Research Article Novel 4-Arm Poly(Ethylene Glycol)...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 507103 11 pageshttpdxdoiorg1011552013507103

Research ArticleNovel 4-Arm Poly(Ethylene Glycol)-Block-Poly(Anhydride-Esters) Amphiphilic Copolymer MicellesLoading Curcumin Preparation Characterizationand In Vitro Evaluation

Li Lv1 Yuanyuan Shen1 Min Li1 Xiaofen Xu1 Mingna Li1

Shengrong Guo1 and Shengtang Huang2

1 School of Pharmacy Shanghai Jiao Tong University Shanghai 200240 China2 School of Pharmacy Hubei University of Science and Technology Xianning 437100 China

Correspondence should be addressed to Shengrong Guo srguosjtueducn and Shengtang Huang hst6511163com

Received 8 April 2013 Accepted 2 June 2013

Academic Editor Ajit S Narang

Copyright copy 2013 Li Lv et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A novel 4-arm poly(ethylene glycol)-block-poly(anhydride-esters) amphiphilic copolymer (4-arm PEG-b-PAE) was synthesizedby esterization of 4-arm poly(ethylene glycol) and poly(anhydride-esters) which was obtained by melt polycondensation of 120572- 120596-acetic anhydride terminated poly(L-lactic acid)Theobtained 4-armPEG-b-PAEwas characterized by 1H-NMRand gel permeationchromatographyThe criticalmicelle concentration of 4-armPEG-b-PAEwas 238 120583gmLThe curcumin-loaded 4-armPEG-b-PAEmicelles were prepared by a solid dispersionmethod and the drug loading content and encapsulation efficiency of the micelles were70 and 852 respectively The curcumin-loaded micelles were spherical with a hydrodynamic diameter of 1519 nm Curcuminwas encapsulated within 4-arm PEG-b-PAE micelles amorphously and released from the micelles faster in pH 50 than pH 74presenting one biphasic drug release pattern with rapid release at the initial stage and slow release later The hemolysis rate of thecurcumin-loaded 4-arm PEG-b-PAE micelles was 318 which was below 5 The IC

50value of the curcumin-loaded micelles

against Hela cells was 1021120583gmL lower than the one of free curcumin (2590 120583gmL)The cellular uptake of the curcumin-loadedmicelles in Hela cell increased in a time-dependent manner The curcumin-loaded micelles could induce G

2M phase cell cycle

arrest and apoptosis of Hela cells

1 Introduction

Biodegradable polyanhydrides and polyesters for pharma-ceutical applications have received considerable interestsin recent decades [1ndash5] Polyanhydrides are a particularlypromising class of polymers for drug delivery due to theirchemical properties and biocompatibility [6 7] The surface-eroding properties of polyanhydrides in aqueous mediummake them desirable for controlled drug release [8 9]Polyanhydride-based wafers that deliver carmustine for thetreatment of brain cancer have been approved by the FDA[10] Polyesters such as poly(lactic acid) poly(D L-lactic-co-glycolic acid) and poly(120576-caprolactone) have been commonly

used to form micelles to deliver a variety of drugs [11]Recently poly(anhydride-esters) copolymers have been syn-thesized in order to combine advantages of polyanhydridesand polyesters [12ndash14] These copolymers possess both thedegradation and surface erosion properties of individualpolyesters and polyanhydrides and may therefore provideextended advantages compared to either polyanhydrides orpolyesters alone

Amphiphilic block copolymers have been used exten-sively in pharmaceutical applications ranging from sustained-release technology to gene delivery [15] In aqueous solutionthey can form micelle structures with a hydrophobic innercore and hydrophilic outer shell in aqueousmedia at or above

2 BioMed Research International

the critical micelle concentration (CMC) The polymericmicelles have unique characteristics such as nanosize andgood thermodynamic stability in physiological conditions[16] Upon the CMC the hydrophobic core regions serveas reservoirs for drugs that have a poor water solubilityPoly(ethylene glycol) (PEG) is often used to build hydrophilicblocks of micelle-forming copolymers because it is bio-compatible highly soluble and hydrated in water [16]Amphiphilic block copolymer composed of PEG and polyan-hydride has been prepared by melt copolymerization By reg-ulating the PEG content polyanhydrides with adjustable sur-face erosion rate can be obtained which are more suitable fora drug-delivery device [17 18] In this study it was expectedthat a novel 4-arm poly(ethylene glycol)-b-poly(anhydride-esters) amphiphilic copolymers (4-armPEG-b-PAE) could beobtained by esterization of 4-arm poly(ethylene glycol) andpoly(anhydride-esters) which was obtained by melt polycon-densation of 120572- 120596-acetic anhydride terminated poly(L-lacticacid)

Curcumin a natural polyphenolic compound isolatedfrom the rhizome of the perennial herb curcuma longa[19ndash21] has been receiving considerable attention becauseof its putative cancer prevention and anticancer activitiesIt was reported that curcumin could interact with manycellular targets such as nuclear factor-kappa B (NF-120581B)and transcription factor activator protein-1 (AP-1) [22ndash24]Curcumin has antiproliferating effect in some cancer celllines of human origin [25] Attractively clinical trials haveproved that curcumin is very safe for humans even at ahigh dose of 12 g per day [26 27] In this study curcuminwas used as a model drug to evaluate the possibility ofthe 4-arm PEG-b-PAE as a nanocarrier The 4-arm PEG-b-PAE was characterized by 1H-NMR and gel permeationchromatography The curcumin-loaded 4-arm PEG-b-PAEmicelles were prepared by a solid dispersion method andthen the characteristics of the cucumin-loaded micelles (egsize 120577-potential encapsulation efficiency and drug loadingcontent) were investigated The in vitro released behaviorof curcumin was studied in phosphate buffer solution (PBSpH 74 or pH 50) and the stability and hemolysis of themicelles were evaluated In vitro cytotoxicity cellular uptakeretardation of cell cycle and induction of apoptosis ofmicelles to Hela cells were also reported

2 Materials and Methods

21 Materials L-Lactide diethylene glycol Sn (II) octoatesuccinic anhydride acetic anhydride and 4-arm poly(ethy-lene glycol) (Mn = 20000) were obtained from Aladdinchemistry Co Ltd (Shanghai China) Curcumin was pur-chased from Alfa-Aesar Dulbeccorsquos modified eaglersquos mediumwas purchased from Invitrogen Corporation (Grand IslandUSA) Penicillin-streptomycin fetal bovine serum (FBS)025 (wv) trypsine 003 (wv) EDTA solution phos-phate buffer solution (PBS) and propidium iodide (PI) werepurchased from Solarbio Science amp Technology (BeijingChina) RNase A and Annexin V-FITCPI were purchasedfrom Beyotime (Suzhou China)

