Research Article Core Cross-linked Star Polymers for...

Research ArticleCore Cross-linked Star Polymers for TemperaturepH ControlledDelivery of 5-Fluorouracil

Elizabeth Saacutenchez-Bustos12 Joseacute M Cornejo-Bravo2 and Angel Licea-Claverie1

1 Instituto Tecnologico de Tijuana Centro de Graduados e Investigacion en Quımica Apartado Postal 1166 22000 Tijuana BCMexico2Facultad de Ciencias Quımicas e Ingenierıa Universidad Autonoma de Baja California Calzada Universidad 1441822390 Tijuana BC Mexico

Correspondence should be addressed to Jose M Cornejo-Bravo jmcornejouabcedumxand Angel Licea-Claverie aliceactectijuanamx

Received 28 September 2015 Revised 31 December 2015 Accepted 10 January 2016

Academic Editor Marinos Pitsikalis

Copyright copy 2016 Elizabeth Sanchez-Bustos et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

RAFT polymerization with cross-linking was used to prepare core cross-linked star polymers bearing temperature sensitive armsThe arms consisted of a diblock copolymer containing N-isopropylacrylamide (NIPAAm) and 4-methacryloyloxy benzoic acid(4MBA) in the temperature sensitive block and poly(hexyl acrylate) forming the second hydrophobic block while ethyleneglycoldimethacrylate was used to form the coreThe acid comonomer provides pH sensitivity to the arms and also increases the transitiontemperature of polyNIPAAm to values in the range of 40 to 46∘C Light scattering and atomic force microscopy studies suggest thatloose core star polymers were obtained The star polymers were loaded with 5-fluorouracil (5-FU) an anticancer agent in valuesof up to 30ww In vitro release experiments were performed at different temperatures and pH values as well as with heating andcooling temperature cycles Faster drug release was obtained at 42∘C or pH 6 compared to normal physiological conditions (37∘CpH 74) The drug carriers prepared acted as nanopumps changing the release kinetics of 5-FU when temperatures cycles wereapplied in contrast with release rates at a constant temperature The prepared core cross-linked star polymers represent advanceddrug delivery vehicles optimized for 5-FU with potential application in cancer treatment

1 Introduction

Recent research reports on stimuli-responsive polymers havebeen focused in the field of polymer topology particularlyin the synthesis of well-defined macromolecules with pre-cisely controlled architecture by incorporating site-specificbranching points and functionalities [1] in addition to theirability to respond to various stimuli like temperature pHor light [2] The features mentioned above are desirable forthe preparation of tailor-made polymeric systems for a broadrange of applications covering the fields of drug deliverydiagnostics biotechnology sensors micromechanical andoptical systems coatings and textiles [3]

Star polymers represent a special case of branchedmacro-molecules and are very attractive for polymer chemists dueto their multiarm topological architecture smaller hydrody-namic size ideal rheological behavior particular bulkiness

and solution properties compared to their linear counterparts[4] Well-defined star polymers can be prepared by growingthe polymer arms from a functional core (core-first) or by theldquoarm-firstrdquo approach where end-functionalized polymericchains are attached to a functional core by specific reactionsor by copolymerization with a cross-linker The ldquoarm-firstrdquoapproach exhibits several advantages over the ldquocore-firstrdquomethodology such as an easier procedure to achieve highermolecular weight star polymers without the formation ofcoupling star structures [5] and the possibility of havingwell-defined arms and combining different types of armsin a single star [6] Star polymers were first synthesizedby the ldquoarm-firstrdquo approach in 1966 using anionic livingpolymerization to obtain arms followed by a reaction with asuitable linking agent to create the star polymer architecture[7] Controlled radical polymerization (CRP) techniquessuch as atom transfer radical polymerization (ATRP) [8]

Hindawi Publishing CorporationJournal of ChemistryVolume 2016 Article ID 4543191 12 pageshttpdxdoiorg10115520164543191

2 Journal of Chemistry

nitroxide mediated radical polymerization (NMP) [9] andreversible addition-fragmentation chain transfer polymeriza-tion (RAFT) have been versatile techniques for the synthesisof star polymers using both ldquoarm-firstrdquo and ldquocore-firstrdquometh-ods [10ndash13] Star polymers can be divided into two structuralcategories homogeneous (homoarm) or mixed (miktoarm)star polymers [6] Arms may be built of homo- co- oreven terpolymers in such a way that the final propertiesof the resulting star-shaped polymers can be adjusted bychoosing the respective chemical structure of arms and coredepending on the required application [14]

Amphiphilic polymers formed by hydrophilic andhydrophobic segments self-aggregate in aqueous solutionsto form micelles and other types of aggregates [15ndash18]Amphiphilic star polymers may also aggregate in a complexfashion depending on their chemical composition relativesize of core to arms number of arms and so forth [19ndash21]

Due to their small size (lt200 nm) star polymers mayaccumulate in tumors or inflamed tissues through theenhanced permeability and retention effect (EPR) [22] Sev-eral pathologies such as inflammation tumor and infarctedtissue show a local decrease in pH (1ndash25 pH units) [23ndash25] The particular pH conditions at pathological sites andalso local heating by 2ndash5∘C in solid tumors can be used toenhance drug release from accumulated or locally adminis-tered stimuli-responsive polymeric materials [26ndash28]

An important issue for the biomedical application oftemperature sensitive stars polymers is to adjust the transitiontemperature (LCST) above normal body temperature thuswhen the nanomaterials are injected into the body they arein the swollen state (37∘C lt LCST) and circulate in bloodvessels but collapse inside cancer tissue (119879tumor gt LCST)due to the hydrophobic change at temperatures above theirLCST [29]The process of collapse (and possible aggregation)leads to drug release inside tumors [30 31] The LCST ofpolyNIPAAm (32∘C) can be increased by copolymerizationof NIPAAm with hydrophilic or ionized monomers [3233] It was demonstrated that copolymerization of NIPAAmwith amphiphilic weak acid monomers allows tuning theLCST of polyNIPAAm above body temperature [13] thisleads in the case of nanohydrogels to maintaining adequateswellingdeswelling properties [34 35]

In a previous study our group has reported the synthe-sis of core cross-linked (CCS) temperature sensitive star poly-mers with a random number of poly(N-isopropylacryl-amide)-b-poly(hexyl acrylate) (PNIPAAm-b-PHA) or PNI-PAAm arms using the RAFT polymerization techniqueFurthermore the release behavior of different drugs fromthose star polymers was also investigated observing that astar polymer with PNIPAAm-b-PHA arms is more effectivein entrapping drugs than a star polymer formed by purePNIPAAm arms since the PHA block forms a hydrophobicintermediate shell over the corewhere hydrophobic drugs canbe entrapped [12] Other examples of PNIPAAm-based starpolymers have been developed for drug delivery applications[21 36ndash42] However most of them have been designed soas to be able to entrap and release the drug due to the abi-lity of PNIPAAm star polymers to self-aggregate [21 36ndash41] which may have the drawback of early aggregate

disruption due to the dilution factor in the bloodstreamresulting in premature drug release CCS star polymers rep-resent intermediate architecture between branched polymerchains and polymer nanoparticles [43] They are larger thanconventional star polymers having a single molecule oratom as a core but smaller than nanomicrogels They havethe advantage of presenting a relatively large core (cargocompartment) depending on the synthetic conditions andthey are surrounded by a large number of polymer arms thatstabilize them making them dispersible in a suitable solventfor the arms and preventing self-aggregation

In the present study the RAFT technique was employedto synthesize poly(NIPAAm-co-amphiphilic weak acid) poly-mers to tailor the LCST in the range of 40 to 46∘C at pH of74 These polymeric chains were further activated to formblock copolymers with HAThe block copolymers were usedto prepare dual sensitive temperature and pH star polymerscross-linked with ethyleneglycol dimethacrylate (EGDMA)These CCS star polymers were tested to load 5-fluorouracil(5-FU) an anticancer drug and to study their drug deliverybehavior under different in vitro conditions

2 Experimental Section

21 Materials The acid monomer 4-methacryloyloxyben-zoic acid (4MBA) the RAFT agent 2-hydroxyethyl 2-phenylacetate dithiobenzoate (DFH) and the free-radicalinitiator 441015840-azobis(4-cyanopentanol) (ACP)were preparedas described in the literature [13] N-Isopropylacrylamide(NIPAAm 97 Aldrich) was purified by recrystallizationfrom n-hexane 441015840-Azobis(4-cyanopentanoic acid) (ACPA98 Fluka) was recrystallized from methanol n-Hexylacrylate (HA Aldrich) and ethyleneglycol dimethacrylate(EGDMA Aldrich) were purified by passing the reagentsthrough an inhibitor remover column for benzoquinones(Aldrich) p-Dioxane (ACS grade Fermont) diethyl ether(ACS grade Fermont) tetrahydrofuran (THF HPLC gradeAldrich) and 5-fluorouracil (5-FU Sigma) were usedwithoutfurther purification

