$ the$ critical$ concentration$ for$ micellisation$ has$ been$ investigated$ recently$ for$ diblock$ EOmBOn$copolymers$ covering$ a$ range$ of$ BO:block$ lengths$ from$ BO7$to$ BO76,$ with$ transitions$ in$ the$ results$ assigned$ to$ the$ onset$ and$ completion$of$unimolecular$micelle$formation$(18).$$However,$the$possibility$of$related$ effects$ in$ the$ micellisation$ of$ BOnEOmBOn$copolymers$ in$ dilute$ solution$ has$ not$ been$ investigated$ since$ the$ range$ of$ hydrophobicity$ has$ been$ much$ restricted$ for$ those! copolymers,$i.e.$from$BO4$to$BO12,$8$to$24$BO$units$per$molecule$(3).$In$this$paper,$we$ describe$the$properties$ of$ BOEOBO$ triblock$copolymers$ with$ longer$ BO$ blocks,$ which$ provide$ a$ range$ of$ hydrophobicity$ more$ comparable$ with$ that$ of$ the$diblocks.$Specifically$we$present$a$direct$comparison$of$the$micellisation$of$triblock$ BOEOBO$ copolymers$ with$ that$ of$ diblock$ EOBO$ copolymers$ over$ a$ wide$ range$ of$ hydrophobicity,$ with$ special$ emphasis$ on$ the$ effect$ of$ collapse$ of$ long$ BO$ blocks$ in$ molecular$solution,$and$on$the$effect$(if$any)$of$splitting$the$total$number$of$BO$units$ between$two$blocks.$ $$ 3.2.3%Experimental%section% % 3.2.3.1%Materials% $ Five$ BOnEOmBOn$copolymers$ with$ narrow$ chain$ length$ distributions$ were$ prepared$and$characterised$using$methods$described$previously$(15,19)$(see$Table$1).$ Copolymer$BO8EO90BO8$was$included$to$overlap$with$the$range$of$copolymers$studied$ previously$and$so$provide$direct$validation$of$values$of$the$cmc$determined$by$different$ techniques.$ % % % % 104 Table%1.$BOnEOmBOn$copolymers$ Copolymer$ 103$Mn$ (g$mol:1)$ Mw/Mn$ BO8EO90BO8$ 0.51$ 1.07$ BO12EO227BO12$ 11.7$ 1.05$ BO14EO378BO14$ 18.6$ 1.12$ BO20EO411BO20$ 21.0$ 1.08$ BO21EO385BO21$ 20.0$ 1.10$ Uncertainties$in$Mn$determination$are$ca.$±$1%.$ $$ 3.2.3.2%Methods% % a. Fluorescence!measurements! ! Values$ of$ the$ critical$ micelle$ concentration$ (cmc)$ were$ obtained$ from$ pyrene$ fluorescence$measurements,$as$described$by$Lee$et!al.$(20).$Pyrene$was$obtained$from$ Sigma:Aldrich$Co.$and$used$as$received.$Stock$solutions$were$prepared$by$dissolving$the$ copolymers$in$Milli:Q$water$and$allowing$24$h$for$complete$dissolution$before$diluting$ to$ desired$ concentrations$ within$ the$ range$ 1:50·∙$ 103$mg$ dm:3.$ Pyrene$ dissolved$ in$ acetone$was$added$to$the$copolymer$solution$and,$after$acetone$evaporation,$24$h$was$ allowed$ for$ equilibration.$ The$ final$ copolymer$ solution$ contained$ 3·∙10:7$mol$ dm: 3$pyrene.$A$Cary$Eclipse$fluorescence$spectrophotometer$equipped$with$a$temperature$ control$Peltier$device$and$a$multi:cell$sample$holder$(Varian$Instruments$Inc.)$was$used$ in$ the$ experiments,$ with$ solution$ temperatures$ kept$ at$ 25$ ±$ 0.1$ ºC.$ The$ excitation$ wavelength$(lex$=$335$nm)$was$the$maximum$intensity$in$the$excitation$spectrum.$The$ fluorescence$spectrum$was$the$average$of$three$scans$and$was$corrected$for$scattering$ using$an$equivalent$blank$solution$before$determining$the$ratio!I1/I3$of$the$first$and$third$ vibronic$peaks.$Reproducibility$was$better$than$2$%.$ $ % 105 b. Isothermal!titration!calorimetry!measurements! ! !Heats$ of$ demicellisation$ were$ measured$ using$ a$ VP:ITC$ titration$ microcalorimeter$ (MicroCal$ Inc.,$ Northampton,$ MA,$ USA.)$ Small$ aliquots$ (5:10$ml)$ of$ stock$ solution$ of$ copolymers$at$concentrations$well$above$the$cmc$were$injected$into$a$known$volume$of$ water$(ca.$1$ml)$held$in$the$cell$of$the$calorimeter,$initially$to$produce$a$solution$well$ below$the$cmc.$Repeated$additions$of$the$stock$solution$gave$the$heat$evolved$(DHi)$as$ a$function$of$copolymer$concentration.$ $$ 3.2.4%Results%and%discussion% $ 3.2.4.1%Determination%of%critical%micelle%concentration% ! !! Figure$ 1a$ shows$ the$ dependence$ of$ the$ ratio$I1/I3$from$ pyrene$ fluorescence$ intensity$ on$ copolymer$ concentration$ (logarithmic$ scale)$ for$ the$ block$ copolymer$ BO8EO90BO8.$As$ indicated,$ the$value$ of$the$ cmc$ at$25$ °C$was$ obtained$ as$0.33$ g$dm: 3$from$ linear$ fitting$ of$ the$ two$ regions$ defined$ when$ the$I1/I3$$ pyrene$ fluorescence$ intensity$starts$to$decrease$abruptly.$As$seen$in$Table$2,$this$value$of$the$cmc$fits$well$ within$the$set$of$values$determined$using$static$light$scattering$in$the$Stonybrook$and$ Manchester$laboratories$for$copolymers$BO4EO40BO4$to$BO12EO260BO12.$Figure$1b$shows$ similar$ plots$ for$ copolymers$ BO14EO378BO14$and$ BO20EO411BO20,$ with$ the$ same$ construction$used$to$obtain$the$values$of$the$cmc$listed$in$Table$2.$The$data$points$for$ the$other$two$copolymers$were$treated$in$the$same$way$but,$for$clarity,$are$omitted$ from$Figure$1.$ $ 3.2.4.2%Influence%of%temperature%on%cmc% % ! !Figure$2$shows$the$limited$effect$of$solution$temperature$(range$25$to$35$°C)$on$ the$concentration$dependence$of$I1/I3$for$copolymers$BO12EO227BO12$and$BO21EO385BO21.$ A$ similar$ insensitivity$ to$ temperature$ has$ been$ reported$(3)$for$ diblock$ and$ triblock$ copolymers$with$15$or$more$$BO$units,$with$consequently$low$values$of$the$van't$Hoff$ enthalpy$of$micellisation,$i.e.! $ ∆micH$=!RT!d$ln(cmc)/d(1/T)$$$$$$$$(1)$ 106 $ approaching$zero.$ 10-3 10-2 10-1 100101 0.7 0.8 0.9 1.0 10-3 10-2 10-1 100101 0.7 0.8 0.9 1.0 I1/I3 a) I1 / I3 concentration /g dm-3 b) $ Figure% 1:$Normalised$ pyrene$ fluorescence$ intensities$ (I1$/!I3)$ for$ copolymers$ a)$ (¿)$ BO8EO90BO8,$b)$(")$BO20EO411BO20,$and$(q)$BO14EO378BO14$in$aqueous$solution$at$25$ºC.$$ $ Isothermal$ titration$ calorimetry$ (ITC)$ was$ used$ to$ confirm$ this$ result.$The$ ITC$ curve$ obtained$ for$ copolymer$ BO12EO227BO12$at$ 20$ ºC$ is$ shown$ in$ Figure$ 3.$ The$ heat$ evolved$ (Hi)$ is$ plotted$ against$ the$ copolymer$ concentration.$ As$ discussed$ previously$(25),$at$concentrations$below$the$cmc$the$concentration$dependence$of$Hi$is$ determined$ by$ dilution$ of$ unimers,$ and$ that$ at$ high$ concentrations$ by$ dilution$ of$ micelles.$ The$ sharp$ increase$ in$Hi$as$ the$ concentration$ is$ increased$ from$ dilute$ to$ concentrated$is$the$enthalpy$of$micellisation,$∆micH$≈$19$kJ$mol:1,$very$similar$in$value$to$ the$ van't$ Hoff$ enthalpy$ reported$ for$ copolymer$ BO12EO260BO12$in$ ref.$ 3.$ Very$ small$ values$of$∆micH$were$recorded$for$the$other$four$block$copolymers.$ $$ % % 113 16.$ (a)$ Brown,$ R.A.;$ Masters,$ A.J.;$ Price,$C.;$ Yuan,$ X.:F.$in$ Comprehensive$ Polymer$ Science,$Vol.$2,$Polymer$Properties,$1989.$Booth,$C.;$Price$C.$Eds.,$Pergamon$Press,$ Oxford,$Ch.$6,$pp.$185:186.$(b)$Tuzar,$Z.;$Kratochvil$P.$Surf.$Colloid$Sci.$1993,%15,$1.$ (c)$Chu,$B.$Langmuir%1995,$11,$414.$ 17.$$$$Cooke,$I.R.;$Williams,$D.R.M.$Macromolecules$2003,%36,$2149.$ 18.$$ Ribeiro,$ M.N.E.P.;$ de$ Oliveira,$ S.A.;$ Ricardo,$ N.M.P.S.;$ Mai,$ S.:M.;$ Attwood,$ D.;$ Yeates,$S.G.;$Booth,$C.$Int.$J.$Pharm.$2008,%362,$193.$ 19.$$ Yang,$Y.:W.;$Yang,$Z.;$Zhou,$Z.:K.;$Attwood,$D.;$Booth,$C.$Macromolecules$1996,%29,$ 670.$ 20.$$Lee,$K.;$Shin,$C.:H.;$Oh,$I.$Arch.$Pharm.$Res.$2003,%26,$653.$ 21.$$Zhou,$Z.;$Chu,$B.;$$Nace,$V.M.$Langmuir$1996,%12,$5016.$ 22.$$Liu,$T.;$ Zhou,$Z.;$ Wu,$C.;$ Schneider,$D.K.;$ Chu,$B.;$ Nace,$ V.M.$ J.$ Phys.$ Chem.$ B.$ 1997,%101,$8808.$ 23.$$Liu,$T.;$Zhou,$Z.;$Wu,$C.;$$Nace,$V.M.;$Chu,$B.$J.$Phys.$Chem.$B.$1998,%102,$2875.$ 24.$$Zhou,$Z.;$Yang,$Y.:W.;$Booth,$C.;$Chu,$B.$Macromolecules$1996,%29,$8357.$ 25.$$ (a)$Taboada,$P.;$$Mosquera,$V.;$Attwood,$D.;$Yang,$Z.;$Booth,$C.$Phys.$Chem.$Chem.$ Phys.!2003,$5,$2625.$(b)$Taboada,$P.;$Velasquez,$G.;$Barbosa,$S.;$Yang,$Z.;$Nixon,$ S.K.;$ Zhou,$ K.;Heatley,$ $ F.;$ Ashford,$ M.;$ Mosquera,$ V.;$ Attwood,$ D.;$ Booth,$ C.$ Langmuir,$2006,$22,$7465.%% 26.$$ Alexandridis,$P.;$Holzwarth,$J.F.;$$Hatton,$T.A.;$Macromolecules$1994,%27,$2414.$ 27.$$ Kelarakis,$A.;$Havredaki,$V.;$Rekatas,$C.J.;$Booth,$C.$Phys,$Chem.$Chem.$Phys.$2001,% 3,$5550.$ 28.$ Irache,$ J.M.;$ Salman,$ H.H.;$ Gomez,$ S.;$ Espuelas,$ S.;$ Gamazo,$ C.$ Frontiers$ Bios.$ 2010,%S2;$876.$ 29.$ Batrakova,$E.$V.;$Kabanov,$A.V.$J.$Controlled$Release$2008,%130,$98.$ 30.$ Cambón,$A.;$Rey:Rico;$A.;$Mistry,$D.;$$Brea,$J.;$Loza,$M.I.;$Attwood,$D.;$Barbosa,$S.;$ Alvarez:Lorenzo,$C.;$Concheiro,$A.;$Taboada,$P.;$Mosquera,$V.$Int.$J.$Pharm.$2013,% 445,$47.$$ ! 114! 3.3!COMPLEX! SELF/ASSEMBLY! OF! REVERSE! POLY(BUTYLENE! OXIDE)!/!POLY(ETHYLENE! OXIDE)/POLY(BUTYLENE! OXIDE)! TRIBLOCK! COPOLYMERS!WITH!LONG!HYDROPHOBIC!AND! EXTREMELY!LENGTHY!HYDROPHILIC!BLOCKS! ! 3.3.1 Abstract! Amphiphilic! block! copolymers! have! emerged! during! last! years! as! a! fascinating! substrate! material! to! develop! micellar! nanocontainers! able! to! solubilize,! protect,! transport!and!release!under!external!or!internal!stimuli!different!classes!of!cargos!to! diseased!cells!or!tissues.!However,!this!class!of!materials!can!also!induce!biologically! relevant!actions!which!complement!the!therapeutic!activity!of!their!cargo!molecules! through!their!mutual!interactions!with!biological!relevant!entities!(cellular!membranes,! proteins,! organelles…),! which,! at! the! same! time,! are! regulated! by! the! nature,! conformation!and!and!state!of!the!copolymeric!chains.!For!this!reason,!in!this!paper! we! investigated! the! self/assembly! process! and! physico/chemcial! properties! of! two! reverse!triblock!poly(butylene!oxide)/poly(ethylene!oxide)/poly(butylene!oxide)! block! copolymers,!BO14EO378BO14!and!BO21EO385BO21,!which!have!been!recently!found!to!be! very! useful! as! drug! delivery! nanovehicles! and! biological! response! modifiers! under! certain!conditions!(A.!Cambón!et#al.#Int.#J.#Pharm.#2013,!445,!47/57)!in!order!to!obtain! a! clear! picture! of! the! solution! behavior! of! this! class! or! block! copolymers! and! to! understand! their! biological! activity.! These! block! copolymers! are! characterised! by! possessing!long!!BO!blocks!and!extremely!lengthy!central!EO!ones!which!provide!them! with!a!rich!rheological!behavior!characterised!by!the!formation!of!flower/like!micelles! with! sizes! ranging! from! 20! to! 40! nm! in! aqueous! solution! and! the! presence! of! intermicellar! bridging! even! at! low! copolymers! concentrations! as! denoted! by! atomic! force! microscopy.! Bridging! is! also! clearly! observed! by! analysing! the! rheological! response! of! these! block! copolymers! both! storage! and! loss! moduli! upon! changes! on! time,! temperature! and! or! concentration.! Strinkingly,! the! relatively! wide! Posisson! distribution! of! copolymeric! chains! make! the! present! copolymers! to! behave! rather! distinctly! to! conventional! associative! thickeners.! The! observed! rich! rheological! behavior! and! their! tunability! make! also! these! copolymers! a! promising! materials! to! configure!drug!gelling!depots.!! ! ! 