22 Synthesis of Copolymers 4-arm PEG-b-poly(anhydride-esters) was synthesized by melt polycondensation as shownin Figure 1 Under nitrogen protection Sn (II) octanoate(100mg in 1mL of toluene) was added to the mixtureof 36 g L-Lactide and 087mL of diethylene glycol Thereaction mixture was heated to 135∘C for 25 minutes Theobtained polymer was dissolved in dichloromethane (DCM)and precipitated in ethanolhexane (2 8 vv) mixture Afterdrying in vacuum for 48 h 08 g succinic anhydride wasadded to the polymer and the mixture was heated to 130∘Cfor 8 hours The obtained polymer was dissolved in DCM50mL of water was introduced and the solution was stirredfor 1 hour The water was separated by a separatory funnelThe polymer was recovered from precipitation in cold diethylether and dried under vacuum After drying the polymerwas heated with 60mL acetic anhydride to 100∘C for 8hours then the mixture was applied vacuum to remove theexcess of acetic anhydride Once all the unreacted aceticanhydride was removed under vacuum the temperature wasincreased to 180∘C for 6 hours The poly L-lactic acid basedpoly(anhydride-esters) was recovered from precipitation ofthe polymer in cold diethyl ether and dried under vacuum

4-arm PEG (00004mol) and poly L-lactic acid basedpoly(anhydride-esters) (00016mol) were added to the reac-tor and thenwere heated to 180∘C under vacuum for 12 hoursAfter polycondensation the polymer was dissolved in DMCand precipitated in cold petroleum benzin and dried undervacuum

23 Characterization of Copolymers

231 1H-NMR 1H-NMR spectra weremeasured on aVarianMercury plus 400 spectrometer with CDCl

3as solvent The

tetramethylsilane (TMS) was used as an internal standard

232 Gel Permeation Chromatography (GPC) GPC mea-surements were performed at 40∘C on aWaters HPLC systemequipped with a model 1525 binary HPLC pump a model2414 refractive index detector and a series of 110 Styragelcolumns (HR

3and HR

4) Tetrahydrofuran was used as an

eluent at a flow rate of 10mLmin The molecular weightswere calculated by using a calibration curve which wasconstructed using polystyrene as standard

233 Critical Micelle Concentration (CMC) A steady-statepyrene fluorescence method was used to determine theCMC of the copolymer according to the method describedpreviously [28] Briefly pyrene was dissolved in acetone andadded to 5mL volumetric flasks to provide a concentrationof 6 times 10minus7M in the final solutions Acetone was thenevaporated and replaced with aqueous polymeric micellesolutions with concentrations ranging from 01 to 100 120583gmLFluorescence spectra were recorded on a Hitachi F-7000spectrophotometer The scans were performed at mediumspeed (240 nmmin) and at a PMT detector voltage of 350VThe emission wavelength was carried out at 395 nm andthe excitation spectra were recorded ranging from 300 to350 nm with both bandwidths set at 5 nm A CMC value

BioMed Research International 3

O

O

O

O

O OO

O

O O

OO O

OO

OO

OO O

O OO O

2 2

O O O O OO O

O O

O

O

O

O

2 2

O O

OO

O

O O O O OO O

O O

O

O

O

O

2 2

O O

m

n

n n

n n

n n

O O O O OO O

O OO

O

O

2 2

O

m

n nC C O

x

4

C C O Hx

4

HO

HO

HOOH

OH

OH

n

135∘CSn(OCOR)2

130∘C

100∘ C

180∘C

180∘C

lt2 mmHg

lt2 mmHg

+

+ CH2CH2O

CH2CH2O

H2

H2

Figure 1 Schematic of synthesis of the 4-arm PEG-b-PAE

was determined from the ratios of pyrene intensities at 337(119868337

) and 335 (119868335

) nm and calculated from the intersectionof two tangent plots of 119868

337119868335

versus log concentrations ofcopolymers

24 Preparation of Micelles The curcumin-loaded micelleswere prepared by a solid dispersionmethod Briefly curcumin(5mg) and 4-arm PEG-b-PAE (50mg) were dissolved in10mL of dichlormethane by sonication The organic solventwas evaporated on a rotary evaporator under reduced pres-sure at 40∘C to obtain a homogenous coevaporation cur-cumincopolymer matrix The resulting matrix was hydratedby adding 20mL of water and then sonicated at 200w for5min the power was pulsed for 5 s every 30 s to minimizeincreases in temperature The formed suspension was cen-trifuged at 4000 rpm for 20min to remove the aggregatedparticles and unencapsulated free curcumin and then filteredthrough a 045 120583m filter (Agela Technologies Inc China)followed by lyophilization for 3 days to get the powdered formofmicelles Empty 4-armPEG-b-PAEmicelles were preparedby the same way

25 Characterization of Polymeric Micelles

251 Particle Size Size Distribution and 120577-Potential Theaverage particle size and polydispersity (PDI) of micelleswere determined by dynamic light scattering (DLS) usinga Malvern Instrument zeta size nano-s at a detection angleof 173∘ 120577-potential was measured using the BC Haveninstruments corporation 90 plus particle size analyzer Thesamples were dispersed in distilled water at 25∘C and dilutedto a suitable density

252 Transmission Electron Microscopy (TEM) MorphologyThemorphological characteristics of polymericmicelles wereexamined by transmission electron microscopy (TEM Tec-nai G

2spirit Biotwin) The curcumin-loaded micelles were

diluted with distilled water and placed on a copper gridcovered with nitrocellulose

253 Evaluation of the Drug Encapsulation Efficiency andDrug Loading Content The amount of encapsulated cur-cumin in the micelles and the drug loading content wereevaluated by direct method Briefly 10mL of acetonitrile was


added to 2mg of the freeze-dried micelles powder in order todisrupt the micelles structure The solution was dried undernitrogen then dissolved inmobile phase centrifuged at a rateof 10000 rpm for 10 minutes and filtered through 045 120583mfilter to obtain a clear solution then the sample solution wasdetermined at 425 nm by using a Shimadzu HPLC systemequipped with an LC-10ADvp pump an SPD-17310 Avp UV-vis detector and a Diamonsil C

18reversed phase column 174

(46mm times 250mm 5120583m) The mobile phase was composedof an acetonitrile3 acetic acid (7525 vv) at a temperatureof 30∘C and a flow rate of 10mLmin The encapsulationefficiency and drug loading content were calculated using (1)as follows

Encapsulation efficiency ()

=

weight of curcumin in micellesweight of feeding curcumin

times 100

Drug loading content ()

=

weight of curcumin in micellesweight of micelles

times 100

(1)

254 Crystallographic Study Crystallographic assay wasperformed on curcumin powders empty 4-arm-PEG-PAEmicelles and the curcumin-loaded 4-arm PEG-b-PAEmicelles free-dried powers by a Rigaku 195Dmax 2200PCdiffractometer using a Cu-K120572 radiation source (40 kV20mA) The samples were scanned over a 2120579 range of 5ndash45∘at a rate of 5∘min