22 Synthesis of Linear Poly(NIPAAm) Arms (Macro-CTAsof PolyNIPAAm) and Statistical Copolymers of NIPAAmwith 4MBA Linear poly(NIPAAm) used to form the armsof star polymers was synthesized with prescribed mole-cular weights via RAFT polymerization (Figure 1(a))Poly(NIPAAm) arms were prepared using molar ratios ofmonomer CTA initiator of 180 10 02 of 132 10 02and of 88 10 02 Statistical copolymers were synthesizedwith various molar percentages of the acid comonomer inorder to adjust the LCST above 37∘C Poly(NIPAAm-co-4MBA) aiming at 9 of 4MBA was synthesized using thesame molar ratios that were used in the synthesis of linearpoly(NIPAAm) considering the total amount of monomersNIPAAm + 4MBA in the molar ratio

The general procedure was performed as follows 0049 g(0147mmol) of DFH was stirred until it was completelydissolved in 30mL of p-dioxane and poured into anampoule containing a magnetic stir bar 285 g (2518mmol)of NIPAAm 02867 g (130mmol) of 4MBA 00074 g

Journal of Chemistry 3

(00295mmol) of ACP and 30mL of p-dioxane were addedto that ampoule under continuous stirring The ampoule wasdegassed by three freeze-thaw cycles alternating betweenvacuum and argon gas The reaction mixture was heated to70∘C under argon atmosphere and the polymerization wasperformed over 16 h The reaction mixture was cooled downto 25∘C and the polymerization product was precipitatedin diethyl ether then redissolved in acetone and finallyprecipitated in cold diethyl ether The purification processwas repeated three times then the solvent was removedunder vacuum and the product was allowed to dry overnightin a vacuum oven at 25∘C

23 Preparation of Linear Blocks of Poly(NIPAAm)-b-poly(hexyl acrylate HA) and Poly(NIPAAm-co-4MBA)-b-poly(HA) Arms (Macro-CTAs of Block Copolymers) Thesame procedure described above for the preparation of linearpoly(NIPAAm) macro-CTAs was followed (Figure 1(b))Briefly 1 g (03435mmol) of PNIPAAm macro-CTA (usedinstead of DFH) was stirred until it was fully dissolved in25mL of p-dioxane and the resulting mixture was addedto an ampoule containing a magnetic stir bar 01799 g(115mmol) of HA monomer 000173 (000687mmol) ofACP and 25mL of p-dioxane were added to the ampouleunder continuous stirring The ampoule was degassed bythree freeze-thaw cycles alternating between vacuum andargon gas The reaction mixture was allowed to warm upto 70∘C under argon atmosphere and the polymerizationwas performed with stirring for 16 h The reaction mixturewas cooled down to 25∘C and the polymerization productwas precipitated in diethyl ether dissolved in acetone andprecipitated again in cold diethyl ether The purificationprocess was repeated three consecutive times The solventwas removed under vacuum and the product was allowed todry overnight in a vacuum oven at 25∘CThe same procedurewas followed for the preparation of block copolymers start-ing with macro-CTAs of poly(NIPAAm-co-4MBA) Thecalculations were always done considering the same macro-CTA to initiator molar ratio and the same volume ofp-dioxane as the solvent of choice

24 Preparation of Star Polymers with a Cross-Linked CoreThe synthesis of star polymers using the ldquoarm-firstrdquo RAFTmethod with cross-linking was adapted from proceduresreported in the literature [12 13 44 45] (Figure 1(c)) Differ-ent molar ratios of cross-linker to arms were used to preparestars polymers with PNIPAAm-b-PHA arms An example isdescribed as follows 06 g (00187mmol) of PNIPAAm-b-PHA macro-CTA was stirred until it was fully dissolved in24mL of p-dioxane and poured into an ampoule contain-ing a magnetic stir bar 0037 g (0187mmol) of EGDMA000094 g (000374mmol) of ACP and 15mL of p-dioxanewere added to the ampoule under continuous stirring Theampoule was degassed by three freeze-thaw cycles alternatingbetween vacuum and argon gas The reaction mixture wasallowed to warm up to 70∘C under argon atmosphere andthe polymerization was performed under stirring for 24 hThe reaction mixture was cooled down to 25∘C and thepolymerization product was precipitated in diethyl ether

redissolved in acetone and precipitated again in cold diethylether The purification process was repeated three times Thesolvent was removed under vacuum and the product wasallowed to dry overnight in a vacuum oven at 25∘C

For the synthesis of star polymers with poly(NIPAAm-co-4MBA)-b-PHA arms a molar ratio of EGDMA macro-CTA= 10 1 was used with macro-CTA poly(NIPAAm-co-4MBA

5)-b-PHA (119872119899= 28570 gmolminus1) and EGDMA

macro-CTA ratio = 20 1 was used with poly(NIPAAm-co-4MBA

9)-b-PHA (119872119899= 21730 gmolminus1) ACPA was used as

the initiator and a 5 1 molar ratio of macro-CTA initiatorwas used in all the cases The polymerization procedure isthe same as the one described above

25 Characterization and Measurements The polymeriza-tion yields were determined gravimetrically The molecularweights and dispersity (Đ = 119872

119908119872119899) were determined by

gel permeation chromatography (GPC) The GPC systemconsists of a Varian 9002 HPLC pump equipped with arefractive index detector (Varian RI-4) a triangle light scat-tering detector (Mini Dawn 120582 = 690 nm Wyatt) and Phe-nomenex separation columns Phenogel 10 120583m 500 A (00H-0643-K0) Phenogel 10 120583m 104 A (00H-0645-K0) Phenogel10 120583m 105 A (00H-0646-K0) in seriesThemobile phase usedin the GPC was tetrahydrofuran (THF HPLC grade) forNIPAAm polymers (homopolymers block copolymers andstar polymers) and THFCH

3COOH (50 1) vv for copoly-

mers block copolymers and star polymers containing acidcomonomer units while the flow rate was 05mLminminus1 at25∘C Monodisperse polystyrene was used as the calibrationstandard to confirm the accuracy of the light scatteringdetector dndc = 0093mLg [46] reported for polyNIPAAmin THF was used for molecular weight determinations ofall polymers without HA For polymers containing HA anaverage dndc value between that of polyNIPAAm and thatreported for polybutylacrylate in THF (0065mLg [47]) wasused namely 0079mLg

Dynamic light scattering (DLS)was used to determine thehydrodynamic diameters (119863

ℎ) Measurements were carried

out at 20∘C using a Zeta-sizer ldquoNano-ZSrdquo from MalvernInstruments (ZEN3500) equipped with a green laser oper-ating at 120582 = 532 nm The angle of measurement was 173∘(backscattering) and the size analysis was performed byCONTIN The reported hydrodynamic diameters were cal-culated using the Stokes-Einstein equation for spheres [48]Reported 119863

ℎvalues were the maxima in size distribution by

volume from CONTIN analysis The scattering intensity as afunction of temperature was also used to determine the phasetransition temperature of the prepared NIPAAm polymersat a given concentration called for the sake of simplicitylower critical solution temperature (LCST) The size analysiswas performed at different temperatures using 3min ofequilibrating time at each temperature in a heating cycleThepolymer concentrationwas 1mgmLminus1 and the value reportedfor the LCST was the temperature at which the scatteringintensity increased sharply The solutions were preparedshaken for 12 h and stored overnight in the refrigerator at4∘C Before measurements the solutions were filtered offusing a 045-micron syringe filter for eliminating dust


OHN

SS

O

DFH (CTA)NIPAAm

OOH +

NN OH + +

CN

CNHO

ACP (initiator)

ONH

SOHOO

SnH

H O

HOOC

X = 4MBA

p-Dioxane

ONH

OHOO n S

S

OO

COOH

Macro-CTA1

Macro-CTA2

x

nX

co

70∘C

H2C

H3CCH3

CH3

H3C

H3C

CH3

CH3

CH3

X=0

(a)

n

Block copolymer 1

macro-CTAs m+ ACP+O

O

Hexyl acrylate

O NH

bO

HO On S

S

OO m

ONH

OHO O

nb

OO

COOH

Block copolymer 2

xS

S

OO m

co

70∘C

H3CCH3

CH3

H3C

CH3

CH3

CH3

CH3 p-Dioxane

(b)

NN

OHO+

CN OOH

CNBlock copolymer

O

OO

O

EGDMA

NIPAAmHAEGDMA cross linker

HOOC

HOOC COOH

HOOCCOOH

70∘ C 70 ∘C

+CH3

CH3

44998400-Azo-bis(4-cyanopentanoic acid)

p-Dioxanep-Diox

ane

(c)

Figure 1 Synthetic routes to obtain pH and temperature sensitive star polymers

1H-NMR spectra were recorded on a Varian Mer-cury 200MHz nuclear magnetic resonance instrument withCDCl

3or CD

3OD as the solvents and tetramethylsilane

(TMS) as the internal reference Atomic force microscopy

(AFM) imaging experiments were performed using an Agi-lent Technologies SPM 5100 microscope equipped withN9520A high resolution scanner The samples were preparedby dissolving the star polymer in a small volume of THF