115! 3.3.2 Introduction! In!the!last!two!decades!a!great!effort!has!been!made!through!the!development! of! a! series! of! nanosized! therapeutic! products! able! to! solubilize! hydrophobic! drugs,! allow! their! sustained! release,! improve! their! pharmacokinetics! and! facilitate! their! access!to!the!site!of!action!(1/5)!The!properties!of!amphiphilic!copolymers!combining! hydrophilic!poly(ethylene!oxide)!units!with!different!types!of!hydrophobic!blocks!have! been! found! to! show! suitable! characteristics! for! fulfill! the! former! requirements,! i.e.! they!can!spontaneously!self/assemble!into!nanoscopic!core/shell!micellar!structures!in! which! the! core! serve! as! reservoir! for! the! hydrophobic! cargo! while! the! corona! is! in! direct!contact!with!the!biological!milieu!providing!“stealthness”!to!evade!scavenging! by! the! mononuclear! phagocyte! system,! which! results! in! larger! circulation! times! and! passive!accumulation!in!solid!tumors!by!the!enhanced!permeation!and!retention!(EPR)! effec.6! Copolymers! which! combine! poly(oxyethylene)! and! poly(oxypropylene)! (EO! =! oxyethylene,!OCH2CH2,!and!PO!=!oxypropylene,!OCH2CH(CH3))!in!a!triblock!structure,! either! direct,! EOmPOnEOm,! or! reverse,! POnEOmPOn! (where! the! subscripts! m! and! n! denote!number/average!block!lengths)!have!been!the!most!extensively!studied!due!to! their!commercial!availability!in!a!very!broad!range!of!compositions,!a!fair!solubilization! capacity!and!sustained!release,!a!good!biocompatibility!of!most!varieties!and!approval! of! some! varieties! by! regulatory! agencies! to! be! used! in! pharmaceutical! formulations! and!medical!devices!(7/9).!Nevertheless,!EOmPOnEOm!or!POnEOmPOn!block!copolymers! possess!several!drawbacks.!For!example,!the!oxyanionic!polymerization!of!propylene! oxide!is!not!straightforward,!the!problem!lying!in!the!transfer!reaction!originated!from! hydrogen!abstraction!rather!than!addition!(10).!For!example,!EOmPOnEOm!copolymers! often! have! a! diblock! component,! detected! as! a! pronounced! shoulder! on! the! high/ elution/volume! side!of!their!gel!permeation! chromatographic! curves,! which! leads! to! variation!in!micellisation!(11/12)!behavior!from!batch!to!batch.!In!addition,!there!exists! an! incomplete! micellisation! of! the! unimers! which! usually! leads! to! self/assembled! nanostructures!with!limited!drug!solubilisation!ability!and!stability!upon!dilution!in!the! bloodstream.! To! circumvent! these! problems,! during! the! last! few! years! a! series! of! more! hydrophobic!block!copolymer!counterparts!with!similar!architecture!but!with!the!PO! segment! replaced! by! a! more! hydrophobic! one! such! as! poly(butylene! oxide)! (PBO),! poly(styrene!oxide)!(PSO)!or!phenylglycidyl! ether! (PG)! have! been!proposed!with!the! aim! of! improving! the! solubilisation! capacities,! release! profiles! and! the! rheological! properties!of!the!polymeric!micelles!for!poorly/water!soluble!drugs!(13/19).! Special!attention!has!been!paid!to!copolymers!with!1,2/butylene!oxide!(BO)!as! the!hydrophobic!monomer.!Transfer!is!not!a!problem!in!the!laboratory!polymerisation! of! BO! (20),! but! this! monomer! (as! PO!does)! adds! to! the! growing! chain! to! give! a! secondary!oxyanion;!also,!the!slow!initiation!of!EO!chains!at!the!secondary!termination! ! 116! may! lead! to! a! broadened! EO/block/length! distribution! (21).! However,! this! effect! is! eliminated! if! BO!blocks! are! polymerised! last! when! forming! EOmBOm! diblock! and! BOnEOmBOn! triblock! copolymers.! Reverse! BOnEOmBOn! triblock! copolymers! have! potential!for!the!control!of!rheological!properties!in!aqueous!systems,!particularly!as! associative! thickeners! ! thanks! to! the! formation! of! transient! micelle! clusters! or! networks!by!bridging!of!extended!chains!between!micelles!as!previously!observed,!for! example,! in! copolymer! BO10EO410BO10! (22/24).! The! larger! relative! hydrophobicity! of! the!BO!block!compared!to!PO!(six/fold!as!estimated!from!the!ratio!of!the!logarithms!of! the! molar! critical! micellar! concentrations,! cmc)! (16)! enables! the! formation! of! polymeric! micelles! at! much! lower! copolymer! concentrations! and! subsequent! larger! solubilised!drug!concentrations!in!the!micelle!core,!providing!excellent! properties!as! drug!delivery!nanocarriers!(25).!In!addition,!these!copolymers!were!demonstrated!to! be! “biologically! active”! in! the! sense! of,! for! example,! enhancing! drug! toxicity! to! cancerous! cells! by! inhibiting! the! P/glycoprotein! P! efflux! pump! mechanism! (25).! Nevertheless,!a!complete!and!detailed!physico/chemical!characterization!of!the!former! class!copolymers!is!still!lacking,!which!might!help!to!obtain!a!better!understanding!of! their! behavior! as! biologically/response! modifiers! and! to! open! up! new! potential! applications!as!injectable!drug!gelling!depots.! ! ! Hence,! in! this! work! we! present! a! deep! characterization! of! the! self/assembly! process! and! the! physico/chemical! properties! of! copolymers! BO14EO378BO14!and! BO21EO385BO21!by!different!techniques!such!as!static!and!dynamic!light!scattering!(SLS! and! DLS,! respectively),! transmission! electron! microscopy! (TEM),! atomic! force! microscopy!(AFM),!and!rheometry.!Both! copolymers!possess!much!longer!BO!blocks! than! previously! analysed! BOnEOmBOn! copolymers.! This! enabled! us! to! observe! the! effects! of! both! the! collapse! of! longer! BO! blocks! in! solution! of!reverse! copolymeric! structures! and! the! splitting! of! BO! units! number! between! two! blocks,! especially! in! dilute!solution!since!the!range!of!hydrophobicity!has!been!much!restricted!for!these! copolymers.!i.e.!from!BO4!to!BO12,!8!to!24!BO!units!per!molecule!(16).! ! 3.3.3! Experimental!section! 3.3.3.1!Materials! Copolymers! were! prepared! by! oxyanionic! polymerisation! of! dry! 1,2/butylene! oxide! initiated! by! polyethylene! glycol! monomer! of! different! molecular! weights! activated! by! mixing! with! KOH! and! heating! while! stirring! under! vacuum! (70! ºC,! 0.1! mmHg,! 100! h)! to! remove! water.! Vacuum! line! and! ampoule! techniques! served! to! exclude!moisture.!Gel!permeation!chromatography!(GPC)!was!used!to!characterise!the! distribution! widths! of! the! products! as! the! ratio! of! mass/average! to! number/average! molar!mass,!i.e.!Mw/Mn!by!using!a!Waters!GPC!system!equipped!with!a!1515!isocratic! ! 117! pump! and! a! 2410! refractive! index! detector! (Waters,! Milford,! MA).! Chloroform! was! used!as!eluent,!and!monodisperse!PEO!was!employed!as!standard.!13C!NMR!spectra! recorded! on! a! Bruker! ARX400! spectrometer! (Bruker,! Milton,! ON,! Canada)! in! deuterated! chloroform! were! used! to! obtain! absolute! values! of! block! length! and! composition,! and! to! verify! block! architecture.! The! general! methods! used! have! been! described! previously! in! detail! (22,26).! Table! 1! summarises! the! molecular! characteristics!of!the!copolymers.! ! Table!1.!Molecular!characteristics!of!the!copolymers.# Polymers! Mn(g/mol)a! Mw!/!Mnb! Mw!(g/mol)! cmc!(g/dm3)c! BO14EO378BO14! 18600! 1.12! 20830! 0.058! BO21EO385BO21! 20000! 1.10! 22000! 0.025! aEstimated!by!NMR;!bEstimated!by!GPC;!Mw!calculated!from!Mn!and!Mw/Mn.!Estimated! uncertainty:!Mn!to!±3!%;!Mw/Mn!to!±0.01.c!Values!from!Ref.!(27).! ! 3.3.3.2! Methods! a. Dynamic#and#static#light#scattering#(DLS#and#SLS)# SLS! intensities! were! measured! by! means! of! an! ALV/5000F! (ALV/GmbH,! Germany)! instrument!with!vertically!polarized!incident! light!(λ!=! 488!nm)! supplied!by!a! diode/ pumped!Nd:YAG!solid/state!laser!(Coherent!Inc.,!CA,!USA)!and!operated!at!2!W,!and! combined!with!an!ALV!SP/86!digital!correlator!with!a!sampling!time!of!25!ns!to!100!ms! (for! DLS).! Measurements! were! made! at! an! angle! θ! =! 90°! to! the! incident! beam,! as! appropriate! for! particles! smaller! than! the! light! wavelength.! The! intensity! scale! was! calibrated! against! scattering! from! toluene.! Solutions! were! filtered! through! Millipore! Millex!filters!(Triton!free,!0.22!µm!porosity)!directly!into!cleaned!scattering!cells!and! allowed!to!equilibrate!at!the!requested!temperature!for!10!min!before!measurement.! Each!experiment!was!repeated!at!least!three!times.!Sampling!time!was!5/10!min!for! each!run!in!order!to!define!an!optimal!correlation!function.!For!DLS,!the!correlation! functions!were!analyzed!by!the!CONTIN!method!to!obtain!the!intensity!distributions!of! decay! rates! (Γ)! (28).! From! the! decay! rate! distributions! the! apparent! diffusion! coefficients! (Dapp! =! Γ/q2,! q# =! (4 π ns/λ)sin(θ/2)! were! derived,! being! ns! the! solvent! refractive! index.! Values! of! the! apparent! hydrodynamic! radius! (rh,app,! radius! of! the! hydrodynamically!equivalent!hard!sphere!corresponding!to!Dapp)!were!calculated!from! the!Stokes/Einstein!equation! !!!!rh,app=!kT/(6π η Dapp)! ! ! ! ! (1)! ! 118! where!k!is!the!Boltzmann!constant!and! η !is!the!viscosity!of!water.! b. Transmission#electron#microscopy#(TEM)# Micellar!solutions!of! both! copolymers!were!applied! dropped! over!carbon/coated! copper!grids,!blotted,!washed,!negatively!stained!with!2%!(w/v)!phosphotungstic!acid,! air/dried,!and!then!examined!with!a!Phillips!CM/12!transmission!electron!microscope! operating!at!an!accelerating!voltage!of!120!kV.! ! c. #Clouding! Copolymer! solutions! were! prepared! by! weighting! the! required! amount! of! each! copolymer! followed! by! the! addition! of! the! same! volume! of! cold! water! (1! mL).! Copolymer! solutions! were! homogenized! under! stirring! at! low! temperature! before! being!stored!at!least!for!one!day!(T!∼!4!ºC)!to!ensure!complete!dissolution.!Clouding! temperatures! (Tcl)! were! determined! by! slowly! heating! (0.2! ºC! min/1)! the! copolymer! solutions!from!0!to!90!