26 In Vitro Release Study In vitro release behavior of cur-cumin from the 4-arm PEG-b-PAE micelles was developedby dialysis method [29] Briefly the curcumin-loaded 4-armPEG-b-PAE micelles (10mg) were dispersed in 3mL PBS(001M pH 74 or pH 50) containing 1 vv of Tween 80 (tomaintain a sink condition) and then placed in a dialysis tube(Snakeskin Pierce USA) with a molecular weight cut-off of3500Da The dialysis tube was suspended in 12mL of releasemedium and placed in a shaking water bath at 37∘C witha shaking speed of 120 rpm At predetermined time pointsthe release medium was completely drawn and replaced withfresh incubation medium The amount of curcumin in therelease medium was determined by HPLC

27 Hemolysis by the Curcumin-LoadedMicelles Briefly freshblood from Sprague-Dawley rat was collected in heparinizedtubes and washed three times with 09 NaCl by centrifuga-tion at 2800 rpm for 5min and suspended in 09NaCl solu-tion (2 vv) Erythrocytes suspension (01mL) was added to09mL of the curcumin-loaded micelles (100120583gmL) 09NaCl solusion (negative control group with 0 hemolysis)and distilled water (positive control group with 100)respectively then incubated for 2 hours in a shaker incubatorat 37∘C After centrifugation at 3000 rpm for 10min theabsorbance of the supernatant was determined at 540 nm

The degree of hemolysis was determined by the followingequation

Hem () =ABS minus ABS

0

ABS100minus ABS

0

times 100 (2)

where ABS100

and ABS0are the absorbances of the solution

at 100 and 0 hemolysis respectively

28 Characteristics of Micelles Uptake The characteristicsof micelles cellular uptake were studied by flow cytometryBriefly Hela cells (30 times 105 cells in 2mL medium) wereseeded in 6-well plates and incubated overnightThemediumwas removed and the cells were washed twice with PBS andincubated in serum-free medium for different times withthe curcumin-loaded 4-arm PEG-b-PAE micelles (curcuminconcentration was 10120583gmL) for 24 h at 37∘C Cells treatedwith only medium were used as control After incubationthe cells were washed three times with ice-cold PBS andthen cells were collected by centrifugation and resuspendedin 05mL PBS The amount of uptake was analyzed by aflow cytometry (BD LSRFortessa Becton Dickinson) afterexcitation with 405 nm argon lasers and detection with a 515ndash545 band-pass filter

29 In Vitro Cytotoxicity The in vitro antitumor activityof the curcumin-loaded micelles and free curcumin wasdetermined by MTT assay Briefly Hela cells (obtained fromShanghai Institute of Cell Biology Chinese Academy ofSciences) in their logarithmic growth were seeded in 96-well plates at a density of 5000 cells per well in a finalvolume of 200 120583L medium and allowed to adhere overnightThe culture medium was carefully replaced with 200 120583L ofmedia containing DMSO-dissolved curcumin (final DMSOconcentration le 01) or the curcumin-loaded micellesat curcumin concentrations ranging from 1 to 40120583gmLCells were incubated with treatments for 72 hours Thenthe viability of the cells was determined using MTT assayThe cells were incubated with 200120583L medium containing05mgmL MTT for another 4 hours allowing the visiblecells to transform the yellow MTT into dark-blue formazancrystals which were dissolved in 200120583L of DMSO Theabsorbance values were measured at 570 nm by an ELISAplate reader (Varioskan Flash) Hela cell viability is defined asthe percent live cells compared with untreated controls Thepercentage of Hela cell viability was calculated as follows

Hela cell viability =A570sample

A570controltimes 100 (3)

In this assay all the experiments were done with tenparallel samples Untreated cells culture medium containing01 DMSO and culture medium containing blank micelleswere also tested as controls

210 Flow Cytometric (FCM) Analysis of Cell Cycle Distri-bution Flow cytometry provides information on cell cyclephases and is sensitive to apoptosis based on bivariate analysisof DNA content For the analysis of cell cycle distribution


Hela cells were seeded at a density of 15 times 105 cellsmL(2mLwell) in 6-well plates 24 hours before the experimentThe cells were exposed to either free curcumin or thecurcumin-loaded micelles at the curcumin concentration of50120583gmL in culture medium and incubated for 72 h in CO

2

incubator at 37∘C Cells treated with only medium were usedas controls After incubation the cells were washed with coldPBS for three timesThe cells were collected by centrifugationandfixedwith 70precooled alcohol overnight at 4∘CBeforemeasurements the cells were washed to eliminate alcoholand then incubated with RNase A (01mgmL) for 30min at37∘C then stained with PI solution (01mgmL) for 30minin the dark After that the distribution of different DNAcontents was analyzed by a flow cytometer (FACS CaliburBD USA) For each cell population at least 10000 cells wereexamined

211 Annexin V-FITCPI Double Staining AnnexinV-FITCPI was used to stain the cells and the percentage of cellapoptosis was determined by flow cytometer Hela cellswere seeded at a density of 15 times 105 cellsmL (2mLwell)in 6-well plates 24 hours before the experiment The cellswere exposed to either free curcumin or the curcumin-loaded micelles at the curcumin concentration of 50120583gmLin culture medium and incubated for 24 h in CO

2incubator

at 37∘CCells treatedwith onlymediumwere used as controlsAt the end of the treatment period the cells were harvestedand washed twice with cold PBS Then 1 times 105 cells werecollected by centrifugation and gently resuspended in 200120583Lbinding buffer thereafter 5120583L Annexin V-FITC and 10 120583L PI(200120583gmL) were added and incubated the cells in the darkfor 15min Finally 300120583L binding buffer was added into thesamples before they were analyzed by a flow cytometer (FACSCalibur BD USA)

212 Statistical Analyses All the experimental data wereexpressed as means plusmn standard deviation (SD) Statisticalanalyses were performed using a Studentrsquos 119905-tests The sig-nificance of differences between groups was considered as119875 lt 005 and very significant for 119875 lt 001

3 Result and Discussion

31 Synthesis and Characterization of Copolymers The 4-arm PEG-b-PAE was synthesized via a five-step syntheticroute as illustrated in Figure 1 L-lactide was polymerized inthe presence of Sn (II) octoate and diethylene glycol yield-ing hydroxyl-terminated poly(L-lactic acid) The hydroxyl-terminated poly(L-lactic acid) was converted to carboxyl-terminated poly(L-lactic acid) by reaction with succinicanhydride And then the carboxyl-terminated poly(L-lacticacid) reacted with an excess of acetic anhydride followedby melt polycondensation to obtain the PAE The 4-armPEG-b-PAE was prepared by esterization of the PAE and4-arm-PEG20k