Table 1 Preparation of linear macro-CTAs at 70∘C in p-dioxane for 12 h

Entries Polymer M CTA Ia 119872119899

(target)(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)

1 PolyNIPAAm 180 1 02 20673b 26100 1008 752 PolyNIPAAm 132 1 02 15249b 14400 1022 703 PolyNIPAAm 88 1 02 10277b 10800 1030 734 PolyNIPAAm 88 1 02 10277b 8200 1040 705 Poly(NIPAAm-co-4MBA

5) 180 1 02 21510c 22000 1157 786 Poly(NIPAAm-co-4MBA

9) 132 1 02 16354c 15200 1149 70aM monomer CTA chain transfer agent I initiatorb119872119899(target) = ([119872]

0[CTA]

0)119872Mon +119872CTAc

119872119899(target) = ([NIPAAm]

0[CTA]

0)119872NIPAAm + ([4MBA]

0[CTA]

0)1198724MBA +119872CTA

and then dispersing it in deionized water The volumesand amounts were adjusted to obtain a concentration of01mgmLminus1 in water Then the polymer solutions werefiltered through a 045120583m syringe filter followed by drop-casting on a freshly cleaved mica wafer and dried in a con-trolled temperature room at 20∘C for 24 h The AFM experi-ments were carried out using the intermittent contact modewith silicon cantilevers at ambient conditions For eachsample three to four images were recorded from the sameslide and the diameter of the assemblies was measured Theaverage value of the assemblies is reported as the averagediameter of the star polymers

26 Drug Loading and In Vitro Drug Release 5-FU (8mg)was dissolved in 15mL of dimethylacetamide (DMAc) whilethe star polymer (4mg) was dissolved in 15mL of DMAcin a separate vial The drug solution was added dropwise tothe star polymer solution with stirring at 20∘C The resultingsolution was poured into a dialysis bag (molecular weightcutoff 12000ndash14000 daltons) and it was subjected to dialysisagainst distilled water for 4 h to remove the nonencapsulateddrug The distilled water was replaced every 30min forthe first 2 h The dialyzed solution was then lyophilizedThe freeze-dried samples of drug-loaded star polymers weredispersed in a phosphate buffer (PBS) at pH of 74 followedby stirring them vigorously for 2 h and then sonicating themfor 15min The loading amount of 5-FU in the star polymerswas determined by measuring the UV absorbance at 267 nmusing a Beckman Coulter spectrometer DU520 The drugloading (DL) and entrapment efficiency (EE) of the starpolymer were calculated as follows

DL = ( MDstarmass of stars +MDstar

) times 100

EE = ( initial MD minusMDsupernatantinitial MD

) times 100

(1)

where MD is the mass of drug (5-FU) and MDstar is themass of drug in the star polymer For the release experimentsthe drug-loaded star polymers were placed into dialysis bags(10mL) and dialyzed against 250mL (sink conditions) ofPBS at pH of 74 or at pH of 6 in a shaking bath (VWRInternational model number 1217) withmild shakingmotion(20 rpm) at 37 and 42∘C respectively At predetermined time

intervals a 1mL aliquot of the released medium was takenout and assayed for 5-FU content and 1mL of fresh buffersolution was replaced into the release media every predeter-mined time intervalThe concentration of 5-FU released fromthe drug delivery systemwasmonitored bymeasuring theUVabsorbance at 267 nmusing a BeckmanCoulter spectrometerDU520The cumulative drug release was calculated from therelationship based on the standard calibration curve All theanalyses were performed in triplicate and the results werereported with an average of three runs

3 Results and Discussion

31 Synthesis of Macro-CTAs The chemical molecular andsize analysis of polyNIPAAm and the statistical copoly-mer poly(NIPAAm-co-4MBA) as macro-CTAs were studiedusing FT-IR 1H-NMR GPC andDLS as analytical toolsThenumber average molecular weights (119872

119899) for polyNIPAAm-

basedmacro-CTAs are defined by theNIPAAm toCTAmolarratio as expected for a controlled radical polymerizationmethod like RAFT The dispersity values (Đ) obtained forpolyNIPAAm were very low (Table 1 entries 1 to 4) demon-strating well behaving RAFT polymerization

A similar behavior can be observed in the case of thepoly(NIPAAm-co-4MBA) statistical copolymers showing Đvalues below 116 (entries 5-6 in Table 1)These values suggestthat the polymerization proceeded in all cases with a highlevel of control The polymerization yields ranged from 70 to78

32 Synthesis of Block Copolymers Themain interest of intro-ducing a second hydrophobic block constituted of n-hexylacrylate (HA) units into polyNIPAAm or poly(NIPAAm-co-4MBA) linear polymers was to synthesize star polymers witha hydrophobic shell over the cross-linked core which areable to retain drugs for transport in physiological conditionsThe hydrophobic PHA block may also lead to star aggregateformation depending on the relative block size and numberof arms of the star Previously the monomer HA had beensuccessfully grown under RAFT polymerization conditionsto form block copolymers with polyNIPAAm to generatepolyNIPAAm-b-polyHA [12] and poly(NIPAAm-co-4MBA)-b-polyHA [13] The molecular weights of the obtained prod-ucts in this work are shown in Table 2


Table 2 Preparation of linear block copolymer arms


(target)i(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)

1 PolyNIPAAm-b-polyHA 22 1 02b 11645 12780 1065 702 PolyNIPAAm-b-polyHA 32 1 02c 15820 17890 1175 703 PolyNIPAAm-b-polyHA 50 1 02d 19930 20200 1137 724 PolyNIPAAm-b-polyHA 32 1 02e 31111 32070 1177 755 Poly(NIPAAm-co-4MBA

9)-b-polyHA 32 1 02f 21160 21730 1030 716 Poly(NIPAAm-co-4MBA

9)-b-polyHA 64 1 02g 25160 25340 1011 657 Poly(NIPAAm-co-4MBA

5)-b-polyHA 32 1 02h 27200 28570 1038 60aReactions in p-dioxane at 70∘C for 16 h M = HA bmacro-CTA is PNIPAAm (119872

119899= 8208 gmol) cmacro-CTA is PNIPAAm (119872

119899= 10820 gmol)

dmacro-CTA is PNIPAAm (119872119899= 14430 gmol) emacro-CTA is PNIPAAm (119872

119899= 26110 gmol) fmacro-CTA is P(NIPAAm-co-4MBA

9) (119872119899=

15160 gmol) gmacro-CTA is P(NIPAAm-co-4MBA9) (119872119899 = 15160 gmol) hmacro-CTA is P(NIPAAm-co-4MBA

5) (119872119899 = 22000 gmol) i119872119899(target) =

([119872]0[macro-CTA]

0)119872Mon +119872macro-CTA

Table 3 Reactions for preparation of star polymers with a random number of arms and a cross-linked poly(EGDMA) core

Entries Star polymer[cross-linker macro-CTA I]a Crosslinker macro-CTA Ia 119872119899 (GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)

1 [PolyNIPAAm-b-polyHA]b-EGDMA 20 1 02 41740 1333 702 [PolyNIPAAm-b-polyHA]c-EGDMA 10 1 02 136500 1200 773 [(Poly(NIPAAm-co-4MBA

9)-b-polyHA)]d-EGDMA 20 1 02 76600 1175 654 [(Poly(NIPAAm-co-4MBA

5)-b-polyHA)]e-EGDMA 10 1 02 100700 1290 70aCross-linker EGDMA macro-CTA block copolymer as macro chain transfer agent I initiator bMacro-CTA of119872

119899= 12780 gmol cMacro-CTA of119872

119899=

32070 gmol dMacro-CTA of119872119899= 21730 gmol eMacro-CTA of119872

119899= 28570 gmol

Results in Table 2 showed that polyNIPAAm was reactedwith differentmolar ratios of HA to form the second block Ineach case a comparison between observed and target molec-ular weights showed that they were similar demonstratinggood RAFT copolymerization control resulting in Đ valuesbelow 12 (entries 1 to 4) Indeed when using the same molarratio of HA to macro-CTA but polyNIPAAm macro-CTAswith different119872

119899values Đ values were not affected (entries

2 and 4) The same results regarding the high level of controlcan be observed for the formation of poly(NIPAAm-co-4MBA)-b-(polyHA) using as macro-CTAs statistical copoly-mers which have different molar ratios of 4MBA and evendifferent lengths of the macro-CTA chain In all the casesgood match was observed between the calculated and theobserved molecular weights with low Đ values (entries 5 to7) The obtained yields were in a range of 60 to 75

33 Synthesis of Star Polymers The star polymers wereprepared from the block copolymers presented in Section 32The block copolymers represent the arms of the star Themolar ratios of cross-linker to arms EGDMA polyNIPAAm-b-polyHAor EGDMA poly(NIPAAm-co-4MBA)-b-polyHAfor the preparation of the corresponding star polymers areshown in Table 3 The molecular weights that were obtainedfrom GPC for the products prepared at 10 1 and 20 1 ratiosare in most of the cases three to four times the molecularweight of the arms These molecular weights are lower thanthe expected values according to literature reports for thistype of star polymer wheremore arms per star were expected