ºC!by!both!visual!inspection!and!detection!of!the!transmitted! light! through! solutions! by! means! of! a!UV/Vis! spectrophotometer! equipped! with! a! temperature!control!Peltier!device!and!a!multi/cell!sample!holder!(Cary!100,!Agilent,! Germany).!A!plot!of!transmitted!intensity!versus!temperature!was!obtained.!The!cloud! point!was!determined!as!the!midpoint!of!an!abrupt!decrease!in!the!transmitted!light! intensity!from!a!plot!of!transmitted!intensity!vs!temperature,!as!previously!described! (24).! d. Rheometry# Solutions!were!prepared!by!weighting!copolymer!and!deionized!water!into!small! tubes!and!subsequent!mixing!in!the!mobile!state!before!being!stored!for!a!day!or!more! at! low! temperature! (ca.! 5! ºC).! Rheological! characterisation! was! carried! out! using! a! controlled! stress! AR2000! rheometer! (TA! instruments,! DE,! USA)! with! Peltier! temperature! control.! Samples! were! investigated! using! cone/plate! geometry! (cone! diameter! 40! mm,! angle! 0.5º)! and! a! solvent! trap! to! maintain! a! water/saturated! atmosphere! around! the! sample! cell! to! avoid! evaporation.! The! temperature! dependence!of!storage!(G)!and!loss!(G´´)!moduli!was!measured!either!by!temperature! scans!at!frequency!f!=!1!Hz!and!heating!rates!of!1!ºC!min/1!or!via!frequency!scans!at! several! temperatures! (1/80ºC).! Experiments! were! carried! out! in! oscillatory! shear! mode,!with!the!strain!amplitude!(A)!maintained!at!a!low!value!(A!<!0.5!%)!by!means!of! the!autostress!facility!of!the!software.!This!ensured!that!measurements!of!G´!and!G´´! were!in!the!linear!viscoelastic!region.!A!dynamic!time!sweep!test!under!A!=!0.5%!and!f! =!1!Hz!was!performed!before!each!frequency!scan!at!a!fixed!temperature!to!ensure! that!the!sample!truly!reached!the!equilibrium!state.! ! ! 119! e. Atomic#force#microscopy#(AFM)! !AFM!images!of!block!copolymer!solutions!were!performed!on!freshly!cleaved!mica! substrates.! The! measurements! were! performed! in! a! JEOL! instrument! (model! JSPM! 4210)! in! noncontact! mode! using! nitride! cantileversNSC15! from! MicroMasch,! U.S.A.! (typical!working!frequency!and!spring!constant!of!325!kHz!and!40!N/m,!respectively).! The!AFM!samples!were!dried!in!air!or!under!a!nitrogen!flow!when!required.!Control! samples!(freshly!cleaved!mica!and!buffer!solution)!were!also!investigated!to!exclude! possible! artifacts.! Topography! and! phase/shift! data! were! collected! in! the! trace! and! retrace!direction!of!the!raster,!respectively.!The!offset!point!was!adapted!accordingly! to!the!roughness!of!the!sample.!The!scan!size!was!usually!500!nm!(aspect!ratio,!1!x!1),! with! a! sample! line! of! 256! points! and! a! step! size! of! 1! μm.! The! scan! rate! was! tuned! proportionally!to!the!area!scanned!and!kept!within!the!0.35=2!Hz!range.!Each!sample! was! imaged! several! times! at! different! locations! on! the! substrate! to! ensure! reproducibility.!Diameters!and!heights!of!copolymer!aggregates!were!determined!by! sectional!analysis!taken!from!the!average!of!several!sections!through!the!aggregates.! 3.3.4!Results!and!discussion! 3.3.4.1!Clouding! The!clouding!and!phase!behavior!of!BOnEOmBOn!copolymers!is!not!completely! resolved!yet!due!to!the!unavailability!of!a!full!range!of!block!lengths.!To!fill!this!gap,! clouding! temperatures! (Tcl)! were! firstly! determined! for! solutions! of! copolymers! BO14EO378BO14! and! BO21EO385BO21!in! the! concentration! range! 0.1/10! wt.%! by! visual! inspection!and!UV/Vis!spectroscopy!following!the!methodology!of!Zhou!et#al!(24).!A! good!agreement!was!observed!between!both!methods.!Figure!1!shows!Tcl!as!a!function! of!copolymer!concentration.!In!the!one/phase!region,!the!cmc!values!were!previously! found! to! be! below! 0.1! mg/mL! for! both! copolymers! (27).! Hence,! the! cloud! point! behavior!can!represented!the!phase!transition!of!a!copolymer!micellar!solution!which! phase! separates! ca.! 20! ºC! above! Tcl.! In! general,! copolymer! BO21EO385BO21! displayed! lower! Tcl#than! BO14EO378BO14! as! expected! for! its! longer! hydrophobic! blocks.!For! BO14EO378BO14,!the!cloud/point!profile!exhibited!a!shallow!minimum!at!50!mg/mL!(at! 60!ºC),whilst!for!BO21EO385BO21!this!minimum!was!observed!at!30!mg/mL!(at!51!ºC);!for! both!copolymers!Tcl#starts!again!to!increase!at!larger!concentrations.!High!Tcl#coincident! with!gel!formation!have!been!also!observed!in!related!systems,!i.e.!aqueous!solutions! of! copolymers! BO12EO114BO12! and! BO12EO227BO12! (23,29,30).!Liu! et# al.(31)#have! investigated! the! effect! of! EO! and! BO! block! lengths! on! Tcl##of! 1! wt.! %! solutions! of! BOnEOmBOn!copolymers!bearing!short!EO!(m#<!40)!and!BO!(n#<!7)!blocks.!These!authors! showed!that!Tcl#decreased!with!increases!in!BO/block!length!at!constant!EO/block!one,! and! it! increased! with! an! increase! in! EO/block! length! at! constant! BO/block! one.! The! results! reported! here! confirm! that! Liu´s! conclusions! can! be! also! applied! to! longer! copolymers! and! higher! concentrations,! i.e.! Tcl# is! lower! the! most! hydrophobic! the! ! 120! copolymer! is.! However,! in! the! present! case! the! copolymers´! behavior! is! largely! influenced!by!their!extremely!long!EO/blocks!which!makes!Tcl##to!increase!if!compared! to! structurally! related! BO12EO114BO12,! BO12EO227BO12! or! BO10EO410BO10! copolymers! previously!studied!(22,32).# ! Figure!1.!Clouding!temperatures!for!copolymers!BO14EO378BO14!(!)!and!BO21EO385BO21! (¢)!obtained!by!UV/vis!spectroscopy.!The!lines!were!drawn!to!guide!the!eye.! ! 3.3.4.2!Population!size!distributions! DLS! measurements! of! BO14EO378BO14! and! BO21EO385BO21!micellar! solutions! at! different!concentrations!were!carried!out!at!10!and!25!°C.!Selected!intensity!fraction! distributions!of!log!rh,app#(rh,app#being!the!apparent!hydrodynamic!radius)!are!illustrated! in! Figure! 2a! for! copolymer! BO21EO385BO21! at! 25! ºC! as! an! example.! For! c# <# cmc,! the! population! distributions! obtained! show! only! a! single! peak! attributed! to! singly! dispersed!copolymer!chains!(rh,app#=!2/3!nm)!(Figure!2a,!red!line).!It!is!conceivable!that! under!these!conditions!the!present!copolymers!can!arrange!the!unimers!in!the!form!of! unimolecular!micelles!in!order!to!prevent!the!contact!of!BO!blocks!with!water!thanks! to! the! flexibility! of! the! central,! very! long! EO! block.! The! low! micellization! enthalpy! values!previously!derived!from!isothermal!titration!calorimetry!(ITC)!would!confirm!the! tightly!packing!of!BO!blocks!in!the!unimer!state!so!that!their!hydrophobic!interaction! with!water!would!be!really!small!(27).!At!c#>#cmc,!several!peaks!are!observed!in!the! intensity/fraction!population!distributions!which!can!correspond!to!unimers!(rh,app#=!2/3! nm),! flower/like! micelles! (rh,app# =! ca.! 8! to! 20! nm)! and! micelle! clusters! formed! by! micellar!bridgening!(rh,app#=!40/60!nm)!(Figure!2a,!black!line).!Owing!to!the!special!chain! architecture! of! BOnEOmBOn/type! block! copolymers,! the! formation! of! flower/like! micelles!would!involve!bending!of!EO!blocks!while!keeping!the!two!terminal!BO!blocks! in!the!same!micellar!core!(an!entropy/loss!process).!Another!possibility!might!be!the! two!BO!blocks!in!one!polymer!chain!would!reside!in!two!adjacent!micelles!and!the!EO! block! would! be! used! as! a! bridge.! This! kind! of! crosslinking! among! the! micelles! can! ! 121! finally!promote!an!open!network!structure!(the!so/called!micellar!clusters),!reflected!in! the!DLS!population!distributions!(Figure!2a,!blue!line).!Peaks!corresponding!to!unimers! and!micelles!were!single!narrow!peaks,!while!those!belonging!to!micellar!clusters!were! broader.! As! the! concentration! increased,! the! latter! peak! became! more! intense! denoting! larger! cluster! sizes.! Population! distributions! also! slightly! shifted! to! smaller! sizes!as!the!temperature!decreases!(not!shown).!This!was!as!expected!provided!that! water!becomes!a!better!solvent!for!micelles!as!the!temperature!is!lowered!and,!hence,! micellar!bridging!(and!hence!clustering)!is!reduced.!On!the!other!hand,!the!shape!of! micelles!was!nearly!spherical!as!observed!by!TEM!and!AFM,!with!their!diameters!(ca.! 27!±!4!and!32!±5!nm!for!BO14EO378BO14!and!BO21EO385BO21!as!calculated!from!TEM,! respectively)!in!fair!agreement!with!those!obtained!from!DLS!data!despite!the!usual! dehydration! of! the! copolymer! corona! and! subsequent! shrinking! of! the! copolymer! structure!upon!solvent!evaporation!during!sample!preparation!(see!Figure!3a).!From! AFM!images!the!protrusion!of!the!EO!corona!can!be!also!observed!showing!a!slightly! less!spherical!micellar!shape!than!in!TEM!images!(Figure!3b).!! ! From! plots! of! 1/rh,app! against! copolymer! concentration! the! micellar! hydrodynamic!radii!(rh)!was!obtained!as!the!intercept!of!each!curve!at!c!=!0!(see!Figure! 2b!and!Table!2).!1/rh,app!is!proportional!to!the!apparent!diffusion!coefficient,!Dapp,!but! without! the! influence! of! temperature! and! solution! viscosity.! The! negative! slopes! of! these! plots! pointed! to! negative! values! of! the! second! virial! (A2)! coefficient;! this! indicates! that! micelles! interact! attractively! by! bridging! (33),! as! confirmed! by! the! protrusions!observed!from!some!micelles!to!others!denoting!intermicellar!bridging!in! AFM!images!(Figure!3c);!van!der!Waals!attraction!and!polymer!depletion!should!not! play!significant!roles!in!the!present!dilute!micellar!systems!(24,26).! !! Figure!2.!a)!Intensity/weighted!population!distributions!obtained!by!DLS!for!copolymer! BO21EO385BO21!in!solution!at!25ºC!(red,!black!and!red!lines!correspond!to!0.4,!2.5!and! 10! mg/mL! solutions,! respectively);! b)! Reciprocal! apparent! hydrodynamic! radius,! 1/rh,app,#against!concentration!for!copolymer!BO21EO385BO21!at!10ºC!(£)!and!25ºC!(¢).! ! 122! Both!the!BO!and!EO!block!lengths!will!affect!intermicellar!interactions:!Longer! BO/end!blocks!should!imply!that!intermicellar!interaction!can!become!stronger,!while! long!central!EO!blocks!can!make!the!BO!blocks!to!be!extended!into!the!solution!more! easily!