The obtained polymers were characterized by 1H-NMRand GPC Figure 2 shows the 1H-NMR spectra of thePAE and the 4-arm PEG-b-PAE It could be found from

b

a

c d

g

fe

ppm

OO O

OOO O O O O

OO O

O On nm

g

ae

b f2 2

c d

12345678

CDCI3

(a)

a

b

c

d

e

4f

a

g

gc d

e h

f

b

hC C O

OO

OO

OO

OO O O

OO

OOO

O O

O n nx m

ppm12345678

CDCI3

a998400d998400

a998400 d998400CH2

H2CH2

(b)

Figure 2 1H-NMR spectra of (a) PAE and (b) 4-arm PEG-b-PAE

Figure 2(a) that the peaks at 120575156ndash160 223 275ndash279 364ndash366 426ndash430 and 513ndash518 ppmwere attributed to the pro-tons of a g c-d e f and b of the PAE structure respectivelyIn Figure 2(b) the sharp peak at 357ndash388 was attributed tomethylene protons of the ndashCH

2CH2Ondash in the 4-arm-PEG

block of the 4-arm PEG-b-PAE Peaks at 150ndash158 166ndash168 210 262ndash278 299 425ndash427 and 502ndash518 ppm wereattributed to the corresponding protons in the PAE block ofthe 4-arm PEG-b-PAE respectively According to the peakareas of Hg He in Figure 2(a) the molecular weight of thePAE was calculatedThe number of repeat units of anhydridewas calculated according to 1H-NMR and the value of thePAE is about 9 Table 1 shows the chemical compositionsand molecular weights of the synthesized polymers Highpolydispersity of the product of coupling looks apparent fromthe PDI of the final polymer sample which increases from 158for PAE copolymer to 265 for 4-armPEG-b-PAE It should be


Table 1 Characterization of the polymers

Sample PAE content in copolymer (wt)a Mn PDIbTheoretical Experimentala

PAE 100 5500 1584-arm PEG-b-PAE 6109 42000 39010 265aCalculated from the 1H-NMR resultsbPDI is the polydispersity of copolymers by GPC

085

09

095

1

105

11

115

12

minus4 minus35 minus3 minus25 minus2 minus15 minus1 minus05

log C

I 337I

335

Figure 3 Plots of intensity ratios 119868337

119868335

from pyrene excitationspectra versus log concentrations of the 4-arm PEG-b-PAE

noted that the polydispersity was determined using the linearpolystyrenemolecular weight standards for calibration in thisstudy which is not very suitable for nonlinear polymers suchas 4-arm PEG-B-PAE Thus it is difficult to compare thepolydispersity data of PAE and 4-armPEG-B-PAE accurately

32 Critical Micelle Concentration Fluorescence spectro-scopy was used to determine the CMC of the copolymerPyrene was used as probe to determine the association andmicellization of the copolymer in solution Pyrene had a verylow solubility in water and a strong hydrophobic character soit preferentially solubilized itself in the hydrophobic regionof micelles The process resulted in a great change of thefluorescence intensity A shift of the peak of the excitationspectra could be observedwith the changing concentration ofmicelles The maximum peak for pyrene in water was about335 nm the peak shifted to 337 nm in the solution of copoly-mer So the CMC could be determined by taking a midpointof the copolymer concentration at which point the ratioof 119868337

119868335

was varied As shown in Figure 3 119868337

119868335

waslow and kept constant at low copolymer concentrations andsharply increased as the copolymer concentration increasedafter the copolymer concentration reached a threshold that isCMC The CMC of the 4-arm PEG-b-PAE was 238 120583gmLwhich was significantly lower than that of low molecularweight surfactants in water [30] and implied the goodthermodynamic stability of the 4-arm PEG-b-PAE micelles

Figure 4 TEM image of the curcumin-loaded 4-arm PEG-b-PAEmicelles

So it can be concluded that the 4-arm PEG-b-PAE canform stable micelles structures at its low concentration afterdilution with a large volume of body fluids

33 Preparation Characterization and Stability of Poly-mericMicelles The4-armPEG-b-PAE could readily produceself-assembled micelles in aqueous solution because of itsamphiphilic property The PAE part could constitute aninternal hydrophobic core while 4-arm-PEG could provide ahydrophilic outer shell of the micelles The curcumin-loadedmicelles were prepared by a solid dispersionmethodThe for-mation of micelles was confirmed by measuring particle sizesize distribution 120577-potential and morphologyThe hydrody-namic diameter and zeta potential values were measured byDLS and are shown inTable 2Thehydrodynamic diameter ofthe particles is expressed in Z-average (nm) and the charge as120577-potential (mV) It shows that the hydrodynamic diametersof empty micelles and the drug loaded micelles are less than200 nm with an acceptably good polydispersity index (PDI lt009) The zeta potential value changes a little after loadingcurcumin into the micelles TEM photograph showed thecurcumin-loadedmicelles were spherical (Figure 4) Becauseof these properties it is assumed that the curcumin-loadedmicelles can effectively accumulate in the tumor region viathe permeability and retention effect and exhibit reduceduptake by reticuloendothelial system [31]


Table 2 Characterization of micelles (119899 = 3)

Micelles Drug loading content () Encapsulation efficiency () Size (nm) PDI 120577-potential (mv)4-arm PEG-b-PAE mdash mdash 1471 plusmn 01 011 plusmn 001 minus99 plusmn 12

Curcumin4-arm PEG-b-PAE 70 plusmn 02 852 plusmn 12 1519 plusmn 06 009 plusmn 005 minus105 plusmn 13

0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)

Figure 5 Hydrodynamic diameters of the curcumin-loaded 4-armPEG-b-PAE micelles versus time (pH 74 PBS 4∘C)

The physical stability of micelles in PBS (pH 74) at4∘C for 5 days was assessed No phase separation and nosignificant change in hydrodynamic diameter of micelleswere observed (Figure 5) These results demonstrated themicelles were physically stable during the study Owing tothe high surfacevolume ratio micelles always tend to formaggregates However due to electrostatic interactions orandstereospecific blockade a stable micelles suspension canalso be obtained The zeta potential of the curcumin-loadedmicelles is approximately minus10mv indicating that micellespresented negative surface charge Meanwhile 4-arm PEG-b-PAE copolymer is amphiphilic and consisted of hydrophilic4-arm PEG blocks and hydrophobic PAE blocks In aqueoussolution the relative difference in hydrophobicity betweenthe 4-arm PEG and the PAE blocks allows the formationof self-assembled micelles Therefore the stability of thecurcumin-loadedmicellesmight bemainly contributed to thestereospecific blockade formedby the hydrophilic 4-armPEGchain located at the surface of the curcumin-loaded micellesand the electrostatic interactions of micelles

Drug encapsulation efficiency and drug loading contentare important factors for drug delivery systems An HPLCassay was used to determine the drug concentration inthe micelles and then calculated the drug loading contentand encapsulation efficiency As it is seen from Table 2 thedrug loading content of the 4-arm PEG-b-PAE micelles was

(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)