[17 43] nevertheless enough to suggest that star polymers areformed Figure 2 shows a comparison between GPC tracesfor a linear copolymer a block copolymer prepared from it(arm) and a star polymer as a result of the final reaction of theblock copolymer with the cross-linker Results reveal narrowsize distributions for the linear and block copolymers and awider distribution for the star polymer while the retentiontime decreases with increasing size as expected

The star polymers were characterized by spectroscopyanalysis FT-IR and 1H-NMR spectra of a 10 1 star productare shown in Figure 3 The spectroscopic analysis confirmedthe incorporation of both HA and EGDMA units into thestar polymer however the EGDMA units were difficult tobe identified separately from HA or 4MBA units (Figures3(a) and 3(b)) The FT-IR spectrum of the star polymer withpoly(NIPAAm-co-4MBA

5)-b-polyHA linear arms showedstrong carbonyl stretching vibration of the polyNIPAAmamide groups at 1641 cmminus1 and a strong carbonyl stretchingband at 1731 cmminus1 corresponding to the ester groups of bothHA and EGDMA units Furthermore stretching vibrationof aromatic C-H at 3074 cmminus1 and at 3295 cmminus1 (O-H)demonstrates the presence of the 4MBA units (Figure 3(a))In the case of the 1H-NMR spectrum the characteristicsignal at 4 ppm corresponds to the methine proton on theisopropyl groups present in polyNIPAAm (signal d) and tothe methylene protons attached to the HA and EGDMA estergroups (signals l and q q1015840) respectively whereas in the caseof the 4MBA units in the linear arms the chemical shifts ofaromatic protons (signals h and i) are observed at 72 and


Table 4 Hydrodynamic diameters and LCST values of star polymers in PBS at pH 6 and in PBS at pH 74

Entries Star polymer PBS pH 6 LCST pH6

PBS pH 74 LCST pH74

119863ℎ

(nm) pdi Temp (∘C) 119863ℎ

(nm) pdi Temp (∘C)

1 [PolyNIPAAm-b-polyHA]a1

-EGDMA20

mdash mdash mdash 6538 plusmn 44 0397 plusmn7eminus3 32

2 [PolyNIPAAm-b-polyHA]b1

-EGDMA10

mdash mdash 30 1088 plusmn 36 0256 plusmn002 30

3 [Poly(NIPAAm-co-4MBA9)-b-polyHA]

c1

-EGDMA20

6274 plusmn062

0257 plusmn1eminus3 36 9468 plusmn 15 0187 plusmn

8eminus3 46


d1

-EGDMA10

1124 plusmn 48 0326 plusmn003 36 17103 plusmn

060302 plusmn002 40

aMacro-CTA of119872119899= 12780 gmol bMacro-CTA of119872


119899= 21730 gmol dMacro-CTA of119872

119899= 28570 gmol

10

08

06

04

02

00

Inte

nsity

()

20 25 30 35 40 45

Time (min)Block copolymerStar polymerLinear arm

Figure 2 GPC traces of poly(NIPAAm-co-4MBA5) (blue)

poly(NIPAAm-co-4MBA5)-b-polyHA (black) and [poly(NIPAAm-

co-4MBA5)-b-polyHA]1-EGDMA

10

(red)

8 ppm respectively other signals are superimposed and wereassigned as shown in that spectrum (Figure 3(b))

The DLS technique was also used to characterize thematerials Figure 4(a) shows the hydrodynamic diame-ters (119863

ℎ) of poly(NIPAAm-co-4MBA

5) poly(NIPAAm-co-4MBA

5)-b-polyHA linear arms and star polymer[poly(NIPAAm-co-4MBA

5)-b-polyHA]1-EGDMA10 all at

20∘C As can be seen the size of the materials increasesin each synthetic step Furthermore the size increases frompolymer arm (sim10 nm) to star polymer (sim100 nm) suggestingthat the star polymer is not a simple star polymer with acongested core

The core cross-linked star architecture may contain aloose core as described in [12] Figure 4(b) shows the lightscattering intensity at different temperatures for thematerialsat pH of 74 At a given temperature an increase in theintensity is observed due to aggregation of the particles We

consider this point as the LCST of the materials As can beobserved the LCST is the same for the copolymer and theblock copolymer whereas in the case of the star polymerit is shifted to lower temperatures The LCST is originatedfrom the poly(NIPAAm-co-4MBA) copolymer first block ofthe star arms and modified slightly by cross-linking withEGDMA

To further investigate the obtained star polymer struc-tures 119863

ℎwas determined in buffer solutions results are

shown in Table 4 The observed hydrodynamic diameterdepends on the size of the arms and on the arms-to-cross-linker ratio The larger the arms the longer the star diameterin agreement with the molecular weight of the stars butalso the smaller the cross-linker-to-arms ratio the longerthe star diameter Lower cross-linker content in the starsynthesis may result in a less congested core which may leadto starstar coupling reactions during polymerization whilehigher cross-linker content in the synthesis may result in amore congested core hindering star-to-star coupling duringthe synthesis In the case of the star polymers containing acidunits that is the star polymer prepared using poly(NIPAAm-co-4MBA

5)-b-polyHA (119872119899= 28570 gmol) arms the diam-

eter (119863ℎ) depends also on the ionization of those acid units

In PBS at pH 74 the tendency of contraction of hydrophobicpolyHA units is counterbalanced by the expansion of armsdue to ionization of 4MBA units It was observed that 119863

ℎ

value of star polymer in PBS at 20∘C is smaller at pH 6than at pH 74 This might be directly related to the factthat at this pH value the 4MBA units are not completelyionized [49] contributing together with polyHAunits to theincrease in the hydrophobic balance resulting in shrinkage ofthe star polymer arms The star polymer reported in entry 3(Table 4) also shows a smaller diameter at pH 6 Thereforethe size of star polymers under certain solvent conditionseither organic or aqueous at certain pH value results frombalance of different contributions from the units present inthe star polymer The effect of pH on the LCST values of theprepared star polymers is also shown in Table 4 In the case ofthe star polymer containing polyNIPAAm-b-polyHA arms inPBS at pH 74 in the absence of 4MBA units (entries 1 and 2)the LCST values are in the range from 30 to 32∘C which aretypical values for the LCST of polyNIPAAm whereas in the


28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000

Wavenumber (cmminus1)

Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone

8 6 4 2Chemical shift (ppm)

0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)

Figure 3 Spectra of star polymer [poly(NIPAAm-co-4MBA)-b-polyHA]1

EGDMA10 (a) FT-IR spectrum (b) 1H-NMR spectrum (MeOD)

30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)

Figure 4 DLS results in PBS at pH 74 (a) hydrodynamic diameters at 20∘C (b) light scattering intensity as a function of temperatureSymbols disclosure poly(NIPAAm-co-4MBA

5)119872119899 = 22000 gmol (◼) poly(NIPAAm-co-4MBA5)-b-polyHA 119872119899 = 28570 gmol (e)

and star polymer [poly(NIPAAm-co-4MBA5)-b-polyHA]1-EGDMA

10

119872119899

= 100700 gmol (998771)

case of star polymers containing [poly(NIPAAm-co-4MBA)-b-polyHA] arms (entries 3 and 4) the observed LCST valueswere in the range from 40 to 46∘C at pH of 74 dependingon the 4MBA content in the arms of the star In Figure 5 theeffect of pH on the LCST values of star polymers is shown

The increase in the LCST at pH 74 can be attributedto the presence of the acid comonomer 4MBA taking intoconsideration the fact that its p119870

119886value is 53 [49] As a

result at pH 74 the majority of monomer units are ionizedincreasing the interaction with water and hence requiring ahigher temperature for chain collapse to occur The results ofthe LCST analysis in PBS at pH 6 are also shown in Table 4In the case of star polymers containing [poly(NIPAAm-co-4-MBA)-b-polyHA] arms (entries 3 and 4) the observed LCST

values are 36∘C which are lower than the LCST at pH 74 (seealso Figure 5)

This is attributed to the decrease in the ionization degreeof 4MBA at pH 6 reducing the interaction with water Theresults imply that this star polymer responds to changes inpH and temperature and that its LCST values at different pHvalues are optimized for drug delivery applications

Further evidence of the star polymers morphologyis obtained from the AFM analysis In Figure 6 we canobserve the morphology of [poly(NIPAAm-co-4MBA

5)-b-polyHA]

1-EGDMA

10and [polyNIPAAm-b-polyHA]

1-

EGDMA10

star polymers The average value was 1001plusmn124 nm of diameter with morphology like a well-definedoval for the star containing the acid units (4MBA) and


400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)

Figure 5 LCST of star polymers star polymer [(PNIPAAm-b-polyHA)

