Synthesis and self-assembly behavior of pH-responsive amphiphilic copolymers containing ketal functional groups

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Research ArticleReceived: 21 September 2009 Revised: 18 October 2009 Accepted: 2 November 2009 Published online in Wiley Interscience: 7 April 2010

(www.interscience.wiley.com) DOI 10.1002/pi.2814

Synthesis and self-assembly behaviorof pH-responsive amphiphilic copolymerscontaining ketal functional groupsDawei Zhang,a,b Hao Zhang,a Jun Nieb∗ and Jing Yanga∗

Abstract

Polymeric micelles that are responsive to pH are particularly attractive for application in drug delivery systems. In this study, onetype of amphiphilic block copolymers with hydrophobic building blocks bearing pH-sensitive ketal groups was designed. In anacidic environment, the polarity transfer from amphiphile to double hydrophile for this copolymer destroyed the driving forceof micelle formation, which triggered the release of encapsulated hydrophobic molecules. The amphiphilic block copolymersmonomethoxy-poly(ethylene glycol)-block-poly(2,2-dimethyl-1,3-dioxolane-4-yl)methyl acrylate (MPEG-block-PDMDMA) wasfabricated by atom transfer radical polymerization using MPEG-Br as macroinitiator. The critical micelle concentration ofvarious compositions of this copolymer in aqueous solution ranged from 4.0 to 10.0 mg L−1, and the partition equilibriumconstant (Kv) of pyrene in micellar solutions of the copolymers varied from 1.61 × 105 to 4.86 × 105. Their overall effectivehydrodynamic diameters from dynamic light scattering measurements were between 80 and 400 nm, and the micellarmorphology showed spherical geometry as investigated using transmission electron microscopy. At pH = 1.0, all of thesepolymeric micelles presented 100% payload release in 24 h of incubation, while at pH = 3.0, nearly 70 and 25% of pyrenewas released for MPEG-block-PDMDMA (44/18) and MPEG-block-PDMDMA (44/25) in 260 h, respectively. The pH-responsiveMPEG-block-PDMDMA polymeric micelles having good encapsulation efficiency for hydrophobic drugs are potential candidatesfor biomedical and drug delivery applications.c© 2010 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: pH-responsive; polymeric micelle; drug delivery carrier; amphiphilic copolymers

INTRODUCTIONRecently, polymeric micelles formed from amphiphilic blockcopolymers have attracted significant attention in the develop-ment of drug delivery systems.1 – 3 In an aqueous environment,the hydrophobic micelle core formed by association of the hy-drophobic blocks of an amphiphilic block copolymer serves asa microenvironment for the incorporation of various hydropho-bic therapeutic compounds; the hydrophilic corona serves as astabilizing interface between the hydrophobic core and exter-nal medium.4 – 6 Such a core–shell architecture of the polymericmicelles means they can be potentially employed as efficient con-tainers for reagents with poor solubility and/or low stability inphysiological environments. Moreover, their suitable dimensions,typically between 20 and 100 nm, mean the micelles can effi-ciently escape renal rapid excretion, as well as avoid componentsof the reticular endothelial system,7 thus increasing the possibilityof accumulation of the micellar carriers at pathological sites andfacilitating potentially passive targeting of drugs to tumors via thewell-accepted enhanced permeation and retention effect.8

Besides improving the circulation effectiveness and decreasingthe systemic toxicity of the drug payload, another important issueconcerning ideal micellar drug carriers is the ability to releasetheir drug load in a controlled manner, upon arrival at the targetsite. This challenge has motivated many researchers to designenvironmentally responsive polymeric micelles and study theirtriggered payload release under various chemical and physical

stimuli including pH, specific molecules, redox, light, ultrasoundand temperature.9 – 14 Compared with external stimuli, change inacidity naturally occurs in the physiological environment. It is wellknown that the extracellular pH of tumors is slightly more acidicthan that of blood and normal tissue.15 Moreover, numerous pHgradients exist in both endosomes (pH = 5.5–6.0) and lysosomes(pH = 4.5–5.0) when micelles are taken up by cells via anendocytosis process which begins near the physiological pH of7.4.16 Therefore, pH-responsive polymeric micelles as particularlypromising drug carriers can be designed to trigger selectively theirpayload release in tumor tissue or within tumor cells.

∗ Correspondence to: Jing Yang, State Key Laboratory of Chemical Resource,College of Life Science and Technology, Beijing University of Chemical andTechnology, Beijing 100029, People’s Republic of China.E-mail: [email protected]

Jun Nie, College of Materials Science and Engineering, Beijing Universityof Chemical and Technology, Beijing 100029, People’s Republic of China.E-mail: [email protected]

a College of Life Science and Technology, Beijing University of Chemical andTechnology, Beijing 100029, People’s Republic of China

b College of Materials Science and Engineering, Beijing University of Chemicaland Technology, Beijing 100029, People’s Republic of China

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Several approaches have been employed for the develop-ment of pH-responsive micelles. One approach is to incorporate‘titratable’ groups such as amines or carboxylic acids into copoly-mers such that micelle formation is tuned by the protonation ofthese groups.17 – 20 Another approach is to incorporate directlyacid-labile linkages such as ortho-ester, hydrazone and cis-acotinylinto the copolymer backbone, such that the cleavage of thelinkages provides sufficient structural changes in the polymer totrigger drug release.21 – 23 Acetal and ketal as acid-labile groupsare of particular importance in this regard. Utilizing their pH-sensitive properties, various polymer-based delivery systems havebeen successfully developed. Frechet’s group exploited one typeof these micelle-forming copolymers, in which trimethoxyben-zylidene acetals as pH-sensitive groups, linked either to the sidechain of poly(aspartic acid) (PAA) segments of poly(ethylene gly-col)–PAA block copolymers or to the periphery of PAA dendronsof linear-dendritic copolymers, could rapidly release encapsulatedNile red or doxorubicin in a mildly acidic environment.24 – 27 Re-cently, Frechet and co-workers further developed pH-responsivepolymer-based delivery vehicles via acetal or ketal bonds embed-ded in polymer main chains. This drug delivery system could notonly trigger the release of a payload in a pH-dependent manner,but also degrade fully into small molecules without leaving a poly-meric residue, which is advantageous for the complete clearanceof polymer carriers from the body and increases the applicabilityto practical systems.28,29 Bulmus and co-workers30 and Zhong andco-workers31 have, respectively, studied core crosslinked micellesconstructed with a divinyl crosslinker having acetal bonds andbiodegradable micelles based on a polycarbonate hydrophobeattaching benzylidene acetal units.