Figure 6 X-ray diffraction spectra of (a) curcumin (b) empty 4-arm PEG-b-PAE micelles (c) the curcumin-loaded 4-arm PEG-b-PAE micelles

70 plusmn 02 and the encapsulation efficiency of the micellewas 852 plusmn 12 which achieve high drug encapsulationefficiency

To examine the crystallinity of micelle-encapsulatedcurcumin XRD analysis was performed on the curcumin-loaded micelles Figure 6 presents the XRD spectra of thecurcumin powders empty 4-arm PEG-b-PAE micelles andthe curcumin-loaded 4-arm PEG-b-PAE micelles It couldbe found that the characteristic diffraction peaks of thecurcumin were absent in the spectrum of the curcumin-loaded micelles which might suggest the curcumin wasencapsulated within the micelles amorphously

34 In Vitro Curcumin Release Figure 7 shows the cumula-tive release profiles of curcumin from the curcumin-loadedmicelles under different pH conditions (pH 50 and 74) at37∘C Curcumin release from the micelles was monitoredfor 9 days Sustained drug release was observed and it wasdependent on the pH of the release mediumThe cumulativerelease of curcumin from the curcumin-loaded micellescould reach up to 995 after 9 days at pH 50 higher thanthat at pH 74 which was 70 Curcumin released from themicelles consisted of two phases An initial fast release forabout 12 h was followed by a sustained released of drug fromthe micelles In Phase 1 the drug adsorbed on the surfaceof the micelles was separated from the carrier material intothe aqueous phase which contributed to the burst releasestage


0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)

Free curcumin (pH 74)Free curcumin (pH 50)

4-arm PEG-b-PAE (pH 74)4-arm PEG-b-PAE (pH 50)

Figure 7 Drug release profiles of the curcumin-loaded 4-arm PEG-b-PAE micelles

35 Hemolysis of Micelles The hemolysis rate of the cur-cumin-loaded 4-arm PEG-b-PAE micelles was 318 plusmn 127which was below 5 indicating that the micelles will notlead to severe hemolysis according to ISO 10993-42002Therefore it is suggested that the curcumin-loaded micelleshave no destructive effect on erythrocyte

36 Cellular Uptake of the Curcumin-Loaded Micelles Cel-lular uptake of the curcumin-loaded micelles in Hela cellwas investigated by flow cytometer To investigate the effectof time on the micelles uptake by Hela cells cells wereincubated with the micelles for various periods and the meanfluorescence intensity of cells was analyzed The results wereshown in Figure 8 The cells untreated with the micelles wereused as negative control to direct autofluorescence It can befound that the histograms of 05 and 2 hours move to rightand the histograms of 4 and 24 hours move much more toright especially the histogram of 24 hours comparing withthe histogram of blank The mean fluorescence intensity ofcells after incubation of 05 2 4 24 hours was 5649 649717395 and 24644 respectivelyThese results demonstrate thatthe cellular uptake of the curcumin-loaded micelles in Helacell increased in a time-dependent manner

37 In Vitro Cytotoxicity As shown in Figure 9 the cytotoxi-city of both the curcumin-loaded micelles and free curcuminto Hela cells at 72 hours showed significant concentrationdependence Half-maximal inhibitory concentrations (IC

50)

of the curcumin-loaded micelles and free curcumin toHela cells were 1021 120583gmL and 2590120583gmL respectivelyFrom these results it can be observed that the curcumin-loaded micelles have better efficiency than free curcuminIn addition the toxicity of the 4-arm PEG-b-PAE used todelivery curcumin was tested No cytotoxicities at the test

100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)

Figure 8 Cellular uptake of the curcumin-loaded 4-arm PEG-b-PAE micelles by Hela cells at 37∘C using flow cytometer (a) Flowhistograms of cellular uptake (b) Mean fluorescence intensity ofHela cells incubated with the curcumin-loaded 4-arm PEG-b-PAEmicelles

range concentrations of the 4-armPEG-b-PAEwere observedat 72 h (data not shown)

38 The Curcumin-Loaded Micelles Induced Cell Cycle Arrestin Hela Cells Figure 10 shows the cell cycle histograms ofHela cells treated with blank control free curcumin andthe curcumin-loaded micelles for 72 h After the cells weretreated with free curcumin or the curcumin-loaded micellesthe percent of cells in G

2M phase increased to 1918 plusmn

043 and 2810 plusmn 021 respectively which differed fromblank control (924 plusmn 178) These data suggested thatthe curcumin-loaded micelles showed more significant cellcycle arrest effect at G

2M phase in Hela cells compared


01020

30

40

5060

70

8090

100

110

1 25 5 10 20 40Curcumin concentration (120583gmL)

Hel

a cel

l via

bilit

y (

of c

ontro

l)

Curcumin4-arm PEG-b-PAE

lowast lowast

lowast

Figure 9 Cytotoxicity of the curcumin-loaded 4-arm PEG-b-PAEmicelles against Hela cells Incubation time was 72 h data werepresented as mean plusmn SD (119899 = 10) lowastSignificant difference betweenthe curcumin-loaded 4-armPEG-b-PAEmicelles and free curcumin(119875 lt 005)

to free curcumin at the same concentration The resultappears to be consistent with that of the in vitro cytotoxicityexperiments

39 Cell Apoptotic Rate Detected by FCM Annexin VPIdouble staining is a sensitive method in detecting apoptosisExternalization of phosphatidyl serine (PS) from the innerside to outer leaflet of the cell membrane is an importantindication of early apoptosis Annexin V possesses a highaffinity towards PS early apoptotic cells can be easily detectedby fluorescently labeled Annexin V Meanwhile PI can detectnecrotic cells due to its permeability through the damaged cellmembranes [32] As shown in Figure 11 after 72 h treatmentthe percentages of early and late apoptosis of the curcumin-loaded micelles-treated cells were 4301 plusmn 260 and 5635 plusmn041 respectively while those of curcumin-treated cellswere 528 plusmn 032 and 8719 plusmn 188 respectively Thetotal apoptosis rates of Hela cells caused by curcuminand the curcumin-loaded micelles were 9247 plusmn 220 and9936 plusmn 056 respectively Obviously in comparison withfree curcumin the curcumin-loaded micelles could promotea higher apoptotic rate in Hela cells at the same doseThe result agreed with that of our in vitro cytotoxicityexperiments

4 Conclusions

Amphiphilic block copolymer 4-arm poly(ethylene glycol)-b-Poly(anhydride-esters) (4-arm PEG-b-PAE) was success-fully synthesized The 4-arm PEG-b-PAE is able to self-assemble in water to form micelles as curcumin nanocarrier

0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt

G1 5754 plusmn 123S 3322 plusmn 235G2 924 plusmn 178

(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)

Figure 10 Flow cytometer analysis of cell cycle distribution Helacells were treated with free curcumin and the curcumin loadedmicelles at the curcumin concentration of 50 120583gmL for 24 h (a)Control (b) free curcumin and (c) the curcumin-loaded 4-armPEG-b-PAE micelles