(34).!Comparison!of!the!present!data!with!those!previously!reported!for!shorter! BOmEOnBOm!copolymers!suggested!that!BO14EO378BO14!and!BO21EO385BO21!displayed!a! stronger! intermicellar! attraction! as! would! correspond! to! reverse! copolymers! with! relatively!long!BO!blocks!and!extremely!lengthy!EO!ones.! !!!!!!!!! ! ! ! ! Figure!3.!a)!TEM!image!of!micelles!formed!by!copolymer!BO21EO385BO21!(scale!bar!500! nm);!b)!AFM!image!of!BO21EO385BO21!micelles!with!a!protruded!corona;!c)!AFM!image! showing!interchain!bridges!between!copolymer!micelles.! ! 3.3.4.3!Micellar!properties! Since! the! hydrodynamic! radii! of! the! present! polymeric! micelles! (Table! 2)! are! small!compared!to!the!light!wavelength,!intraparticle!interference!can!be!neglected.! Clustering! at! higher! copolymer! concentrations! changes! this! picture,! but! here! we! focused!on!the!behavior!in!the!dilute!regime.!Debye!plots,!i.e.!plots!based!on!! a!)! b)! c)! ! 227! 6!ppm!due!to!its!four!equivalent!protons.!Figure!6!c)!shows!a!real!NMR!spectrum!of!a! reverse!triblock!PBOVPEOVPBO!PBO!copolymer!(10).!! ! ! ! Figure!6.!NMR!spectra!of!two!hydrocarbon!chains:!a)!an!ethanol!molecule!(CH3CH2OH),!b)! an!ethylene!molecule!(CH2CH2)!and!c)!a!PBO20PEO411PBO20!triblock!copolymer!chain!(6).! ! ! 5.3!UVVVis!SPECTROSCOPY! ! Ultraviolet!and!visible!(UVVVis)!absorption!spectroscopy!is!a!technique!based!on! the! measurement! of! light! absorbed! by! a! sample! (11).! When! an! atom! or! molecule! absorbs! energy,! electrons! are! promoted! from! their! ground! state! to! an! excited! state.! Molecules!can!only!absorb!radiant!energy!in!finite!units!or!quanta,!which!correspond!to! the!energy!difference!between!the!ground!and!excited!states.!This!energy,!E,!carried!by! any!one!quantum!is!proportional!to!its!frequency!of!oscillation,!𝐸=ℎ𝜈=!! !!,!where!ν!is! the!frequency,!λ!the!related!wavelength,!and!h!the!Planck's!constant.! ! ! 228! In!addition!to!electronic!excitation,!the!atoms!within!a!molecule!can!rotate!and! vibrate!regarding!each!other.!These!vibrations!and!rotations!also!have!discrete!energy! levels,!which!can!be!considered!as!being!packed!on!top!of!each!electronic!level.!UVVVis! molecular! spectroscopy! describes! the! excitation! of! a! valence! electron! of! a! molecule! upon! energy! absorption! from! the! electromagnetic! radiation! which! is,! thereby,! transferred!from!one!energy!level!to!other!more!energetic!one.!An!electronic!transition! consists!of!the!promotion!of!an!electron!from!a!molecular!orbital!in!the!ground!state!to! an!unoccupied!orbital!by!absorption!of!a!photon.!The!molecule!is,!then,!said!to!be!in!an! excited!state.!! ! The!wavelength!range!a!spectrophotometer!scans!usually!goes!from!200!to!1100! nm.! The! experimental! data! ususally! are! plotted! as! the! transmitted/incident! intensity! ratio!versus!the!wavelength!of!incident!radiation.!! ! Absorption! of! ultraviolet! and! visible! light! in! organic! molecules! is! restricted! to! certain! functional! groups! (chromophores)! that! contain! valence! electrons! of! low! excitation!energy.!The!spectrum!of!a!molecule!containing!these!chromophores!is!rather! complex! as! the! superposition! of! atomic! rotational! and! vibrational! transitions! on! the! electronic!transitions!provides!a!combination!of!overlapping!lines.;!hence!the!resulting! spectrum!appears!as!a!continuous!absorption!band.! ! Ultraviolet!radiation!having!wavelengths!less!than!200!nm!is!difficult!to!handle,! and!is!seldom!used!as!a!routine!tool!for!structural!analysis.!UVVVis!light!causes!primarily! electronic!excitation!by!promoting!the!outer!electrons!of!lower!orbitals!to!higher!energy! levels!and,!then,!it!is!sometimes!called!electronic%spectroscopy.!The!easily!accessible!part! of! this! region! (wavelengths! of! 200! to! 800! nm)! shows! energy! absorption! only! if! conjugated!πVelectron!systems!are!present.!There!are!a!number!of!possible!electronic! transitions,! as! shown! in! Figure! 7,! called!𝑛→𝜋∗,!𝜋→𝜋∗,!𝑛→𝜎∗,!𝜋→𝜎∗,!𝜎→𝜋∗,! and! 𝜎→𝜎∗!(12).! The! energy! of! these! electronic! transitions! follows,! generally,! next! order:! 𝑛→𝜋∗!<!!𝜋→𝜋∗!<!𝑛→𝜎∗!<𝜋→𝜎∗<!𝜎→𝜋∗!<!𝜎→𝜎∗.! Of! the! six! transitions! outlined,! only! the! two! lowest! energetic! ones! (𝑛→𝜋∗!and!𝜋→𝜋∗)! are! achieved! by! energies! ranging!between!200!to!800!nm.!The!last!four!types!of!electronic!transitions!required! higher!energy!inputs,!below!200!nm!corresponding!to!the!far!ultraviolet!region!of!the! electromagnetic!spectrum!(12,13).! ! ! 229! ! ! Figure! 7.!Scheme!showing! differences! between!ground!and! singlet! and!triplet!excited! states!(9).!! ! ! A!𝜎!orbital!can!be!formed!either!from!two!s!atomic!orbitals,!from!one!s!and!one!p,! or!from!two!p!atomic!orbitals!having!a!collinear!symmetry!axis.!The!bond!formed!in!this! way! is! called! a!𝜎!bond.!𝜋!orbitals! are! formed! from! two! p! atomic! orbitals! overlapping! laterally;!the!resulting!bond!is!called!a!𝜋!bond.!For!example,!in!ethylene!(CH2=CH2)!the! two! carbon! atoms! are! linked! by! one!𝜎!and! one!𝜋!bond.! Absorption! of! appropriate! energy! can! promote,! for! example,! one! of! the!𝜋!electrons! to! an! antiVbonding! orbital! denoted!as!𝜋∗;!then,!the!transition!is!called!𝜋→𝜋∗.!A!molecule!may!also!possess!nonV bonding! electrons! located! on! heteroatoms! such! oxygen! or! nitrogen;! then,! the! corresponding! molecular! orbitals! are! called!𝑛!orbitals.! Promotion! of! a! nonVbonding! electron! to! an! antiVbonding! orbital! is! also! possible,! and! the! associated! transition! is! denoted! by!𝑛→𝜋∗.! Hence,! molecules! containing! a! nonVbonding! electron,! such! as! oxygen,!nitrogen,!sulphur,!or!halogens,!often!exhibit!absorption!in!the!UV!region!(13).!! ! When! one! of! the! two! electrons! of! opposite! spins! (belonging! to! a! molecular! orbital!of!a!molecule!in!the!ground!state)!is!promoted!to!a!molecular!orbital!of!higher! energy,! its! spin! is,! in! principle,! unchanged! so! that! the! total! spin! quantum! number! (𝑆=𝑠!!with!𝑠!=+! !!or!𝑠!=−! !)! is! zero.! Because! of! the! multiplicities! of! both! the! ground! and! excited! states! (𝑀=2𝑆+1)! are! equal! to! 1,! both! are! called! singlet% states! (usually!denoted!𝑆!!for!the!ground!state,!and!𝑆!,!𝑆!,...!for!the!excited!states,!see!figure! 5.7).!The!corresponding!transition!is!called!a!singletVsinglet!transition.!A!molecule!in!a! singlet!excited!state!may!undergo!conversion!into!a!state!where!the!promoted!electron! has!changed!its!spin;!as!a!consequence,!there!are!two!electrons!with!parallel!spins,!and! ! 230! the!total!spin!quantum!number!is!1,!and!the!multiplicity!is!3.!Such!state!is!called!a!triplet! state!because!it!corresponds!to!three!states!of!equal!energy.!According!to!Hund’s!rule,! the!triplet!state!has!lower!energy!than!the!singlet!state!of!the!same!configuration!(12).! ! The!probability!of!transitions!is!also!strongly!influenced!by!bond!conjugation.!A! conjugation!enhancement!brings!the!highest!occupied!and!lowest!unoccupied!molecular! orbitals! closer! together.! The! energy! (ΔE)! required! to! do! this! electron! promotion! is! therefore!lower,!and!the!wavelength!that!provides!this!energy!is!longer!correspondingly.! Conjugation! of! double! and! triple! bonds! shifts! the! absorption! maximum! to! longer! wavelengths,! and! extending! conjugation! generally! results! in! increased! bathochromic! (longer!wavelength)!and!hyperchromic!(greater!absorbance)!shifts!in!absorption!spectra.! ! The!solvent!in!which!the!absorbing!species!are!dissolved!also!has!an!effect!on!the! resulting!spectrum!of!the!species.!Peaks!resulting!from!n!to!π*!transitions!are!shifted!to! shorter! wavelengths! (blueVshifted)! with! increasing! solvent! polarity.! This! arises! from! increased!solvation!of!the!lone!pair!of!electrons,!which!lowers!the!energy!of!the%n!orbital.! Often,! the! opposite! effect! (i.e.! a! redVshift)! is! observed! for! π! to! π*! transitions.! This! is! produced! by! attractive! polarization! forces! between! the! solvent! and! the! absorbing! molecule,!which!lower!the!energy!levels!of!both!the!excited!and!unexcited!states.!This! effect!is!greater!for! the! excited! state,!and!the!energy! difference! between!the!excited! and!unexcited!states!is!slightly!reduced,!resulting!in!a!small!redVshift.!This!also!influences! n!to!π*!transitions!but!is!overshadowed!by!the!blueVshift!resulting!from!solvation!of!lone! electron!pairs.! ! !!!!!! ! ! Figure!8.!Scheme!of!UVVvis!spectrometer!optical!path!with!its!constitutive!elements,!and! examples!of!absorbance!spectra.! ! 231! In!summary,!when!sample!molecules!are!exposed!to!light!having!an!energy!that! matches!a!possible!electronic!transition!within!a!molecule,!some!of!the!light!energy!will! be! absorbed! as! electrons! are! promoted! to! higher! energy! orbitals.! An! optical! spectrometer! records! the! wavelengths! at! which! absorption! occurs! together! with! the! extent! of! absorption! at! each! wavelength.! Figure! 8! shows! a! scheme! of! the! underlined! excitation! mechanism! done! in! a! spectrophotometer! and! the! resulting! information! provided.!! ! ! 5.4!FLUORESCENCE!SPECTROSCOPY! ! As! mentioned! before,! energy! absorption! by! electrons! in! the! ground! state! promotes!them!to!higher!energy!levels,!called!excited!states.!Once!a!molecule!is!excited! by! the! absorption! of! a! photon,! this! can! return! its! ground! state! through! several! desV excitation!processes,!as!fluorescence!emission,!internal!energy!conversion!(i.e.!by!heat! radiation),!intersystem!crossing!(possibly!followed!by!phosphorescence!emission),!intraV molecular!charge!transfer!and/or!conformational!change.! ! Fluorescence! is! a! property! that! some! materials! can! exhibit! upon! energy! absorption!in!the!wavelength!range!from!XVray!to!UV!(λ!~!0.01V400!nm);!this!process!is! followed!by!desVexcitation!in!the!form!of!light!emission!in!the!visible!range!(λ!~!400V700! nm).!As!a!consequence,!there!may!exist!a!meaningful!difference!between!absorbed!and! emitted! energies! since! a! highVenergy! photon! can! be! absorbed! whilst! a! low! energy! photon! can! be! emitted.! However,! there! is! not! a! violation! of! the! conservation! law,! because! the! energy! difference! is! dissipated! in! the! form! of! heat! owing! to! molecular! vibrations!in!the!excited!state.!! ! Figure!9!shows!the!vibrational!bands!in!absorption!and!fluorescence!spectra.!The! singlet!electronic!states!are!denoted!as!S0!