1

-EGDMA10

] in PBS at pH 74 (e) and in PBS at pH 6 (I)star polymer [poly(NIPAAm-co-4MBA)-b-polyHA]

1

-EGDMA10

inPBS at pH 6 (◻) and in PBS at pH 74 (◼)

9627 plusmn 172 nm for the star without acid units The sizesobtained by AFM are in good agreement with the DLSmeasurements (1124 plusmn 48 at pH sim 6 of aqueous dispersionand 1088 plusmn 36 nm resp) the slightly smaller sizes by AFMcan be attributed to the drying process of the star polymerson the mica surface before measurement

34 Drug Loading and Release Experiments For the drugloading and release experiments two star polymers werechosen with similar arm size and star architecture the starsprepared with a 10 1 ratio of EGDMA arms were selectedsince their arms are comparable in size 119872

119899is 32070 gmol

for polyNIPAAm-b-polyHA arms and 28570 gmol forpoly(NIPAAm-co-4MBA

5)-b-polyHA arms As previouslydiscussed the LCST of the corresponding star polymers withpolyNIPAAm-b-polyHA arms was 32∘C whilst the LCSTof the [(polyNIPAAm-co-4MBA

5)-b-polyHA]1-EGDMA10

star was close to 40∘C at pH of 74 and 36∘C at pH of 6 whichare suitable for a dual sensitivity drug delivery system Theselected star polymers were loaded with 5-FU by the dialysismethod The dialysis time plays an important role in theyield of loaded star polymers avoiding excess of nonbounddrug As the optimized dialysis time a 4 h period was usedto yield 305 ww loading with 25 efficiency for the acidcontaining star polymer and 253 ww loading and 16efficiency for the star polymer without acid groups Figure 7shows a comparison of release profiles of 5-FUunder differenttemperature and pH conditions The free drug diffuses outof the dialysis bag in approximately 30min (68 cumulativerelease) at 37∘C in medium at pH of 74 In the case of thestar that contains the acid monomer 4MBA at pH 74 and37∘C (T lt LCST) a burst of 20 release at the beginning ofthe process (20min) was observed which may be related tothe fact that 5-FUmolecules are entrapped and distributed inthe arms of the star and also at the interface between core

and shell However the drug release is halted to 25 evenafter 26 h while at 42∘C (T = LCST) the cumulative releaseincreases to approximately 70 after 26 h At pH 6 and 37∘C(T gt LCST at these conditions) cumulative release of about84 in 26 hwas observedOn the other hand the starwithoutacid units (4MBA) shows cumulative release at 26 h of about90 at 37∘C and 87 at 42∘C (T gt LCST) respectively Bothtemperatures are much higher than the LCST of that starTheeffect that the pH has on the release rate is clearly visible inthe case of the star polymer containing 4MBA units

The kinetics of release depend on the difference betweenthe experimental temperature and the LCST in other wordsthey depend on how large this difference is The slowestrelease rate was obtained when the experimental temperaturewas lower than the LCST In this case the PHA hydrophobicshell over the core of the star polymer containing 4MBAunits retained 75 of the 5-FU loaded In the case of thestar without 4MBA units the release temperature was 5∘Chigher than the LCST the star polymer tends to precipitategenerating release channels for 5-FU

Drug release studies with temperature jump cyclesbetween 37∘C and 42∘C are shown in Figure 8 Increasingthe temperature from 37∘C to 42∘C results in an increaseof release rate for the star without acid units (T gt LCST atboth temperatures) indicating a temperature effect on drugdiffusivity However the temperature release control is moreefficient for the polymeric star containing the acidmonomerswhen it goes from T lt LCST at 37∘C and pH 74 to T = LCSTat 42∘C and the same pH

In this experiment it was also observed that the starpolymer with arms containing acid units (4MBA) acts as ananopump since the temperature cycles increased the rateof release Altogether 100 drug release was attained after65 h of applying temperature cycles whilst at a constanttemperature the cumulative release was 70 after 26 h at42∘C (pH 74)

At pH of 6 faster release than at pH of 74 was observedboth at 37∘C (Figure 8) A jump in temperature to 42∘Cproduces even faster release at both pH values delivering thepayload in the first temperature cycle almost completely atpH 6 but not at pH 74 These results are very encouragingin regard to the potential application as release carriersof the temperature and pH sensitive star polymers thatwere prepared considering that release occurs at conditionsfound in some pathologies such as in tumors Based onthese results the star polymer containing acid units namely[poly(NIPAAm-co-4MBA

5)-b-polyHA]1-EGDMA10(119872119899=

100700 gmol) represents an advanced drug carrier exhibit-ing tunable release kinetics for 5-FU that can be modifiedby increasing the temperature above the normal body tem-perature at physiological pH either by a pathology or byexternal induction andor at lower pH than physiological pHin the site of delivery at normal body temperature as foundin tumors or in endosomes

4 Conclusions

It was shown that it is possible to synthetize star polymerswith well-defined arms in specific composition by combining


(a) (b)

Figure 6 AFM topography images of star polymers (a) [poly(NIPAAm-co-4MBA5)]1-EGDMA

10

(b) [polyNIPAAm-b-polyHA]1

-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30

Figure 7 Release of 5-fluorouracil from different star polymersat constant temperatures free 5-fluorouracil in PBS at pH 6 at37∘C (I) and at pH 74 at 37∘C (◼) and at 42∘C (◻) star polymer[poly(NIPAAm-co-4MBA)-b-polyHA]

1

-EGDMA10

in PBS at pH 6at 37∘C (e) and at pH 74 at 37∘C (998787) and at 42∘C (998810) star polymer[polyNIPAAm-b-polyHA]

1

-EGDMA10

in PBS at pH 74 at 37∘C (998771)and 42∘C (998779)

the RAFT technique with the use of cross-linkers The ratioof cross-linker to macro-CTA (arms) is critical in orderto obtain the desired product The prepared star polymerscontaining (polyNIPAAm-b-polyHA) arms in different sizesand composition with a cross-linked core showed the sameLCST as linear polyNIPAAm (30ndash32∘C) The RAFT processallows for the incorporation of amphiphilic weak acids intothe polyNIPAAm block producing pH and temperaturesensitive materialsThe LCST of the star polymers containing

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C

Figure 8 Release of 5-fluorouracil from star polymer[poly(NIPAAm-119888119900-4MBA)-119887-polyHA]

1

-EGDMA10

in PBSat pH 74 (◼) and PBS at pH 6 (◻) and from star polymer[polyNIPAAm-b-polyHA]

1

-EGDMA10

in PBS at pH 74 (I) withtemperature jump cycles from 37 to 42∘C

[poly(NIPAAm-co-4MBA)-b-polyHA] arms was tailored tovalues in the range of 40 to 46∘C at pH of 74 dependingon 4MBA content and about 36∘C at pH of 6 The polymericstars can be loaded with 5-FU up to 30ww In vitro releaseexperiments showed that the star polymermaterials release 5-FU at higher rates when the temperature is above their LCSTThe loaded star polymers behave as nanopumps with offonrelease if subjected to temperature cycles around the LCSTFrom these findings we conclude that the temperature sensi-tive star polymers with an adjusted LCST containing 4MBA


are promising intelligent drug carriers with the advan-tages of temperature and pH adjustable release kinetics

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Council of Scienceand Technology of Mexico (CONACYT) Grants CB2010-C01-157173 CB2012-C01-178709 and INFR2014-C01-224284Elizabeth Sanchez-Bustos thanks CONACYT for PHD fel-lowship (no 208593) Furthermore the technical support inNMR analysis by I A Rivero and A Ochoa-Teran and inAFM analysis by P Navarro is gratefully acknowledged Theauthors are indebted to L A Licea-Higgs for English cor-rections in the paper

References

[1] D Kuckling and AWycisk ldquoStimuli-responsive star polymersrdquoJournal of Polymer Science Part A Polymer Chemistry vol 51no 14 pp 2980ndash2994 2013

[2] M A Cohen-Stuart W T S Huck J Genzer et al ldquoEmergingapplications of stimuli-responsive polymer materialsrdquo NatureMaterials vol 9 no 2 pp 101ndash113 2010

[3] H Gao and K Matyjaszewski ldquoSynthesis of functional poly-mers with controlled architecture by CRP of monomers in thepresence of cross-linkers from stars to gelsrdquo Progress in PolymerScience vol 34 no 4 pp 317ndash350 2009

[4] N Hadjichristidis M Pitsikalis and H Iatrou ldquoPolymers withstar-related structuresrdquo inMacromolecular Engineering PreciseSynthesis Materials Properties Applications K MatyjaszewskiY Gnanou and L Leibler Eds chapter 6 pp 909ndash971 Wiley-VCH Weinheim Germany 2007

[5] A Blencowe J F Tan T K Goh and G G Qiao ldquoCore cross-linked star polymers via controlled radical polymerisationrdquoPolymer vol 50 no 1 pp 5ndash32 2009