The critical micelles concentration of the 4-arm PEG-b-PAEwas 238 120583gmL which implied the good thermodynamic sta-bility of 4-arm PEG-b-PAE micelles Curcumin was releasedfrom the 4-arm PEG-b-PAE micelles faster in pH 50 thanpH 74 presenting one biphasic drug release pattern withrapid release at the initial stage and slow release later Thecellular uptake of the the curcumin-loaded micelles in Helacell increased in a time-dependent manner The curcuminloaded PAE-b-PEG micelles had higher toxicity to Hela cellsthan free curcumin which could induce G

2M phase arrest

and apoptosis of Hela cells


016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005

149plusmn 1291plusmn

8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)

Figure 11 Cell apoptotic rate detected by flow cytometer Hela cells were treated with curcumin and the curcumin-loaded micelles at thecurcumin concentration of 50120583gmL for 72 h (a) Blank control (b) Free curcumin and (c) the curcumin-loaded 4-arm PEG-b-PAEmicelles

Authorsrsquo Contribution

Li Lv Yuanyuan Shen and Min Li are equal contributors

Conflict of Interests

The authors declare that there is no conflict of interestswith the trademarks mentioned in the paper They have nofinancial and personal relationships with other people ororganizations that can inappropriately influence their work

Acknowledgments

This work is supported by National Natural ScienceFoundation of China (NSFC) (Grant no 81171439) NationalBasic Research Program of China (973 Program Grant2010CB529902) and the National Key Technology RampDProgram of the Ministry of Science and Technology(2012BAI18B01) The authors are grateful to InstrumentalAnalysis Center of Shanghai Jiao Tong University andProfessor Che for their technical support in XRD and TEMexaminations

References

[1] N Kumar R S Langer and A J Domb ldquoPolyanhydrides anoverviewrdquo Advanced Drug Delivery Reviews vol 54 no 7 pp889ndash910 2002

[2] U Edlund and A C Albertsson ldquoPolyesters based on diacidmonomersrdquo Advanced Drug Delivery Reviews vol 55 no 4 pp585ndash609 2003

[3] J P Jain S Modi A J Domb and N Kumar ldquoRole of poly-anhydrides as localized drug carriersrdquo Journal of ControlledRelease vol 103 no 3 pp 541ndash563 2005

[4] L S Nair and C T Laurencin ldquoBiodegradable polymers as bio-materialsrdquo Progress in Polymer Science vol 32 no 8-9 pp 762ndash798 2007

[5] V Zabaleta G Ponchel H Salman M Agueros C Vauthierand J M Irache ldquoOral administration of paclitaxel with pegy-lated poly(anhydride) nanoparticles permeability and phar-macokinetic studyrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 81 no 3 pp 514ndash523 2012

[6] L Huntimer J H W Welder K Ross et al ldquoSingle immu-nization with a suboptimal antigen dose encapsulated intopolyanhydride microparticles promotes high titer and avidantibody responsesrdquo Journal of Biomedical Materials ResearchB vol 101 no 1 pp 91ndash98 2013

[7] Y Liang L Xiao Y Zhai C Xie L Deng and A Dong ldquoPrepa-ration and characterization of biodegradable poly(sebacic anhy-dride) chain extended by glycol as drug carrierrdquo Journal ofApplied Polymer Science vol 127 no 5 pp 3948ndash3953 2013

[8] A Gopferich and J Tessmar ldquoPolyanhydride degradation anderosionrdquoAdvancedDrugDelivery Reviews vol 54 no 7 pp 911ndash931 2002

[9] M Martina and D W Hutmacher ldquoBiodegradable polymersapplied in tissue engineering research a reviewrdquo PolymerInternational vol 56 no 2 pp 145ndash157 2007

[10] W Dang T Daviau P Ying et al ldquoEffects of GLIADEL waferinitial molecular weight on the erosion of wafer and release ofBCNUrdquo Journal of Controlled Release vol 42 no 1 pp 83ndash921996

[11] G Gaucher M H Dufresne V P Sant N Kang D Maysingerand J Leroux ldquoBlock copolymer micelles preparation char-acterization and application in drug deliveryrdquo Journal of Con-trolled Release vol 109 no 1ndash3 pp 169ndash188 2005


[12] R F Storey and A E Taylor ldquoSynthesis of novel biodegradablepoly(ester-anhydride)srdquo Journal of Macromolecular Science Avol 34 no 2 pp 265ndash280 1997

[13] B A Pfeifer J A Burdick and R Langer ldquoFormulationand surface modification of poly(ester-anhydride) micro- andnanospheresrdquo Biomaterials vol 26 no 2 pp 117ndash124 2005

[14] R A Hakala H Korhonen V V Meretoja and J V SeppalaldquoPhoto-cross-linked biodegradable poly(ester anhydride) net-works prepared from alkenylsuccinic anhydride functionalizedpoly(120576-caprolactone) precursorsrdquo Biomacromolecules vol 12no 7 pp 2806ndash2814 2011

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

[16] N Zhang S R Guo H Q Li L Liu Z H Li and J R GuldquoSynthesis of three types of amphiphilic poly(ethylene glycol)-block-poly(sebacic anhydride) copolymers and studies of theirmicellar solutionsrdquoMacromolecular Chemistry and Physics vol207 no 15 pp 1359ndash1367 2006

[17] S Hou L K McCauley and P X Ma ldquoSynthesis and erosionproperties of PEG-containing polyanhydridesrdquo Macromolecu-lar Bioscience vol 7 no 5 pp 620ndash628 2007

[18] F Najafi and M N Sarbolouki ldquoBiodegradable micellespolymersomes from fumaricsebacic acids and poly(ethyleneglycol)rdquo Biomaterials vol 24 no 7 pp 1175ndash1182 2003

[19] R K Maheshwari A K Singh J Gaddipati and R C SrimalldquoMultiple biological activities of curcumin a short reviewrdquo LifeSciences vol 78 no 18 pp 2081ndash2087 2006

[20] S M Oliveira I F Amaral M A Barbosa and C C TeixeiraldquoEngineering endochondral bone in vitro studiesrdquo Tissue Engi-neering A vol 15 no 3 pp 625ndash634 2009

[21] L Song Y Shen J Hou L Lei S Guo and C Qian ldquoPolymericmicelles for parenteral delivery of curcumin preparation char-acterization and in vitro evaluationrdquo Colloids and Surfaces Avol 390 no 1ndash3 pp 25ndash32 2011

[22] S Singh and B B Aggarwal ldquoActivation of transcription factorNF-120581b is suppressed by curcumin (diferuloylmethane)rdquo TheJournal of Biological Chemistry vol 270 no 42 pp 24995ndash25000 1995

[23] A B Kunnumakkara P Anand and B B Aggarwal ldquoCurcumininhibits proliferation invasion angiogenesis and metastasisof different cancers through interaction with multiple cellsignaling proteinsrdquo Cancer Letters vol 269 no 2 pp 199ndash2252008