(the!fundamental!electronic!state),!S1,!S2,!...! with!different!vibrational!levels!associated!with!each!electronic!state.!It!is!important!to! note!that!energy!absorption!is!very!fast!(in!the!order!of!ms)!regarding!all!other!processes! (there!is!no!concomitant!shift!of!nuclei!according!to!the!FranckVCodon!principle)!(12,14).! The!absorption!process!starts!from!the!fundamental! vibrational!energy!level,!S0,!since! most!of!molecules!are!in!this!state!at!room!temperature.!Absorption!of!a!photon,!hence,! can! bring! a! molecule! to! one! of! the! upper! vibrational! levels! (S1,! S2,! ...).! Emission! of! photons! accompanying! the! S1!→! S0! relaxation! is! called! fluorescence.! The! transition! between! the! ground! state! and! the! excited! state! (0Vtransition)! is! usually! the! same! for! absorption!and!fluorescence.!However,!the!fluorescence!spectrum!is!located!at!higher! ! 232! wavelengths!than!the!absorption!one!as!a!result!of!the!energy!loss!in!the!excited!state! due!to!vibrational!relaxation.!! ! ! ! Figure!9.!Scheme!of!the!vibrational!bands!in!absorption!and!fluorescence!spectra!(15).! ! ! According! to! the! Stokes! rule,! the! fluorescence! emission! wavelength! should! be! always! larger! than! that! the! absorption! one.! However,! the! absorption! spectrum! partly! overlaps!the!fluorescence!spectrum!in!most!cases,! i.e.!a!fraction!of!light!is!emitted!at! shorter!wavelengths!than!the!absorbed!light.!Such!an!observation!seems!to!be,!at!first,! in!contradiction!with!the!energy!conservation!principle.!However,!such!energy!defect!is! compensated!by!the!fact!that!a!small!fraction!of!molecules!is!in!a!higher!vibrational!level! in!the!ground!state!as!well!as!in!the!excited!state!at!room!temperature!(12,!14).! ! In!general,!differences!between!the!vibrational!levels! are!similar!in!the!ground! and! excited! states! so! that! the! fluorescence! spectrum! often! resembles! the! first! absorption!band.!The!gap,!expressed!in!wavenumber,!between!the!maximum!of!the!first! absorption!band!and!the!fluorescence!maximum!is!called!the!Stokes!shift.! ! It!should!be!noted!that!photon!emission!is!as!fast!as!photon!absorption.!However,! excited!molecules!remain!in!the!S1!state!for!a!certain!time!(a!few!tens!of!picoseconds!to! a!few!hundreds!of!nanoseconds!depending!on!the!type!of!molecule!and!its!surrounding! medium)!before!emitting!a!photon!or!undergoing!other!relaxation!processes.!Thus,!after! excitation! of! a! population! of! molecules! by! a! very! short! light! pulse,! the! fluorescence! intensity! decreases! exponentially! with! a! characteristic! time,! reflecting! the! average! ! 233! lifetime!of!the!molecules!in!the!S1!excited!state.!! ! As! a! consequence! of! the! strong! influence! of! the! local! environment! or! surrounding!medium!on!fluorescence!emission,!fluorescent!molecules!are!currently!used! for!physicochemical,!biochemical!and!biological!investigation.!For!example,!fluorescent! molecules! are! added! to! other! systems! to! follow! their! behavior! upon! increases! of! concentration,!temperature,!amount!of!added!salt,!etc!(14,15).! ! Figure!10!shows!the!components!of!a!conventional!spectrofluorimeter.!The!light! source! is! commonly! a! highVpressure! xenon! arc! lamp,! which! offers! the! advantage! of! a! continuous!emission!from!250!nm!to!the!infrared!region.!A!monochromator!is!used!to! select!the!excitation!wavelength.!Fluorescence!is!collected!at!right!angles!with!respect!to! the! incident! beam! and! detected! through! the! monochromator! by! a! photomultiplier.! Automatic!scanning!of! wavelengths! is!achieved!by! motorized! monochromators,!which! are!controlled!by!electronic!devices!and!the!computer,!in!which!data!are!stored.!There!is! an!optical!module!which!contains!several!parts:!a!sample!holder,!shutters,!polarizers!if! necessary,!and!a!beam!splitter!consisting!of!a!quartz!plate!reflecting!a!few!per!cent!of! the! exciting! light! towards! a! quantum! counter! or! a! photodiode.! A! quantum! counter! usually!consists!of!a!triangular!cuvette!which!contains!a!concentrated!solution!of!a!dye! whose!fluorescence!quantum!yield!is!independent!of!the!excitation!wavelength.! ! ! ! Figure!10.!Spectrofluorimeter!setup,!fluorescence!spectrophotometer!and!experimental! curves!obtained!through!this!technique.! ! ! ! 234! 5.4.1!The!pyrene!method! ! Studies! based! on! the! fluorescence! properties! of! several! probes,! as! ethidium! bromide,! methylene! blue,! congo! red,! etc.! has! been! extensively! used! in! biophysical! studies!of!molecular!aggregates!because!their!fluorescence!is!strongly!dependent!on!the! surrounding!media!(16,17).!To! analyze! micellization! processes,!the!most!common! dye! used!is!pyrene!due!to!their!characteristic!structure!and!differential!behavior!in!several! solvents.! The! main! physicoVchemical! properties! that! make! pyrene! really! useful! as! fluorophore!are!its!long!halfVlife!as!monomer!and!its!propensity!to!form!excimers.!The! absorption! and! emission! spectrum! of! pyrene! have! been! extensively! studied,! and! the! maximum!values!at!determined!wavelengths!have!been!related!to!its!vibrational!modes,! as!seen!in!Table!5!(19).! ! Table!5.!Relation!between!the!principal!pyrene!vibrational!bands!at!their!corresponding! wavelengths,! λ,! the! frequency! of! vibrations,! ν,! the! distance! from! the! 0V0! line,! the! corresponding! Raman! assignation,! and! the! vibrational! mode! with! the! corresponding! symmetry.!! Peak% λ,%nm% ν,%cmJ1% Distancy% from%0J0% line% Asignation% (IR/Raman)% Vibrational%mode% and%symmetry% I! 372.51! 26!845! 0! 0!–!0! ! 378.23! 26!439! 406! 0!–!406!(R)! ag!(ω)! II! 378.95! 26!389! 456! 0!–!456!(IR)! b1g!(τ)! 379.58! 26!345! 500! 0!–!500!(IR)! b1g!(τ)! III! 383.03! 26!108! 737! 0!–!737!(IR)! b1g!(κ)! 384.00! 26!042! 803! 0!–!803! ag!(κ)! 387.99! 25!774! 1071! 0!–!1071!(R)! ag!(δ)! IV! 388.55! 25!737! 1108! 0!–!1108!(R)! b1g! 389.08! 25!702! 1143! 0!V1143!(R)! ag!(δ)! 390.42! 25!613! 1232! 0!–!1232!(R)! ag!(δ)! 391.80! 25!523! 1322! 0!V!1322!(R)! (ag!+b1g)(κ)! 392.49! 25!478! 1367! 0!–!1367!(R)! b1g! 392.85! 25!455! 1390! 0!–!1390!(R)! ag!(ω)! V! 393.09! 25!439! 1406! 0!–!1406!(R)! ag!(ω)! 395.34! 25!295! 1551! 0!–!1551!(R)! ag! 396.04! 25!250! 1595! 0!–!1595!(R)! b1g!(ω)! ! ! ! 235! Pyrene! has! a! characteristic! five! peakVspectrum,! being! the! first! and! third! peaks! those! exhibiting! larger! sensitivity! due! to! their! stronger! dependence! with! the! surrounding!medium!(Figure!11).!Pyrene!is!mainly!hydrophobic,!so!if!added!in!very!small! amounts! to! a! polymer! solution,! the! micellization! process! of! such! polymer! upon! concentration!or!temperature!changes!can!be!followed.!Plotting!the!ratio!between!the! first! and! the! third! fluorescence! peaks! (I1/I3)! versus! ! polymer! concentration,! a! characteristic! plot! is! obtained.! The! transition! zone! between! the! two! plateau! regions! corresponds!to!the!concentration!range!where!micellization!occurs,!termed!the!critical! micellar!concentration!(CMC).! ! ! ! Figure!11.!Left!image:!Fluorescence!pyrene!spectrum!in!ethanol.!Right!image:!Plot!of!I1/I3! versus!log(c)!to!derive!the!micellization!region!and!the!critical!micelle!concentration!(18).!! ! ! ! 5.5!References! ! 1.! Thomas,!O.;!Burgess,!C.!UVJvisible%Spectrophotometry%of%Water%and%Wastewater,! 2007!,!Elsevier!BV!.! 2.!de!Broglie,!L.!Foundations!Phys.!1970,!1,!5.! 3.! http://www.espectrometria.com/tipos_de_espectrometra! 4.!Resonancia% Magnética% Nuclear% de% Protón:% Aplicaciones% en% Química% Orgánica,% Cursos%en%Internet%del%Departamento%de%Química%Orgánica%y%BioJOrgánica%de%de% la%UNED.% 5.! Jacobsen,!N.E.!NMR%Spectroscopy%Explained:%Simplified%Theory,%Applications%and% Examples% for% Organic% Chemistry% and% Structural% Biology.! 2007,! Hoboken,! N.J.:! WileyVInterscience.! 6.!Keeler,!J.!!Understanding%NMR%spectroscopy.!2010,!Chichester:!Wiley.! 7.! Mirau,! P.A.,! A% practical% Guide% to% Understanding% the% NMR% of% Polymers.! 2005,! Hoboken,!N.J.:!WileyVInterscience.! 8.! Hamley,!I.W.,!The%Physics%of%Block%Copolymers.!Oxford!science!publications.!1998,! Oxford:!Oxford!University!Press.! ! 236! 9.! Pavia,! D.L.,! Introduction% to% Spectroscopy.! 2009,! Belmont,! Calif.:! Brooks/Cole,! Cengage!Learning.! 10.! Cambón,! A.;!AlatorreVMeda,!M.;!Juarez,! J.;! Topete,! A.;!Mistry,!D.;!Attwood,! D.;! Barbosa,!S.;!Taboada,!P.;!Mosquera,!V.!J.!Colloid!Interface!Sci.!2011,!361,!154.! 11.!User% Guidelines% and% Standard% Operating% Procedure% for% the% Cary% 50% UVJvis% Spectrophotometer,%Laurier!Reseach!Instrumentation.% 12.! Valeur,!B.,!Molecular%Fluorescence:%Principles%and%Applications.!2013,!Weinheim:! WileyVVCH.! 13.! Bernath,!P.F.!Spectra%of%Atoms%and%Molecules.!2005,!!Oxford!Univ.!Press.! 14.! Van! Holde,! K.E.J.W.C.H.P.S.,! Principles% of% Physical% Biochemistry.! 1998,! Upper! Saddle!River,!N.J.:!Prentice!Hall.! 15.! Lakowicz,!J.R.,!Principles%of%Fluorescence%Spectroscopy.!1999,!New!York:!Kluwer! Academic.! 16.! Radda,!G.!K.;!!Vanderkooi,!J.!Biochim.!Biophys.!Acta,!1972,!265,!509.! 17.!Wehry,!E.L.!Modern%Fluorescence%Spectroscopy.!1976,!New!York:!Plenum!Press.! 18.! Lianos,!P.;!Georghiou,!S.!Photochem.!Photobiol.!1979,!30,!355.! 19.! Kalyanasundaram,!K.;!Thomas,!J.K.!J.!Am.!Chem.!Soc.!1977,!99,!2039.! ! ! ! 243! ! In!order!to!determine!the!molecular!weight!(Mw),!the!radius!of!gyration#(Rg),!and! the! second! virial! coeficcient# (A2)! it! is! necessary! to! prepare! several! dilute! solutions! of! different! concentration! and! measure! the! scattering! intensities! data! at! different! scattering!angles!(30,31).! ! 5.7.2!DYNAMIC!LIGHT!SCATTERING! ' In!dynamic!light!scattering,!the!diffusive!motion!of!particles!in!solution!gives!rise! to! fluctuations! in! the! scattered! light! intensity! on! the! microsecond! timescale.! This! technique! is! one! of! the! most! popular! methods! used! to! determine! particle! sizes! by! measuring! the! temporal! fluctuations! of! the! scattered! light! intensity.! Roughly,! this! is! made! by! focusing! a! monochromatic! light! beam,! such! as! a! laser,! on! a! solution! with! particles! in! Brownian! motion;! this! causes! a! Doppler! shift! when! the! light! “hits”! the! moving!particle,!changing!the!light!wavelength.!This!change!is!related!to!the!particle!size! (30,32).! ! ! ' Figure'16.!Illustration!of!an!intensity!autocorrelation!function.! ! In!the!scope!of!DLS,!temporal!fluctuations!are!usually!analyzed!by!means!of!the! intensity! autocorrelation! function! (ACF).! In! the! time! domain,! the! correlation! function! usually! decays! with! time! (Figure! 