[6] A Vora K Singh and D C Webster ldquoA new approach to3-miktoarm star polymers using a combination of reversibleaddition-fragmentation chain transfer (RAFT) and ring open-ing polymerization (ROP) via lsquoClickrsquo chemistryrdquo Polymer vol50 no 13 pp 2768ndash2774 2009

[7] J-G Zilliox D Decker and P Rempp ldquoHerstellung von lsquoStern-polymerenrsquo auf anionischem Wegerdquo Angewandte Chemie vol78 no 11 p 614 1966

[8] KMatyjaszewski and J Xia ldquoAtom transfer radical polymeriza-tionrdquo Chemical Reviews vol 101 no 9 pp 2921ndash2990 2001

[9] C J Hawker A W Bosman and E Harth ldquoNew polymersynthesis by nitroxidemediated living radical polymerizationsrdquoChemical Reviews vol 101 no 12 pp 3661ndash3688 2001

[10] R T A Mayadunne J Jeffery G Moad and E Rizzardo ldquoLiv-ing free radical polymerization with reversible addition-frag-mentation chain transfer (RAFT polymerization) approachesto star polymersrdquoMacromolecules vol 36 no 5 pp 1505ndash15132003

[11] A B Lowe and C L McCormick ldquoReversible addition-frag-mentation chain transfer (RAFT) radical polymerization and

the synthesis of water-soluble (co)polymers under homoge-neous conditions in organic and aqueous mediardquo Progress inPolymer Science vol 32 no 3 pp 283ndash351 2007

[12] J Alvarez-Sanchez A Licea-Claverıe J M Cornejo-Bravo andC W Frank ldquoStar polymers with random number of tem-perature sensitive arms and crosslinked poly(EGDMA)-coreand their application to drug deliveryrdquo Reactive and FunctionalPolymers vol 71 no 11 pp 1077ndash1088 2011

[13] LA Picos-Corrales A Licea-Claverie JMCornejo-BravoK-F Arndt and S Schwarz ldquoWell-definedN-isopropylacrylamidedual-sensitive copolymers with LCST asymp 38∘C in differentarchitectures linear block and star polymersrdquoMacromolecularChemistry and Physics vol 213 no 3 pp 301ndash314 2012

[14] G Lapienis ldquoStar-shaped polymers having PEO armsrdquo Progressin Polymer Science vol 34 no 9 pp 852ndash892 2009

[15] J Li J Ren Y Cao and W Yuan ldquoSynthesis of biodegradablepentaarmed star-block copolymers via an asymmetric BIS-TRIScore by combination of ROP and RAFT from star architecturesto double responsive micellesrdquo Polymer vol 51 no 6 pp 1301ndash1310 2010

[16] X Li Y Qian T Liu et al ldquoAmphiphilic multiarm star blockcopolymer-based multifunctional unimolecular micelles forcancer targeted drug delivery and MR imagingrdquo Biomaterialsvol 32 no 27 pp 6595ndash6605 2011

[17] S Aryal M Prabaharan S Pilla and S Gong ldquoBiodegradableand biocompatiblemulti-arm star amphiphilic block copolymeras a carrier for hydrophobic drug deliveryrdquo International Journalof Biological Macromolecules vol 44 no 4 pp 346ndash352 2009

[18] M L Adams A Lavasanifar and G S Kwon ldquoAmphiphilicblock copolymers for drug deliveryrdquo Journal of PharmaceuticalSciences vol 92 no 7 pp 1343ndash1355 2003

[19] E He C Y Yue and K C Tam ldquoAssociation behavior of star-shaped pH-responsive block copolymer four-arm poly(eth-ylene oxide)-b-poly(methacrylic acid) in aqueous mediumrdquoLangmuir vol 25 no 9 pp 4892ndash4899 2009

[20] A Kowalczuk B Trzebicka S Rangelov M Smet and ADworak ldquoStar macromolecules with hyperbranched poly(ary-lene oxindole) cores and polyacid arms synthesis and solutionbehaviorrdquo Journal of Polymer Science Part A Polymer Chemistryvol 49 no 23 pp 5074ndash5086 2011

[21] Q Bian Y Xiao and M Lang ldquoThermoresponsive biotinylatedstar amphiphilic block copolymer synthesis self-assembly andspecific target recognitionrdquo Polymer vol 53 no 8 pp 1684ndash1693 2012

[22] H Kobayashi R Watanabe and P L Choyke ldquoImproving con-ventional enhanced permeability and retention (EPR) effectswhat is the appropriate targetrdquo Theranostics vol 4 no 1 pp81ndash89 2014

[23] K Engin D B Leeper J R Cater A J Thistlethwaite LTupchong and J D Mcfarlane ldquoExtracellular ph distributionin human tumoursrdquo International Journal of Hyperthermia vol11 no 2 pp 211ndash216 1995

[24] I F Tannock and D Rotin ldquoAcid pH in tumors and its potentialfor therapeutic exploitationrdquoCancer Research vol 49 no 16 pp4373ndash4384 1989

[25] J C Garbern E Minami P S Stayton and C E Murry ldquoDeliv-ery of basic fibroblast growth factor with a pH-responsiveinjectable hydrogel to improve angiogenesis in infarctedmyocardiumrdquo Biomaterials vol 32 no 9 pp 2407ndash2416 2011

[26] A M Ponce Z Vujaskovic F Yuan D Needham and M WDewhirst ldquoHyperthermia mediated liposomal drug deliveryrdquo


International Journal of Hyperthermia vol 22 no 3 pp 205ndash213 2006

[27] W Xiong W Wang Y Wang et al ldquoDual temperaturepH-sensitive drug delivery of poly(N-isopropylacrylamide-co-acrylic acid) nanogels conjugated with doxorubicin for poten-tial application in tumor hyperthermia therapyrdquo Colloids andSurfaces B Biointerfaces vol 84 no 2 pp 447ndash453 2011

[28] A Chilkotia M R Drehera D E Meyera and D RaucherbldquoTargeted drug delivery by thermally responsive polymersrdquoAdvanced Drug Delivery Reviews vol 54 no 5 pp 613ndash6302002

[29] J E ChungMYokoyamaMYamato TAoyagi Y Sakurai andT Okano ldquoThermo-responsive drug delivery from polymericmicelles constructed using block copolymers of poly(N-isopro-pylacrylamide) and poly(butylmethacrylate)rdquo Journal of Con-trolled Release vol 62 no 1-2 pp 115ndash127 1999

[30] L Zhang G Rui Y Mi J Xiqun and L Baorui ldquoThermoand pH dual-responsive nanoparticles for anti-cancer drugdeliveryrdquo Advanced Materials vol 19 no 19 pp 2988ndash29922007

[31] F M Winnik A R Davidson G K Hamer and H KitanoldquoAmphiphilic poly(N-isopropylacrylamides) prepared by usinga lipophilic radical initiator synthesis and solution propertiesin waterrdquoMacromolecules vol 25 no 7 pp 1876ndash1880 1992

[32] M A Ward and T K Georgiou ldquoThermoresponsive polymersfor biomedical applicationsrdquo Polymers vol 3 no 3 pp 1215ndash1242 2011

[33] L Klouda and A G Mikos ldquoThermoresponsive hydrogels inbiomedical applicationsrdquo European Journal of Pharmaceuticsand Biopharmaceutics vol 68 no 1 pp 34ndash45 2008

[34] A Serrano-Medina J M Cornejo-Bravo and A Licea-Claverıe ldquoSynthesis of pH and temperature sensitive core-shellnanomicrogels by one pot soap-free emulsion polymeriza-tionrdquo Journal of Colloid and Interface Science vol 369 no 1 pp82ndash90 2012

[35] CObeso-Vera JMCornejo-BravoA Serrano-Medina andALicea-Claverie ldquoEffect of crosslinkers on size and temperaturesensitivity of poly(N-isopropylacrylamide) microgelsrdquo PolymerBulletin vol 70 no 2 pp 653ndash664 2013

[36] W-Q Chen H Wei S-L Li et al ldquoFabrication of star-shaped thermo-sensitive poly(N-isopropylacrylamide)-cholicacid-poly(120576-caprolactone) copolymers and their self-assembledmicelles as drug carriersrdquo Polymer vol 49 no 18 pp 3965ndash3972 2008

[37] H Wei W-Q Chen C Chang et al ldquoSynthesis of star blockthermosensitive poly(l-lactide)-star block-poly(N-isopropy-lacrylamide-co-N-hydroxymethylacrylamide) copolymers andtheir self-assembledmicelles for controlled releaserdquoThe Journalof Physical Chemistry C vol 112 no 8 pp 2888ndash2894 2008

[38] S J T Rezaei M R Nabid H Niknejad and A A EntezamildquoFolate-decorated thermoresponsive micelles based on star-shaped amphiphilic block copolymers for efficient intracellularrelease of anticancer drugsrdquo International Journal of Pharma-ceutics vol 437 no 1-2 pp 70ndash79 2012

[39] K Miao H Liu and Y Zhao ldquoThermo pH and reduction res-ponsive coaggregates comprising AB