[24] E O Farombi S Shrotriya and Y J Surh ldquoKolaviron inhibitsdimethyl nitrosamine-induced liver injury by suppressingCOX-2 and iNOS expression via NF-120581B andAP-1rdquo Life Sciencesvol 84 no 5-6 pp 149ndash155 2009

[25] K Mehta P Pantazis T McQueen and B B Aggarwal ldquoAnti-proliferative effect of curcumin (diferuloylmethane) againsthuman breast tumor cell linesrdquo Anti-Cancer Drugs vol 8 no5 pp 470ndash481 1997

[26] A L Chen C H Hsu J K Lin et al ldquoPhase I clinical trial ofcurcumin a chemopreventive agent in patients with high-riskor pre-malignant lesionsrdquoAnticancer Research vol 21 no 4 pp2895ndash2900 2001

[27] C D Lao M T Ruffin IV D Normolle et al ldquoDose escalationof a curcuminoid formulationrdquo BMC Complementary andAlternative Medicine vol 6 article 10 2006

[28] C L Zhao M A Winnik G Riess andM D Croucher ldquoFluo-rescence probe techniques used to study micelle formation in

water-soluble block copolymersrdquo Langmuir vol 6 no 2 pp514ndash516 1990

[29] Y Wang C Wang C Gong et al ldquoPolysorbate 80 coated poly(120576-caprolactone)-poly (ethylene glycol)-poly (120576-caprolactone)micelles for paclitaxel deliveryrdquo International Journal of Phar-maceutics vol 434 no 1-2 pp 1ndash8 2012

[30] K Y Lee I C Kwon Y H Kim W H Jo and S Y JeongldquoPreparation of chitosan self-aggregates as a gene deliverysystemrdquo Journal of Controlled Release vol 51 no 2-3 pp 213ndash220 1998

[31] H Xin X Jiang J Gu et al ldquoAngiopep-conjugated poly(ethy-lene glycol)-co-poly(120576-caprolactone) nanoparticles as dual-targeting drug delivery system for brain gliomardquo Biomaterialsvol 32 no 18 pp 4293ndash4305 2011

[32] H Lou L Gao X Wei et al ldquoOridonin nanosuspension en-hances anti-tumor efficacy in SMMC-7721 cells and H22 tumorbearingmicerdquoColloids and Surfaces B vol 87 no 2 pp 319ndash3252011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


the critical micelle concentration (CMC) The polymericmicelles have unique characteristics such as nanosize andgood thermodynamic stability in physiological conditions[16] Upon the CMC the hydrophobic core regions serveas reservoirs for drugs that have a poor water solubilityPoly(ethylene glycol) (PEG) is often used to build hydrophilicblocks of micelle-forming copolymers because it is bio-compatible highly soluble and hydrated in water [16]Amphiphilic block copolymer composed of PEG and polyan-hydride has been prepared by melt copolymerization By reg-ulating the PEG content polyanhydrides with adjustable sur-face erosion rate can be obtained which are more suitable fora drug-delivery device [17 18] In this study it was expectedthat a novel 4-arm poly(ethylene glycol)-b-poly(anhydride-esters) amphiphilic copolymers (4-armPEG-b-PAE) could beobtained by esterization of 4-arm poly(ethylene glycol) andpoly(anhydride-esters) which was obtained by melt polycon-densation of 120572- 120596-acetic anhydride terminated poly(L-lacticacid)

Curcumin a natural polyphenolic compound isolatedfrom the rhizome of the perennial herb curcuma longa[19ndash21] has been receiving considerable attention becauseof its putative cancer prevention and anticancer activitiesIt was reported that curcumin could interact with manycellular targets such as nuclear factor-kappa B (NF-120581B)and transcription factor activator protein-1 (AP-1) [22ndash24]Curcumin has antiproliferating effect in some cancer celllines of human origin [25] Attractively clinical trials haveproved that curcumin is very safe for humans even at ahigh dose of 12 g per day [26 27] In this study curcuminwas used as a model drug to evaluate the possibility ofthe 4-arm PEG-b-PAE as a nanocarrier The 4-arm PEG-b-PAE was characterized by 1H-NMR and gel permeationchromatography The curcumin-loaded 4-arm PEG-b-PAEmicelles were prepared by a solid dispersion method andthen the characteristics of the cucumin-loaded micelles (egsize 120577-potential encapsulation efficiency and drug loadingcontent) were investigated The in vitro released behaviorof curcumin was studied in phosphate buffer solution (PBSpH 74 or pH 50) and the stability and hemolysis of themicelles were evaluated In vitro cytotoxicity cellular uptakeretardation of cell cycle and induction of apoptosis ofmicelles to Hela cells were also reported

2 Materials and Methods

21 Materials L-Lactide diethylene glycol Sn (II) octoatesuccinic anhydride acetic anhydride and 4-arm poly(ethy-lene glycol) (Mn = 20000) were obtained from Aladdinchemistry Co Ltd (Shanghai China) Curcumin was pur-chased from Alfa-Aesar Dulbeccorsquos modified eaglersquos mediumwas purchased from Invitrogen Corporation (Grand IslandUSA) Penicillin-streptomycin fetal bovine serum (FBS)025 (wv) trypsine 003 (wv) EDTA solution phos-phate buffer solution (PBS) and propidium iodide (PI) werepurchased from Solarbio Science amp Technology (BeijingChina) RNase A and Annexin V-FITCPI were purchasedfrom Beyotime (Suzhou China)

22 Synthesis of Copolymers 4-arm PEG-b-poly(anhydride-esters) was synthesized by melt polycondensation as shownin Figure 1 Under nitrogen protection Sn (II) octanoate(100mg in 1mL of toluene) was added to the mixtureof 36 g L-Lactide and 087mL of diethylene glycol Thereaction mixture was heated to 135∘C for 25 minutes Theobtained polymer was dissolved in dichloromethane (DCM)and precipitated in ethanolhexane (2 8 vv) mixture Afterdrying in vacuum for 48 h 08 g succinic anhydride wasadded to the polymer and the mixture was heated to 130∘Cfor 8 hours The obtained polymer was dissolved in DCM50mL of water was introduced and the solution was stirredfor 1 hour The water was separated by a separatory funnelThe polymer was recovered from precipitation in cold diethylether and dried under vacuum After drying the polymerwas heated with 60mL acetic anhydride to 100∘C for 8hours then the mixture was applied vacuum to remove theexcess of acetic anhydride Once all the unreacted aceticanhydride was removed under vacuum the temperature wasincreased to 180∘C for 6 hours The poly L-lactic acid basedpoly(anhydride-esters) was recovered from precipitation ofthe polymer in cold diethyl ether and dried under vacuum