16),! and! a! faster! dynamics! leads! to! a! faster! decorrelation!of!the!scattered!intensity!trace.!It!can!be!shown!that!for!a!random!process! the!intensity!ACF!is!the!Fourier!transform!of!the!power!spectrum!and,!therefore,!DLS! measurements! can! be! equally! wellKperformed! in! the! spectral! domain.! In! fact,! DLS! experiments!were!initially!discussed!in!terms!of!the!broadening!of!the!spectrum!peak!of! ! 244! monochromatic! light! due! to! Doppler! shifts! experienced! by! propagating! light! waves! scattered!by!moving!particles!(33).!! ! To! detect! the! intensity! fluctuation! with! time,! a! DLS! system! requires! an! autocorrelator!on!top!of!a!regular!SLS!system,!as!shown!in!Figure!17.!The!pulse!amplifier! discriminator! converts! the! analogic! signal! of! the! photodetector,! I(t),! in! a! digitalized! signal,! which! is! further! processed! by! the! autocorrelator! into! the! autocorrelation! function.! ! ! ! Figure'17.!Sketch!of!a!dynamic!light!scattering!system.!! ! ! Figure! 18a! illustrates! how! the! intensity! (I)! varies! with! time! (t).! I(t)! fluctuates! around!its!mean!value,! <I>.! Motions! of!particles!(i.e.!polymer!molecules)! and! solvent! molecules!contribute!to!the!change!of!I(t)!with!time.!This!apparently!noisy!signal!carries! the!information!about!particles!motions.! ! ! 245! ! ! Figure' 5.18.! a)! Light! scattering! intensity! I(t)! fluctuates! around! its! mean! value! 𝐼.! b)! Autocorrelation!function! 𝐼𝑡𝐼𝑡+𝜏!is!obtained!as!the!longKtime!average!for!various! delay! times,τ.! The! autocorrelation! function! decays! from! 𝐼!!to! 𝐼!!over! time.! The! amplitude!of!the!decaying!component!is! ∆𝐼!.! ! ! The!autocorrelator!calculates!the!product!average!of!two!scattering!intensities! I(t)!and!I(t+ τ )!measured!at!two!different!times!separated!by!a!delay!time,! τ .!The!average! product! 𝐼𝑡𝐼𝑡+𝜏!is!called!the!correlation!function!of!I(t),!or!the!intensityKintensity! correlation!function.!The!correlator!converts!I(t)!into! 𝐼𝑡𝐼𝑡+𝜏!over!a!long!period!T.! Hence,!we!can!write!(25,35):! ! 𝐼𝑡𝐼𝑡+𝜏=lim!→! ! ! 𝐼𝑡𝐼𝑡+𝜏𝑑𝑡 ! !!!!!!!!!!!!!!!!!!!!!!!!(13)! ! The!autocorrelation!function!of!I(t)!(Figure!18a)!is!shown!in!figure!5.18!b).!When! τ ! =! 0,! 𝐼𝑡𝐼𝑡+𝜏=𝐼!.! With! increasing! τ ,! 𝐼𝑡𝐼𝑡+𝜏!decays! to! an! asymptotic! level!(baseline),! 𝐼!.! ! Because! the! scattering! intensity! I(t)! fluctuates! around! a! mean! value! 𝐼,! it! is! convenient! to! separate! its! fluctuating! component,!∆𝐼𝑡,! as!𝐼𝑡=𝐼+∆𝐼𝑡.! The! correlation!function!can!be!rewritten!as!(26):!! ! 𝐼𝑡𝐼𝑡+𝜏=𝐼!+∆𝐼𝑡∆𝐼𝑡+𝜏!!!!!!!!!!!!!!!!!!!!!!!!(14)! ! 246! ! Division!of! 𝐼𝑡𝐼𝑡+𝜏!by! 𝐼!!leads!to!the!intensity!autocorrelation!function:! ! ! 𝐼𝑡𝐼𝑡+𝜏𝐼!=1+∆𝐼𝑡∆𝐼𝑡+𝜏𝐼!=1+𝑓 !𝑔!𝜏!!! !!!!!!(15)! ! ! where!𝑓 !≡∆𝐼!𝐼!!(the! coherent! factor),! and!𝑔!𝜏!is! the! normalized! intensity! autocorrelation!function:! ! 𝑔!𝜏≡∆𝐼𝑡∆𝐼𝑡+𝜏∆𝐼!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(16)! ! fc! depends! on! the! coherence! of! the! light! reaching! the! photodetector.! The! measured! intensity!correlation!function!is!related!to!the!field!correlation!function!by!the!Siegert´s! relation!(34):! ! 𝑔!𝜏=1+𝑏𝑔!𝜏!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(17)! ! where!𝑔!𝜏,!the!field!ACF,!is:! ! 𝑔!𝜏=𝐸∗𝑡𝐸(𝑡+𝜏)𝐸(𝑡)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(18)! ! being!𝐸∗𝑡!the!electric!field!conjugate!function.!For!monodisperse!spherical!particles:! ! 𝑔!𝑡=𝑒𝑥𝑝 −Γ𝜏!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(19)! ! where! Γ !is!the!characteristic!delay!rate,!which!is!related!to!the!translational!diffusion! coefficient,!D,!of!a!solute!by!means!of!the!expression:! ! !!𝛤=𝑞!𝐷!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!(20)! ! D! is! frequently! used! to! determine! the! hydrodynamic! radius,! RH,! of! the! constituent! particles!by!using!the!StokesKEinstein!equation!(34):! ! H R T B k D πη 6 = !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! !!!!!(21)! ! where! kB! is! the! Boltzmann! constant,! T! the! absolute! temperature! and! η ! the! liquid! viscosity.! The! hydrodynamic! radius! obtained! by! DLS! represents! an! ideal! hard! sphere! ! 247! that!diffuses!with!the!same!speed!as!the!particle!under!examination.!Actually,!particles! are! solvated! and! the! radius! calculated! from! the! particle! diffusion! corresponds! to! the! size!of!the!dynamic!solvated!particle.! ! 5.7.3'References' ' 25.! Schärtl,! W.! Light# Scattering# from# Polymer# Solutions# and# Nanoparticle# Dispersions.!2007,!Springer.! 26.! Xu,!R.!Particle#Characterization#Light#Scattering#Methods.!!2002,!Springer.! 27.! Sun,!S.F.!Physical#Chemistry#of#Macromolecule:#Basic#Principles#and#Issues.!1994,! New!York:!Wiley.! 28.! Young,!R.J.,!Introduction#to#Polymers.!1981,!Chapman!and!Hall.! 29.! Huglin,!M.B.,!Light#Scattering#from#Polymer#Solutions.!1972,!!Academic!Press.! 30.! Holoubek,!J.!J.!Quant.!Spectrosc.!Radiat.!Transfer.!2007,!106,!104.! 31.! Uchegbu,! I.F.;! Schätzlein,! A.G.,! Polymers# in# Drug# Delivery.! 2006,! CRC,! Taylor! &! Francis.! 32.! Murphy,!R.M.!Curr.!Opin.!Biotechnol.!1997,!8,!25.! 33.! Kokhanovsky,! A.A.! Light# Scattering# Reviews# 4:# Single# Light# Scattering# and# Radiative#Transfer.!!2009,!Springer! 34.! Burchard,!W.P.G.D.,!Light#Scattering#from#Polymers.!1983,!SpringerKVerlag.! ! ! ! 248! 5.8!ISOTERMAL!TITRATION!CALORIMETRY! Isothermal!titration!calorimetry!(ITC)!is!a!physical!technique!used!to!determine! the!thermodynamics!of!chemical!interactions.!!The!basis!of!the!method!rely!on!the!fact! that!heat!is!either!generated!or!absorbed!when!substances!bind,!so!the!ITC!equipment! directly! measures! the! heat! released! or! absorbed! during! a! mixing! process.!The! experimental!setup!is!plotted!in!Figure!19!(35).!! ! Figure'19.!Right:!ITC!equipment.!Left:!Main!parts!of!an!ITC!instrument:!jacket,!injector,! sample´s!cell!and!reference!cell.!! Briefly,! a! syringe! containing! one! solution! is! titrated! into! a! cell! containing! a! different! solution.! When! the! two! solutions! (or! species)! interact,! the! heat! variation! causes!a!difference!in!temperature!respect!to!a!reference!cell.!The!energy!that!the!ITC! equipment!apply!to!maintain!the!reference!cell!at!the!same!temperature!as!the!sample´s! cell! is! equivalent! to! that! involved! in! the! mixing! process.! The! process! is! done! in! an! completely! isolated! cell,! maintaining! constant! both! volume! and! pressure!so! that! the! variation! in! internal! energy! (i.e.,! heat)! involved! corresponds! to! the! enthalpy! of! the! system.!Measurement!of!the!enthalpy!(∆H)!allows!the!accurate!determination!of!binding! constants! (KB),! reaction! stoichiometry! (n)! and! entropy! (ΔS)! (36).! Figure! 20! shows! a! typical!thermogram!obtained!by!an!ITC!experiment.! ! 249! ! ! Figure' 20.!Typical!plots!of!experimental!ITC!data.!Each!peak!represents!a!heat!change! associated!with!the!injection!of!a!small!volume!of!sample!into!the!ITC!reaction!cell.!Top:! Raw!ITC!data.!Bottom:!Binding!isotherm!(35).! Thermodynamic! data,! specifically! enthalpy! (ΔH)! and! entropy! (ΔS),! reveal! the! forces! that! drive! complex! formation! and! their! mechanism! of! action! (36Y38).! The! thermodynamic!data!provide!information!on!conformational!changes,!hydrogen!bonding,! hydrophobic! interactions,! and! chargeYcharge! interactions! of! the! involved! species.!ITC! data!permits!to!distinguish!if!the!interaction!arise!from!electrostatic!interaction!or!are! driven!by! hydrophobic!forces.!!In!addition,! if!the!system!absorbs!or!releases!heat! the! process!is!called!endothermic!or!exothermic,!respectively.!! As!well!as!binding!processes!between!different!materials!in!solution,!aggregation! processes!can!also!be!followed!by!ITC!due!to!energy!changes!in!this!type!of!process.!For! example,!mIcellization!in!aqueous!solution!is!an!endothermic!process!that!also!can!be! followed! by! ITC! analysis.! One! of! the! most! common! method! consist! of! diluting! a! concentrated! sample;! because! it! is! a! reversible! process,! the! demicellization! test! is! a! reliable! analyses! of! the! micellization! process! that! permits! to! obtain! the! thermal! parameters!of!the!process.!! ! 250! The!Origin!software!provides!six!builtYin!curve!fitting!models!for!ITC!data!analysis:! one!set!of!identical!sites,!two!sets!of!identical!sites,!sequential!binding!sites,!competitive! binding,! dissociation! and! enzyme! assays! (35).! Each! fitting! model! has! a! unique! set! of! fitting! parameters.! For! the! one! set! of! identical! sites! model! these! parameters! are! N! (number!of!sites),!K!(binding!constant!in!MY1),!and!ΔH!(heat!change!in!cal/mole).!A!fourth! parameter,!ΔS!(entropy!change!in!cal/mole/deg)!is!calculated!from!ΔH!and!K.!The!model! for!one!set!of!sites!will!work!for!any!number!of!sites!n!if!all!of!them!have!the!same!K!and! ΔH.! If! a! macromolecule! has! sites! with! two! different! values! of! K! and/or! ΔH,! then! the! model! with! two! sets! of! identical! sites! must! be! used.! These! two! models! employ! the! following!equation!that!incorporates!the!Langmuir!isotherm!binding!equilibrium!for!“i”! independent! sites! of! association,! where! Q! is! the! heat! per! injection,! M! is! the! macromolecule! concentration,! V! is! the! volume! of! the! cell,! n! and! ΔH! are! the! stoichiometry!and!enthalpy!of!interactions,!respectively,!and!Θ!is!the!fraction!of!ligand! bound!to!the!macromolecule!(39):! ! 𝑄=𝑀𝑉 𝑛!! !𝐻𝑖++++!(22)! ! with!the!subYindice!“i”!indicating!the!corresponding!binding!sites.! ! One!can!solve!the!last!equation!for!Θ!using!the!equilibrium!equations!for!binding! constants!Ki,!being!X+the!concentration!of!ligand!and![X]!the!concentration!of!free!ligand! (35,39):! 𝐾𝑖=𝜃 1−𝜃[𝑋]!!!!!!!!!!!!!(23)! 𝑋=𝑋−𝑀𝑛!𝜃! ! ! ' One!and!two!binding!site!are!the!most!employed!methods!for!analyzing!ITC!data.! ! ! 5.8.1'References' ! 35.! ITC+Data+Analysis+in+Origin.+Tutorial+Guide+Version+7.0.+2004.+ 36.! Haines,!P,!Principles+of+Thermal+Analysis+and+Calorimetry.!2002,!Cambridge:!Royal! Society!of!Chemistry.! 