2

C2

star terpolymers formulti-triggered release of doxorubicinrdquo Polymer Chemistry vol5 no 10 pp 3335ndash3345 2014

[40] L Saleh-Ghadimi M Fathi and A A Entezami ldquoHet-eroarm star-shaped poly (N-isopropylacryamide-co-itaconicacid) copolymer prepared by glucose core as ATRP initiatorrdquo

International Journal of Polymeric Materials and PolymericBiomaterials vol 63 no 5 pp 246ndash255 2014

[41] F Xu J-W Xu B-X Zhang and Y-L Luo ldquoSelf-assemblymicelles from novel tri-armed star C3-(PS-b-PNIPAM) blockcopolymers for anticancer drug releaserdquo AIChE Journal vol 61no 1 pp 35ndash45 2015

[42] S-P Rwei Y-Y Chuang T-FWayW-Y Chiang and S-P HsuldquoPreparation of thermo- and pH-responsive star copolymersvia ATRP and its use in drug release applicationrdquo Colloid andPolymer Science vol 293 no 2 pp 493ndash503 2015

[43] Q Chen X Cao Y Xu and Z An ldquoEmerging synthetic stra-tegies for core cross-linked star (CCS) polymers and appli-cations as interfacial stabilizers bridging linear polymers andnanoparticlesrdquoMacromolecular Rapid Communications vol 34no 19 pp 1507ndash1517 2013

[44] J Ferreira J SyrettMWhittaker DHaddleton T P Davis andC Boyer ldquoOptimizing the generation of narrow polydispersitylsquoarm-firstrsquo star polymers made using RAFT polymerizationrdquoPolymer Chemistry vol 2 no 8 pp 1671ndash1677 2011

[45] J Liu H Duong M R Whittaker T P Davis and C BoyerldquoSynthesis of functional core star polymers via RAFT polymer-ization for drug delivery applicationsrdquo Macromolecular RapidCommunications vol 33 no 9 pp 760ndash766 2012

[46] R Salgado-Rodrıguez A Licea-Claverıe and K-F ArndtldquoRandom copolymers of N-isopropylacrylamide and meth-acrylic acid monomers with hydrophobic spacers pH-tunabletemperature sensitivematerialsrdquoEuropean Polymer Journal vol40 no 8 pp 1931ndash1946 2004

[47] J Brandrup E H Immergut and E A Grulke Eds PolymerHandbook JohnWileyamp Sons NewYork NYUSA 4th edition1999

[48] K-F Arndt and G Muller Polymer Charakterisierung CarlHanser Munchen Germany 1996

[49] A Licea-Claverıe E Rogel-Hernandez J A Lopez-Sanchez LA Castillo-Arambula J M Cornejo-Bravo and K F Arndt ldquoAfacile synthesis route for carboxyaryl-methacrylates a way toobtain aromatic polyelectrolytesrdquoDesignedMonomers and Poly-mers vol 6 no 1 pp 67ndash80 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


nitroxide mediated radical polymerization (NMP) [9] andreversible addition-fragmentation chain transfer polymeriza-tion (RAFT) have been versatile techniques for the synthesisof star polymers using both ldquoarm-firstrdquo and ldquocore-firstrdquometh-ods [10ndash13] Star polymers can be divided into two structuralcategories homogeneous (homoarm) or mixed (miktoarm)star polymers [6] Arms may be built of homo- co- oreven terpolymers in such a way that the final propertiesof the resulting star-shaped polymers can be adjusted bychoosing the respective chemical structure of arms and coredepending on the required application [14]

Amphiphilic polymers formed by hydrophilic andhydrophobic segments self-aggregate in aqueous solutionsto form micelles and other types of aggregates [15ndash18]Amphiphilic star polymers may also aggregate in a complexfashion depending on their chemical composition relativesize of core to arms number of arms and so forth [19ndash21]

Due to their small size (lt200 nm) star polymers mayaccumulate in tumors or inflamed tissues through theenhanced permeability and retention effect (EPR) [22] Sev-eral pathologies such as inflammation tumor and infarctedtissue show a local decrease in pH (1ndash25 pH units) [23ndash25] The particular pH conditions at pathological sites andalso local heating by 2ndash5∘C in solid tumors can be used toenhance drug release from accumulated or locally adminis-tered stimuli-responsive polymeric materials [26ndash28]

An important issue for the biomedical application oftemperature sensitive stars polymers is to adjust the transitiontemperature (LCST) above normal body temperature thuswhen the nanomaterials are injected into the body they arein the swollen state (37∘C lt LCST) and circulate in bloodvessels but collapse inside cancer tissue (119879tumor gt LCST)due to the hydrophobic change at temperatures above theirLCST [29]The process of collapse (and possible aggregation)leads to drug release inside tumors [30 31] The LCST ofpolyNIPAAm (32∘C) can be increased by copolymerizationof NIPAAm with hydrophilic or ionized monomers [3233] It was demonstrated that copolymerization of NIPAAmwith amphiphilic weak acid monomers allows tuning theLCST of polyNIPAAm above body temperature [13] thisleads in the case of nanohydrogels to maintaining adequateswellingdeswelling properties [34 35]

In a previous study our group has reported the synthe-sis of core cross-linked (CCS) temperature sensitive star poly-mers with a random number of poly(N-isopropylacryl-amide)-b-poly(hexyl acrylate) (PNIPAAm-b-PHA) or PNI-PAAm arms using the RAFT polymerization techniqueFurthermore the release behavior of different drugs fromthose star polymers was also investigated observing that astar polymer with PNIPAAm-b-PHA arms is more effectivein entrapping drugs than a star polymer formed by purePNIPAAm arms since the PHA block forms a hydrophobicintermediate shell over the corewhere hydrophobic drugs canbe entrapped [12] Other examples of PNIPAAm-based starpolymers have been developed for drug delivery applications[21 36ndash42] However most of them have been designed soas to be able to entrap and release the drug due to the abi-lity of PNIPAAm star polymers to self-aggregate [21 36ndash41] which may have the drawback of early aggregate

disruption due to the dilution factor in the bloodstreamresulting in premature drug release CCS star polymers rep-resent intermediate architecture between branched polymerchains and polymer nanoparticles [43] They are larger thanconventional star polymers having a single molecule oratom as a core but smaller than nanomicrogels They havethe advantage of presenting a relatively large core (cargocompartment) depending on the synthetic conditions andthey are surrounded by a large number of polymer arms thatstabilize them making them dispersible in a suitable solventfor the arms and preventing self-aggregation

In the present study the RAFT technique was employedto synthesize poly(NIPAAm-co-amphiphilic weak acid) poly-mers to tailor the LCST in the range of 40 to 46∘C at pH of74 These polymeric chains were further activated to formblock copolymers with HAThe block copolymers were usedto prepare dual sensitive temperature and pH star polymerscross-linked with ethyleneglycol dimethacrylate (EGDMA)These CCS star polymers were tested to load 5-fluorouracil(5-FU) an anticancer drug and to study their drug deliverybehavior under different in vitro conditions

2 Experimental Section

21 Materials The acid monomer 4-methacryloyloxyben-zoic acid (4MBA) the RAFT agent 2-hydroxyethyl 2-phenylacetate dithiobenzoate (DFH) and the free-radicalinitiator 441015840-azobis(4-cyanopentanol) (ACP)were preparedas described in the literature [13] N-Isopropylacrylamide(NIPAAm 97 Aldrich) was purified by recrystallizationfrom n-hexane 441015840-Azobis(4-cyanopentanoic acid) (ACPA98 Fluka) was recrystallized from methanol n-Hexylacrylate (HA Aldrich) and ethyleneglycol dimethacrylate(EGDMA Aldrich) were purified by passing the reagentsthrough an inhibitor remover column for benzoquinones(Aldrich) p-Dioxane (ACS grade Fermont) diethyl ether(ACS grade Fermont) tetrahydrofuran (THF HPLC gradeAldrich) and 5-fluorouracil (5-FU Sigma) were usedwithoutfurther purification

22 Synthesis of Linear Poly(NIPAAm) Arms (Macro-CTAsof PolyNIPAAm) and Statistical Copolymers of NIPAAmwith 4MBA Linear poly(NIPAAm) used to form the armsof star polymers was synthesized with prescribed mole-cular weights via RAFT polymerization (Figure 1(a))Poly(NIPAAm) arms were prepared using molar ratios ofmonomer CTA initiator of 180 10 02 of 132 10 02and of 88 10 02 Statistical copolymers were synthesizedwith various molar percentages of the acid comonomer inorder to adjust the LCST above 37∘C Poly(NIPAAm-co-4MBA) aiming at 9 of 4MBA was synthesized using thesame molar ratios that were used in the synthesis of linearpoly(NIPAAm) considering the total amount of monomersNIPAAm + 4MBA in the molar ratio