4-arm PEG (00004mol) and poly L-lactic acid basedpoly(anhydride-esters) (00016mol) were added to the reac-tor and thenwere heated to 180∘C under vacuum for 12 hoursAfter polycondensation the polymer was dissolved in DMCand precipitated in cold petroleum benzin and dried undervacuum

23 Characterization of Copolymers

231 1H-NMR 1H-NMR spectra weremeasured on aVarianMercury plus 400 spectrometer with CDCl

3as solvent The

tetramethylsilane (TMS) was used as an internal standard

232 Gel Permeation Chromatography (GPC) GPC mea-surements were performed at 40∘C on aWaters HPLC systemequipped with a model 1525 binary HPLC pump a model2414 refractive index detector and a series of 110 Styragelcolumns (HR

3and HR

4) Tetrahydrofuran was used as an

eluent at a flow rate of 10mLmin The molecular weightswere calculated by using a calibration curve which wasconstructed using polystyrene as standard

233 Critical Micelle Concentration (CMC) A steady-statepyrene fluorescence method was used to determine theCMC of the copolymer according to the method describedpreviously [28] Briefly pyrene was dissolved in acetone andadded to 5mL volumetric flasks to provide a concentrationof 6 times 10minus7M in the final solutions Acetone was thenevaporated and replaced with aqueous polymeric micellesolutions with concentrations ranging from 01 to 100 120583gmLFluorescence spectra were recorded on a Hitachi F-7000spectrophotometer The scans were performed at mediumspeed (240 nmmin) and at a PMT detector voltage of 350VThe emission wavelength was carried out at 395 nm andthe excitation spectra were recorded ranging from 300 to350 nm with both bandwidths set at 5 nm A CMC value


O

O

O

O

O OO

O

O O

OO O

OO

OO

OO O

O OO O

2 2

O O O O OO O

O O

O

O

O

O

2 2

O O

OO

O

O O O O OO O

O O

O

O

O

O

2 2

O O

m

n

n n

n n

n n

O O O O OO O

O OO

O

O

2 2

O

m

n nC C O

x

4

C C O Hx

4

HO

HO

HOOH

OH

OH

n

135∘CSn(OCOR)2

130∘C

100∘ C

180∘C

180∘C

lt2 mmHg

lt2 mmHg

+

+ CH2CH2O

CH2CH2O

H2

H2



) and 335 (119868335


337119868335














=


times 100


=


times 100

(1)






0

ABS100minus ABS

0

times 100 (2)

where ABS100











2



2incubator






b

a

c d

g

fe

ppm

OO O

OOO O O O O

OO O

O On nm

g

ae

b f2 2

c d

12345678

CDCI3

(a)

a

b

c

d

e

4f

a

g

gc d

e h

f

b

hC C O

OO

OO

OO

OO O O

OO

OOO

O O

O n nx m

ppm12345678

CDCI3

a998400d998400

a998400 d998400CH2

H2CH2

(b)









085

09

095

1

105

11

115

12


log C

I 337I

335


119868335




119868335


119868335









0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)




(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)






0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


O

O

O

O

O OO

O

O O

OO O

OO

OO

OO O

O OO O

2 2

O O O O OO O

O O

O

O

O

O

2 2

O O

OO

O

O O O O OO O

O O

O

O

O

O

2 2

O O

m

n

n n

n n

n n

O O O O OO O

O OO

O

O

2 2

O

m

n nC C O

x

4

C C O Hx

4

HO

HO

HOOH

OH

OH

n

135∘CSn(OCOR)2

130∘C

100∘ C

180∘C

180∘C

lt2 mmHg

lt2 mmHg

+

+ CH2CH2O

CH2CH2O

H2

H2



) and 335 (119868335


337119868335














=


times 100


=


times 100

(1)






0

ABS100minus ABS

0

times 100 (2)

where ABS100











2



2incubator






b

a

c d

g

fe

ppm

OO O

OOO O O O O

OO O

O On nm

g

ae

b f2 2

c d

12345678

CDCI3

(a)

a

b

c

d

e

4f

a

g

gc d

e h

f

b

hC C O

OO

OO

OO

OO O O

OO

OOO

O O

O n nx m

ppm12345678

CDCI3

a998400d998400

a998400 d998400CH2

H2CH2

(b)









085

09

095

1

105

11

115

12


log C

I 337I

335


119868335




119868335


119868335









0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)




(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)






0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






=


times 100


=


times 100

(1)






0

ABS100minus ABS

0

times 100 (2)

where ABS100











2



2incubator






b

a

c d

g

fe

ppm

OO O

OOO O O O O

OO O

O On nm

g

ae

b f2 2

c d

12345678

CDCI3

(a)

a

b

c

d

e

4f

a

g

gc d

e h

f

b

hC C O

OO

OO

OO

OO O O

OO

OOO

O O

O n nx m

ppm12345678

CDCI3

a998400d998400

a998400 d998400CH2

H2CH2

(b)









085

09

095

1

105

11

115

12


log C

I 337I

335


119868335




119868335


119868335









0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)




(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)






0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

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l via

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of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



2



2incubator






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1

105

11

115

12


log C

I 337I

335


119868335




119868335


119868335









0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)




(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)






0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





085

09

095

1

105

11

115

12


log C

I 337I

335


119868335




119868335


119868335









0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)




(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)






0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





0

20

40

60

80

100

120

140

160

180

200

0 1 2 3 4 5

Dia

met

er (n

m)

Time (days)




(a)

(b)

(c)

5 10 15 20 25 30 35 40 45Two-Theta (deg)






0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

20

40

60

80

100

120

Cum

ulat

ive r

elea

se ra

te (

)

50 100 150 200 2500Time (h)







50)


100101 102 103 104 105

AmCyan-A

Cou

nt

0

55

111

166

221

Blank05 h2 h

4 h24 h

(a)

0

5000

10000

15000

20000

25000

30000

05 2 4 24

Fluo

resc

ent

Time

56496497

17395

24644

(b)








01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


01020

30

40

5060

70

8090

100

110


Hel

a cel

l via

bilit

y (

of c

ontro

l)


lowast lowast

lowast




4 Conclusions


0 256 512 768 1024FL2-A

0

140

281

421

562

Cou

nt


(a)

0 256 512 768 1024FL2-A

0

158

316

473

631

Cou

nt


(b)

0 256 512 768 1024FL2-A

0

149

298

447

596

Cou

nt


DateFitDiploid G1

Diploid S

Diploid G2

(c)



2M phase arrest



016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


016 29

232

05

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Data005


8103plusmn

457plusmn

(a)

023 188

145

032

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date010


724plusmn

528plusmn

(b)

039

06

041024

100

100

101

101

102

102

103

103

104

104

FL1-H

FL3-

H

Date014


051plusmn

4301plusmn

(c)






Acknowledgments


References







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Novel 4-Arm Poly(Ethylene Glycol)...

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Transcript of Research Article Novel 4-Arm Poly(Ethylene Glycol)...