37.! Kelarakis,! A.,! Havredaki,! V.;! Rekatas,! C.J.;! Booth,! C.! Phys.!Chem.!Chem.!Phys.! 2001,!3,!5550.! 38.! Brown,! M.E.! Handbook+ of+ Thermal+ Analysis+ and+ Calorimetry+ Principles+ and+ Practice.!!1998,!Elsevier.! 39.! Flory,!P.J.!Principles+of+Polymer+Chemistry.!1953,!!Cornell!University.! ! 251! 5.9!SURFACE!TENSION! Surface! tension! is! a! property! of! liquids! owimg! to! the! cohesive! nature! of! their! molecules.!Molecules!in!bulk!are!isotropically!surrounded!by!neighbouring!ones,!which! involves!a!zero!net!force!over!them!(see!Figure!21).!Conversely,!molecules!at!surfaces! have!at!least!one!part!of!the!proximal!neighbours!in!contact!to!another!surface,!which! provokes!a!different!force!balance.!! ! By!changing!the!surface!tension!of!a!liquid,!different!properties!are!accessible.!As! an!example,!hot!water!has!a!lower!surface!tension,!which!makes!it!easy!to!pass!through! the! clothes! fibres! and! obtain! better! results! when! cleaning.! When! adding! salt! or! surfactants!to!water,!the!surface!tension!of!the!solution!is!also!changed,!and!this!is!the! basis!of!soaping.! ! ! ! Figure'21.!Surface!tension!in!water!molecules.! ! ! Surfactants! have! a! great! affinity! for! surfaces! owing! to! their! amphiphilic! character.! Hydrophilic! chains! tend! to! aggregate! on! surfaces,! where! the! energy! they! need! is! lower! and,! as! a! consequence,! the! surface! energy! (or! surface! tension)! is! minimised.!The!same!behaviour!is!also!observed!for!amphiphilic!polymers!in!solution,! especially!in!water!(see!Figure!22).!As!the!aggregation!properties!of!polymers!depend! on!concentration!as!well!as!surfactants!do,!changes!in!the!solution!structure!containing! polymers! can! be! followed! by! surface! tension! measurements.! The! critical! micelle! concentration! (CMC)! of! amphiphilic! systems! can! be! determined! by! measuring! their! surface!tension!(γ)!as!a!function!of!concentration,!being!such!behaviour!denoted!by!a! fairly!sharp!decrease!in!a!γ!vs.!log!(c)!plot.!! ! ! 252! ! ! Figure'22.!Behaviour!of!amphiphilic!copolymers!in!water!solution!as!the!concentration! raises!and!the!corresponding!change!in!surface!tension.! ! ! There! are! several! methods! for! determining! the! surface! tension! of! amphiphilic! polymer!solutions!such!as!the!Du!Noüy!ring,!the!Wilhelmy!plate,!the!pendant!drop,!the! bubble!pressure!or!the!sessile!drop!methods!(40). ! ! 5.9.1!WILHELMY!PLATE!METHOD! ! ' The!Wilhelmy!plate!method!measures!the!force!(F)!with!which!a!platinum!plate! of!known!perimeter!(L=$l+d)!is!pulled!downwards!by!an!interface.!The!surface!tension! force,$L γ cos θ !,!is!equal!to!the!weight!of!the!liquid!meniscus!adsorbed!onto!the!plate!and! detected!by!a!balance!(2),!which!can!be!related!through!the!following!equation:! ! 𝑤=2(𝑙+𝑑)𝛾𝑐𝑜𝑠𝜃!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(24)! ! where!w!is!the!meniscus’!weight!detected!by!the!balance,! θ !is!the!contact!angle!defined! by!the!meniscus!shape!on!the!wet!platinum!surface!plate,$l!is!the!width!and!d!the!plate! thickness,! respectively! (Figure! 23).! Considering! θ $very! small! and! the! plate! thickness! negligible!compared!with!its!width,!the!former!expression!can!be!simplified!to:! ! 𝑤=2𝛾𝑙!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(25)! ! ! 259! A!scanning!electron!microscope!(Figure!28)!consists!of!an!electron!gun!at!the!top! of!the!column,!which!creates!a!divergent!electron!beam.!In!the!column,!which!is!under! high!vacuum!conditions,!a!series!of!magnetic!apertures!focuses!the!electron!beam,!and! an! electrostatic! field! drives! the! electrons! through! a! small! spot,! called! crossover,! and! accelerates! them! through! the! column! until! the! sample! chamber,! where! the! electron! beam!interacts!with!the!sample.!The!signals!resulting!from!the!beamRsample!interaction! are!monitored.!Finally,!SEM!constructs!a!virtual!image!from!the!signal!emitted!from!the! sample!by!scanning!the!electron!beam!line!by!line!through!a!rectangular!(raster)!pattern! on!the!sample!surface.!The!scan!pattern!defines!the!area!represented!in!the!image.!At! any!time,!the!beam!illuminates!only!a!single!point!in!the!pattern.!As!the!beam!moves,! the!signals!it!generates!vary!in!strength,!reflecting!structural/morphological!differences! in!the!sample!(51).! ! ! ! Figure(28.(Left:!SEM!schematic!diagram.!Right:!Real!SEM!picture.! ! ! 5.10.3!ATOMIC!FORCE!MICROSCOPY!(AFM)( ( ! AFM! is! part! of! a! large! family! of! instruments! termed! as! scanning! probe! microscopes!(SPM).!The!common!factor!in!all!SPM!techniques!is!the!use!of!a!very!sharp! tip!probe,!which!is!scanned!across!a!surface!of!interest.!The!interactions!between!the! ! 260! probe! and! the! surface! are! able! to! produce! a! highRresolution! image! of! the! sample! (potentially!up!to!the!subRnanometre!scale)!depending!on!the!technique!and!sharpness! of!the!probe!tip.!For!AFM,!the!probe!usually!interacts!directly!with!the!surface!probing! the! repulsive! and! attractive! forces,! which! exist! between! the! probe! and! the! sample! surface.!This!serves!to!produce!a!high!resolution!threeRdimensional!topographic!image! of!the!latter.!The!great!versatility!of!AFM!makes!possible!measurements!in!air!or!fluid! environments!rather!than!in!high!vacuum,!which!allows!the!imaging!of!polymeric!and! biological! samples! in! their! native! states.! In! addition,! it! is! highly! adaptable,! with! tip! probes!being!able!to!be!chemically!functionalised!to!allow!quantitative!measurements!of! interactions!between!many!different!types!of!materials!(43).! An!AFM!instrument!(Figure!29)!consists!of!a!sharp!tip!probe!mounted!at!the!apex! of!a!flexible!cantilever,!made!of!Si!or!Si3N4.!The!cantilever!itself!or!the!sample!surface!is! mounted!on!a!piezoRcrystal,!which!allows!the!position!of!the!probe!to!be!shifted!respect! to!the!surface.!The!movement!in!this!direction!is!conventionally!referred!to!as!the!ZRaxis.! The!deflection!of!the!cantilever!is!monitored!by!changes!in!the!path!of!a!laser!light!beam! deflected!from!the!upper!sideRend!of!the!cantilever!recorded!by!a!photodetector!(43).! ! Figure(29.!Left:!Typical!AFM!setup.!Right:!AFM!picture.! ! ! 5.10.4(References( ( 41.! Herman,!B.L.J.J.,!Optical"Microscopy:"Emerging"Methods"and"Applications.!1993,!! Academic!Press.! 42.! Murphy,!D.B.!Fundamentals"of"Light"Microscopy"and"Electronic"Imaging.!!2001,! John!Wiley!&!Sons.! ! 261! 43.! Bowen,!W.R.H.N.,!Atomic"Force"Microscopy"in"Process"Engineering:"Introduction" to" AFM" for" Improved" Processes" and" Products.! 2009,! ! Elsevier/ButterworthR Heinemann.! 44.! Haynes,!R.,!Optical"Microscopy"of"Materials.!1984,!!Springer.! 45.! Moorehead,!W.!Scanning!2004,!26,!204.! 46.! Buffington,!A.!Optical"Microscopy.!!2012.! 47.! Corle,!T.R;!Kino,!G.S.!Confocal"Scanning"Optical"Microscopy"and"Related"Imaging" Systems.!!1996,!Academic!Press.! 48.! Fultz,! B.H.J.M.,! Transmission" Electron" Microscopy" and" Diffractometry" of" Materials.!2008,!!Springer.! 49.! Kaupp,!G.!Atomic"Force"Microscopy,"Scanning"NearFfield"Optical"Microscopy"and" Nanoscratching"Application"to"Rough"and"Natural"Surfaces.!!2006,!Springer.! 50.! Goldstein,! J.Y.H.,! Practical" Scanning" Electron" Microscopy:" Electron" and" Ion" Microprobe"Analysis.!1975,!Plenum!Press.! 51.!Wells,!O.C.,!Scanning"Electron"Microscopy.!1974,!McGrawRHill.! ! ! ! 262! 5.11!RHEOLOGY! ! Rheology!involves!the!study!of!matter!deformation!and!flow!due!to!compressive! stresses! acting! onto! it.! Particularly,! it! refers! to! the! behaviour! of! materials! when! a! mechanical! force! is! applied! on! (52).! Rheology! includes! three! main! concepts! such! as! force,!deformation!and!time.!Irreversible!flows,!reversible!elastic!deformations!or!their! combination! (viscoelasticity)! can,! therefore,! model! and! describe! a! rheological! phenomenon!under!certain!assumptions.!The!type!of!deformation!depends!on!the!state! of!matter;!for!example,!gases!and!liquids!will!flow!when!a!force!is!applied!whilst!solids! will!deform!by!a!fixed!amount!and,!then,!back!to!their!original!shape!when!the!force!is! removed.!In!the!case!of!polymers,!the!rheological!and!mechanical!properties!affect!the! polymers! molecular! properties,! such! as! their! molecular! mass,! molecular! mass! distribution,!conformation,!architecture!and!crystallinity!(53,54).! A!typical!rheometer!measures!the!velocity!of!displacement!of!the!moving!surface! and!the!force!exerted!on!one!of!the!surfaces.!Most!of!rheometers!are!based!on!rotary! motion! and! use! one! of! the! three! following! geometries! (Figure! 30)! (52):! concentric! cylinder,!cone!and!plate,!and!parallel!disk.!In!most!cases!the!same!rotary!instrument!can! use!all!three!of!these!flow!geometries.!To!generate!the!needed!motion,!they!typically! use! actuators! like! a! hydraulic! piston! or! ball! screws! found! in! standard! tensile! testing! machines!for!solids.!Solenoids!or!other!electromechanical!actuators!are!often!used!for! small!amplitudes!and!low!forces.!There!are!two!basic!designs!of!rheometers:!controlled! stress!ones,!where!the!stress!is!applied!electrically!via!a!motor!measuring!the!strain;!and! controlled!strain!instruments,!in!which!a!strain!is!imposed!and!the!stress!is!computed! from!the!deformation!of!a!calibrated!spring!system!(54).! ! ! ! Figure! 30.! Schematic! diagram! of! basic! tool! geometries! for! a! rotational! rheometer:! a)! concentric!cylinder,!b)!cone!and!plate,!c)!parallel!plate.! ! 263! 5.11.1!Viscoelasticity! ! Many!materials!can!be!classified!as!solid!or!fluids,!displaying!elastic!and!viscous! behaviour,! respectively.! Viscoelastic! materials! such! as! polymers! combine! the! characteristics!of!both!elastic!and!viscous!materials!depending!on!the!experimental!time! scale.! Application! of! relatively! long! duration! stress! may! cause! some! flow! and! irrecoverable!deformation,!while!a!rapid!shearing!would!induce!an!elastic!response!in! some!polymeric!fluids.!Then,!a!classification!of!these!materials!should!take!into!account! the! timescale! of! the! measurement! relative! to! the! characteristic! time! of! the! material.! This!classification!is!given!by!the!Deborah!number!