The general procedure was performed as follows 0049 g(0147mmol) of DFH was stirred until it was completelydissolved in 30mL of p-dioxane and poured into anampoule containing a magnetic stir bar 285 g (2518mmol)of NIPAAm 02867 g (130mmol) of 4MBA 00074 g






















OHN

SS

O

DFH (CTA)NIPAAm

OOH +

NN OH + +

CN

CNHO

ACP (initiator)

ONH

SOHOO

SnH

H O

HOOC

X = 4MBA

p-Dioxane

ONH

OHOO n S

S

OO

COOH

Macro-CTA1

Macro-CTA2

x

nX

co

70∘C

H2C

H3CCH3

CH3

H3C

H3C

CH3

CH3

CH3

X=0

(a)

n

Block copolymer 1

macro-CTAs m+ ACP+O

O

Hexyl acrylate

O NH

bO

HO On S

S

OO m

ONH

OHO O

nb

OO

COOH

Block copolymer 2

xS

S

OO m

co

70∘C

H3CCH3

CH3

H3C

CH3

CH3

CH3

CH3 p-Dioxane

(b)

NN

OHO+

CN OOH

CNBlock copolymer

O

OO

O

EGDMA


HOOC

HOOC COOH

HOOCCOOH

70∘ C 70 ∘C

+CH3

CH3


p-Dioxanep-Diox

ane

(c)



3or CD







(target)(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)




0[CTA]

0)119872Mon +119872CTAc

119872119899(target) = ([NIPAAm]

0[CTA]

0)119872NIPAAm + ([4MBA]

0[CTA]

0)1198724MBA +119872CTA




) times 100


) times 100

(1)












(target)i(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)






119899= 10820 gmol)



9) (119872119899=


5) (119872119899 = 22000 gmol) i119872119899(target) =

([119872]0[macro-CTA]

0)119872Mon +119872macro-CTA



Đ(119872119908

119872119899

) Yield (wt)





119899=


119899= 28570 gmol











PBS pH 74 LCST pH74

119863ℎ




-EGDMA20



-EGDMA10



c1

-EGDMA20

6274 plusmn062


8eminus3 46


d1

-EGDMA10


060302 plusmn002 40




119899= 28570 gmol

10

08

06

04

02

00

Inte

nsity

()

20 25 30 35 40 45





10

(red)
















ℎ



28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000


Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone


0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)



30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)




10

119872119899

= 100700 gmol (998771)



119886value is 53 [49] As a





5)-b-polyHA]

1-EGDMA


1-

EGDMA10



400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















OHN

SS

O

DFH (CTA)NIPAAm

OOH +

NN OH + +

CN

CNHO

ACP (initiator)

ONH

SOHOO

SnH

H O

HOOC

X = 4MBA

p-Dioxane

ONH

OHOO n S

S

OO

COOH

Macro-CTA1

Macro-CTA2

x

nX

co

70∘C

H2C

H3CCH3

CH3

H3C

H3C

CH3

CH3

CH3

X=0

(a)

n

Block copolymer 1

macro-CTAs m+ ACP+O

O

Hexyl acrylate

O NH

bO

HO On S

S

OO m

ONH

OHO O

nb

OO

COOH

Block copolymer 2

xS

S

OO m

co

70∘C

H3CCH3

CH3

H3C

CH3

CH3

CH3

CH3 p-Dioxane

(b)

NN

OHO+

CN OOH

CNBlock copolymer

O

OO

O

EGDMA


HOOC

HOOC COOH

HOOCCOOH

70∘ C 70 ∘C

+CH3

CH3


p-Dioxanep-Diox

ane

(c)



3or CD







(target)(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)




0[CTA]

0)119872Mon +119872CTAc

119872119899(target) = ([NIPAAm]

0[CTA]

0)119872NIPAAm + ([4MBA]

0[CTA]

0)1198724MBA +119872CTA




) times 100


) times 100

(1)












(target)i(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)






119899= 10820 gmol)



9) (119872119899=


5) (119872119899 = 22000 gmol) i119872119899(target) =

([119872]0[macro-CTA]

0)119872Mon +119872macro-CTA



Đ(119872119908

119872119899

) Yield (wt)





119899=


119899= 28570 gmol











PBS pH 74 LCST pH74

119863ℎ




-EGDMA20



-EGDMA10



c1

-EGDMA20

6274 plusmn062


8eminus3 46


d1

-EGDMA10


060302 plusmn002 40




119899= 28570 gmol

10

08

06

04

02

00

Inte

nsity

()

20 25 30 35 40 45





10

(red)
















ℎ



28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000


Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone


0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)



30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)




10

119872119899

= 100700 gmol (998771)



119886value is 53 [49] As a





5)-b-polyHA]

1-EGDMA


1-

EGDMA10



400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




(target)(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)




0[CTA]

0)119872Mon +119872CTAc

119872119899(target) = ([NIPAAm]

0[CTA]

0)119872NIPAAm + ([4MBA]

0[CTA]

0)1198724MBA +119872CTA




) times 100


) times 100

(1)












(target)i(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)






119899= 10820 gmol)



9) (119872119899=


5) (119872119899 = 22000 gmol) i119872119899(target) =

([119872]0[macro-CTA]

0)119872Mon +119872macro-CTA



Đ(119872119908

119872119899

) Yield (wt)





119899=


119899= 28570 gmol











PBS pH 74 LCST pH74

119863ℎ




-EGDMA20



-EGDMA10



c1

-EGDMA20

6274 plusmn062


8eminus3 46


d1

-EGDMA10


060302 plusmn002 40




119899= 28570 gmol

10

08

06

04

02

00

Inte

nsity

()

20 25 30 35 40 45





10

(red)
















ℎ



28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000


Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone


0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)



30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)




10

119872119899

= 100700 gmol (998771)



119886value is 53 [49] As a





5)-b-polyHA]

1-EGDMA


1-

EGDMA10



400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




(target)i(gmol)

119872119899

(GPC)(gmol)

Đ(119872119908

119872119899

) Yield (wt)






119899= 10820 gmol)



9) (119872119899=


5) (119872119899 = 22000 gmol) i119872119899(target) =

([119872]0[macro-CTA]

0)119872Mon +119872macro-CTA



Đ(119872119908

119872119899

) Yield (wt)





119899=


119899= 28570 gmol











PBS pH 74 LCST pH74

119863ℎ




-EGDMA20



-EGDMA10



c1

-EGDMA20

6274 plusmn062


8eminus3 46


d1

-EGDMA10


060302 plusmn002 40




119899= 28570 gmol

10

08

06

04

02

00

Inte

nsity

()

20 25 30 35 40 45





10

(red)
















ℎ



28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000


Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone


0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)



30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)




10

119872119899

= 100700 gmol (998771)



119886value is 53 [49] As a





5)-b-polyHA]

1-EGDMA


1-

EGDMA10



400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




PBS pH 74 LCST pH74

119863ℎ




-EGDMA20



-EGDMA10



c1

-EGDMA20

6274 plusmn062


8eminus3 46


d1

-EGDMA10


060302 plusmn002 40




119899= 28570 gmol

10

08

06

04

02

00

Inte

nsity

()

20 25 30 35 40 45





10

(red)
















ℎ



28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000


Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone


0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)



30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)




10

119872119899

= 100700 gmol (998771)



119886value is 53 [49] As a





5)-b-polyHA]

1-EGDMA


1-

EGDMA10



400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


28774

30744 2934

32954

2973

97545

17308

89184

84109

75749

125311062

6918

13666 11307

1387511606

16413

1459115398

100

95

90

85

80

75

70

65

60

55

4000 3500 3000 2500 2000 1500 1000


Tran

smitt

ance

()

(a)

c h iq q998400

d lb k

j oa f

Acetone


0

MeOH e g g998400 g998400998400 p

coa b

O OO O

O

OO

OO

Me Me

Me

Me

Me

NH

Me

ee d

ch

nx

mf

g

b

j k

i

l

o

o

p

q

COOH

q998400g998400998400

g998400

H3C

(b)



30

20

10

0

Volu

me m

ean

()

10 100 1000

Dh (nm)

(a)

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

0

20

20 25 30 35 40 45 50

Temperature (∘C)

Inte

nsity

mea

n (n

m)

(b)




10

119872119899

= 100700 gmol (998771)



119886value is 53 [49] As a





5)-b-polyHA]

1-EGDMA


1-

EGDMA10



400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


400

300

200

100

0

Inte

nsity

mea

n

20 25 30 35 40 45 50

Temperature (∘C)


1

-EGDMA10


1

-EGDMA10




119899is 32070 gmol










5)-b-polyHA]1-EGDMA10(119872119899=


4 Conclusions



(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b)


10


-EGDMA

10

100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 5 10 15 20 25 30


1

-EGDMA10


1

-EGDMA10



100

80

60

40

20

0

Cum

ulat

ive r

elea

se (

)

Time (h)0 1 2 3 4 5 6 7 8 9

37∘C 42

∘C 37∘C 42

∘C 37∘C


1

-EGDMA10


1

-EGDMA10







Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Acknowledgments


References











































2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















2

C2






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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