(De),!which!is!a!dimensionless!number! that!characterize!the!fluidity!of!materials!under!specific!flow!conditions.!Formally,!the! Deborah! number! is! defined! as! the! ratio! of! the! stress! relaxation! time! and! the! characteristic! time! scale! of! an! observation.! It! incorporates! both! the! elasticity! and! viscosity! of! the! material.! At! low! Deborah! numbers,! De!<! 1,! the! material! behaves! in! a! more!fluidYlike!manner,!with!an!associated!Newtonian!viscous!flow.!So,!when!De!=!1!the! material! will! display! both! viscous! and! elastic! behaviour,! and! it! is! described! as! viscoelastic.!At!high!Deborah!numbers,!De!>!1,!the!material!behavior!changes!to!a!nonY Newtonian! regime,! increasingly! dominated! by! elasticity! and! demonstrating! solidYlike! behavior;!by!contrast!for!De!<!1!the!material!behaves!in!a!more!fluidYlike!manner,!with! an!associated!Newtonian!viscous!flow!(53,54).! ! ! E η σσ η E a) b) ! ! Figure!31.!a)!Maxwell’s!and!b)!Voigt’s!models.! ! Pure! elastic! solid! behaviour! may! be! exemplified! by! a! Hook’s! spring,! and! pure! viscous!flow!can! be!exemplified! by!the! behaviour!of!a! dashpot,!which! is!essentially!a! piston!moving!in!a!cylinder!of!a!Newtonian!fluid.!The!use!of!mechanical!models!such!as! the!spring! and!dashpot!as!analogues!of! the!behaviour! of!real!materials!enables!us! to! describe! very! complex! experimental! behaviours! by! simple! combination! of! models,! where!the!spring!purely!exhibits!an!elastic!effect!(as!a!Hookean!solid),!and!the!dashpot! exhibits! purely! a! viscous! effect! (as! a! viscous! fluid).! Nevertheless,! the! viscoelasticity! ! 264! cannot!be!described!accurately!by!neither!spring!nor!dashpot!alone,!but!a!combination! of!both.!Among!all!models,!Maxwell’s!and!KelvinYVoigt’s!models!are!the!most!frequently! used!(Figure!31)!(54).!! ! As! shown! in! Figure! 31a,! Maxwell! suggested! a! simple! combination! of! both! elements,!in!which!one!spring!is!attached!to!one!dashpot!in!series.!Because!the!material! possesses!the!ability!to!flow,!some! inertial!relaxation!will! occur!and!less!force!will!be! required!upon!time!to!sustain!the!deformation.!The!goal!in!the!Maxwell’s!model!is!to! calculate! how! the! stress! varies! with! time,! or! expressing! the! stress! in! terms! of! the! constant!strain!to!describe!the!timeYdependent!modulus.!When!a!force!is!acting!on!the! Maxwell’s!model,!the!spring!is!downwards!at!𝑡=0!(in!one!dimensional!flow),!and!the! stressYstrain!relation!for!the!spring!(Hookean!material)!may!be!described!by!(54):! ! 𝜎=𝛾𝐺!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(26)! ! where!𝜎!is!the!applied!stress,!𝛾!is!the!strain,!and!G!the!elastic!modulus.!Conversely,!the! stress! response! of! the! dashpot! with! a! viscous! Newtonian! fluid! to! an! applied! deformation!rate!may!be!described!as:! ! 𝜎=𝜂𝛾!!!!!!!!!!!!!!!!!!!!!! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(27)! ! where!𝛾=𝑑𝛾 𝑑𝑡!is!the!strain!rate!and!𝜂,!the!viscous!response!of!the!dashpot.!! ! In! the! Maxwell’s! element,! both! the! spring! and! the! dashpot! support! the! same! stress!and,!therefore:! ! 𝜎=𝜎!" +𝜎!"#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(28)! where!𝜎!" !and!𝜎!"#!are! the!stresses!on! the!spring!and! dashpot,! respectively.!However,! the! overall! strain! and! the! strain! rates! are! the! sum! of! the! elemental! strain! and! strain! rates,!respectively,!that!is:! ! 𝛾=𝛾!" +𝛾!"#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(29)! !! 𝛾=𝛾!" +𝛾!"#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(30)! ! where!𝛾!"!!is! the! total! strain! rate,! while!𝛾!" !and!𝛾!"#!are! the! strain! rates! of! the! spring! and!dashpot,!respectively.!Therefore,!the!total!strain!of!the!spring!and!dashpot!at!any! time!𝑡!is!the!sum!of!that!of!the!spring!and!the!dashpot.!Then,!for!a!Maxwell’s!model!the! strain!rates!can!be!written!as:! ! ! 265! 𝛾=𝜎𝐺+𝜎𝜂!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(31)! ! By!rheometry!we!can!measure!the!response!of!a!material!to!an!oscillating!stress! or! strain,! so! it! is! considered! as! a! mechanical! spectroscopy.! When! a! sample! is! constrained!in,!for!example,!a!cone!and!plate!assembly,!an!oscillating!strain!at!a!given! frequency! can! be! applied! to! the! sample.! After! an! initial! startYup! period! due! to! a! transient!sample!state!a!stress!develops!in!direct!response!to!the!applied!strain.!If!the! strain! has! an! oscillating! value! with! time! the! stress! must! also! be! oscillating.! We! can! represent!these!two!wave!forms!as!in!Figure!32.!The!elastic!and!viscous!effects!are!out! of!phase!by!an!angle!δ!(55).! ! ! ! Figure!32.!An!oscillating!strain!and!the!stress!response!for!a!viscoelastic!material.! ! All!the!information!about!the!response!of!the!sample!at!the!specified!frequency! is!contained!within!these!wave!forms.!However,!this!information!is!not!in!a!usable!form.! What! we! would! really! prefer! is! to! have! a! few! representative! terms! such! as! the! relaxation! time! and! elasticity! or! viscosity! of! the! sample! in! order! to! characterize! the! material´s!properties.!In!order!to!obtain!this!information!some!mathematical!operations! are!required.!Two!key!constant!features!can!be!utilized:!! • The!first!one!is!the!maximum!stress,!𝜎,!divided!by!the!maximum!strain,!𝛾,!which! is!constant!for!a!given!frequency!𝜔.!This!ratio!is!called!the!complex!modulus!𝐺∗:! ! 𝐺∗𝜔=𝜎𝛾!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(32)! 𝜔!is! the! radial! frequency,! which! is!2𝜋𝑓,! where!𝑓!is! the! applied! frequency! ! 266! measured!in!Hz.!! ! • The!other!feature!constant!with!time!at!any!given!frequency!is!𝛿!(rad).!! ! These! two! values,! 𝐺∗!and! 𝛿,! are! characteristics! of! the! material.! It! is! straightforward!to!visualise!the!situation!where!an!elastic!solid!is!placed!in!a!cone!and! plate!geometry.!When!a!tangential!displacement!is!applied!to!the!lower!plate!a!strain!in! the!sample!is!produced.!That!displacement!is!transmitted!directly!through!the!sample.! The!upper!cone!will!react!proportionally!to!the!applied!strain!to!give!a!stress!response.! An! oscillating! strain! will! give! an! oscillating! stress! response! that! is! in! phase! with! the! strain,!so!𝛿!will!be!zero.!However,!if!we!have!a!Newtonian!liquid,!the!peak!stress!will!be! out! of! phase! by!𝜋2!rad! as! the! peak! stress! is! proportional! to! the! strain! rate.! In! summary,! if! we! have! a! viscoelastic! material! part! of! the! energy! is! stored! and! another! part! dissipated;! the! stored! contribution! will! be! in! phase! whilst! the! dissipated! or! loss! contribution!will!be!out!of!phase!respect!to!the!applied!strain.! ! In!order!to!describe!the!material!properties!as!a!function!of!frequency!we!need! to!use!Eq.!33.!This!equation!describes!the!relation!between!the!stress!and!the!strain.! However,! it! is! most! convenient! to! express! the! applied! sinusoidal! wave! in! the! exponential!form!of!a!complex!number!notation:! ! 𝛾∗=𝛾!𝑒!"#;!𝛾∗=𝑖𝜔𝛾!𝑒!"# =𝑖𝜔𝛾∗!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(33)! ! Now,!the!stress!response!lags!by!the!phase!angle!𝛿:! ! 𝜎∗=𝜎!𝑒!!"!!;!𝜎∗=𝑖𝜔𝜎!𝑒!!"!!=𝑖𝜔𝜎∗!!!!!!!!!!!!!!!!!!!!!!!!!(34)! ! Substituting!the!complex!stress!and!strain!into!the!constitutive!equation!for!a!Maxwell! fluid,!the!resulting!relation!is!given!by:! ! 𝛾∗=𝜎∗ 𝐺+𝜎∗ 𝜂!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(35)! ! Using!eq.!31!and!32!in!eq.!33!and!rearranging!we!have:! ! 𝛾∗ 𝐺𝜎∗=1+𝐺 𝑖𝜔𝜂!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(36)! ! Thus,!arrangement!of!this!expression!gives!the!complex!modulus!and!frequency:! ! ! 267! 𝐺 𝐺∗𝜔=1+1 𝑖𝜔𝜏!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(37)! or:! ! 𝐺∗𝜔=𝐺!"# !!!"# !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(38)! ! where! 𝜏=𝐺𝜂!is! the! characteristic! relaxation! time! This! expression! describes! the! variation!of!the!complex!modulus!with!frequency!for!the!Maxwell!model.!It!is!normal!to! separate!the!real!and!imaginary!components!of!this!expression!to!give:! ! 𝐺∗𝜔=𝐺!𝜔−𝑖𝐺!! 𝜔!!!!!!!!! ! !!!!!!!!!!!!!!!!!(39)! ! Then:! 𝐺!𝜔=𝐺!" ! !!!" !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(40)! ! 𝐺!! 𝜔=𝐺!" !!!" !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(41)! ! where! 𝐺!𝜔!is! an! inYphase! elastic! modulus! with! energy! storage! in! the! periodic! deformation,! called! the! dynamic! storage! modulus.!𝐺!! 𝜔!is! an! outYofYphase! elastic! modulus!associate!with!the!energy!dissipation!as!heat,!called!the!dynamic!loss!modulus.! ! These!expressions!describe!the!frequency!dependence!of!the!stress!with!respect! to! the! strain.! It! is! normal! to! represent! them! as! two! moduli! that! determine! the! component! of! stress! inYphase! with! the! applied! strain! (storage! modulus)! and! the! component! outYofYphase! by! 900.! In! an! experiment,! the! amplitudes! of! the! oscillation! input! (𝛾!)! and! output! (𝜎!)! and! the! phase! angle! (𝛿)! are! measured.! Therefore,! each! oscillatory! shear! flow! measured! at! a! given!𝜔!provides! two! independent! quantities,! amplitude!ratio!and!phase!angle:! ! 𝐺∗𝑖𝜔 =𝜎!𝛾!=𝐺′𝜔!+𝐺′′ 𝜔!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!(42)! ! tan 𝛿=𝐺′′ 𝜔𝐺′𝜔!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(43)! ! The! storage! and! loss! moduli! are! subtle! descriptions! of! the! material! properties! of! a! system.! These! two! properties! are! related! to! the! phase! angle! and! complex! modulus.! These! are! both! functions! of! the! applied! frequency! and! represent! an! alternative! description!of!the!system.! ! ! 268! 𝐺!!=𝐺∗𝜔cos 𝛿,!!!!!!!!!𝐺!! !=𝐺∗𝜔sin 𝛿!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!(44)! ! The! phase! angle! changes! with! the! frequency,! from! 90! degrees! at! low! frequency! to! 0! degrees! at! the! high! frequency! limit;! thus,! as! the! frequency! increases! the! sample! becomes! more! elastic,! and! the! phase! difference! between! the! stress! and! the! strain! reduces.! ! 5.11.2!References! 52.! Barnes,!H.A.!A#Handbook#of#Elementary#Rheology.!2000,!University!of!Wales,! Institute!of!NonYNewtonian!Fluid!Mechanics.! 53.! Goodwin,!J.W.;!Hughes!R.W.!Rheology#for#Chemists:#An#Introduction,!RSC! Publishing.! 54.! Guinebretière,!R.!!X>Ray#Diffraction#by#Polycrystalline#Materials.!2007,!Wiley.! 55.! Hiemenz,!P.C.,!Polymer#Chemistry:#The#Basic#Concepts.!1984,!CRC!Press.! !