Although great progress has been achieved in the developmentof pH-sensitive micelles as drug delivery agents, the design andsynthesis of novel and efficient pH-sensitive carriers are stillextremely important. In this paper, we report our efforts onthe synthesis of one type of amphiphilic block copolymers, i.e.monomethoxy-poly(ethylene glycol)-block-poly(2,2-dimethyl-1,3-dioxolane-4-yl)methyl acrylate (MPEG-block-PDMDMA), and thepH-responsive behavior of micelles of this polymeric material.Due to the hydrophobic PDMDMA block of the copolymercontaining ketal pendant groups, these diblock copolymersare stable in a neutral aqueous environment. Under acidicconditions, ketal removal from the hydrophobic blocks leadsto the polarity transition of the copolymer from amphiphile todouble hydrophile, which destroys the driving force of micellarself-assembly and triggers payload release. In particular, acetoneas the hydrolyzate of ketal deprotection, a compound on theUS Food and Drug Administration (FDA) Generally Recognized asSafe (GRAS) list, has good biocompatibility. With the advantagesof good biocompatibility and convenient synthesis, MPEG-block-PDMDMA may become a potential candidate for the encapsulationand delivery of hydrophobic drugs in the human body.

EXPERIMENTALMaterialsMonomethoxy-poly(ethylene glycol) (MPEG2000), purchased fromFluka, was dried by azeotropic distillation with toluene, the residualtoluene being removed under high vacuum prior to use. CuBr pur-chased from Aldrich was purified by stirring in acetic acid overnight,followed by washing with ethanol and diethyl ether, and dryingin vacuum. Triethylamine and methylene dichloride (CH2Cl2) weredehydrated with KOH and CaCl2 overnight, respectively, and

distilled. Toluene was dried using sodium with benzophenone ascolor indicator. All of the purified solvents and reagents mentionedabove were stored in solvent storage flasks prior to use. All otherreagents (2-bromopropionyl bromide and pentamethyldiethylen-etriamine (PMDETA) purchased from Aldrich, glycerol, acryloylchloride, p-toluenesulfonic acid, CuBr2, diethyl ether and hexanes)were used as received without further treatment.

General characterization1H NMR and 13C NMR spectra were obtained with a 400 MHz NMRinstrument (Bruker Corporation, Germany) at room temperatureusing CDCl3 as solvent. The chemical shifts were measured againstthe solvent signal of CDCl3 as internal standard. The molecularweights of the polymers were determined with a Waters 515–2410gel permeation chromatography (GPC) instrument equipped witha Styragel HT6E-HT5-HT3 chromatographic column followinga guard column and a differential refractive index detector.The sample solution was filtered with a 0.45 µm syringe filterprior to injection. The measurements were performed usingtetrahydrofuran (THF) as the eluent at a flow rate of 1.0 mL min−1

at 30 ◦C and a series of low-polydispersity polystyrene standardsfor calibration of the columns. Fluorescence spectra were recordedwith a Hitachi F-4500 fluorescence instrument (Hitachi High-Technologies Corporation, Tokyo, Japan) at room temperatureranging from 25 to 30 ◦C. UV-visible spectra were recordedwith a Hitachi U-3010 UV-visible spectrometer (Hitachi High-Technologies Corporation, Tokyo, Japan).

Synthesis of amphiphilic block copolymerSynthesis of (2,2-dimethyl-1,3-dioxolane-4-yl)methyl acrylate(DMDMA) monomerThe synthesis of the monomer, involving two steps, was modifiedbased on reported methods.32 For the first step, isopropylideneglycerol (IPG) was prepared by reacting glycerol (9.2 g, 100.0 mmol)with acetone (30 mL) at 80 ◦C in the presence of p-toluenesulfonicacid (0.32 g, 2.0 mmol) as catalyst. After 48 h azeotropic distillation,sodium acetate (0.22 g, 2.6 mmol) was added to terminatethe reaction. The reaction solution was filtered, and excessacetone was removed by rotary evaporation. Pure liquid IPG wascollected under reduced-pressure distillation (40 ◦C/0.37 mmHg)in 72.6% yield. 1H NMR (400 MHz, CDCl3), δ (ppm): 4.24 (m, 1H,CH2CHCH2), 3.79–4.04 (m, 2H, HOCH2CHCH2), 3.58–3.72 (m, 2H,HOCH2CHCH2), 1.99 (br, 1H, OH), 1.41 (d, 6H, C(CH3)2).

For the second step, the preparation of DMDMA was carried outby the reaction of IPG (16.8 g, 130.0 mmol), acryloyl chloride (16.3 g,180.0 mmol) and triethylamine (18.2 g, 180.0 mmol) in anhydrousCH2Cl2 (80 mL) in an ice bath for 14 h. The reaction solution wasfiltered and sequentially washed with 0.1 N HCl, 0.1 N NaOH anddistilled water. The dried and concentrated organic mixture waspurified by reduced-pressure distillation (50 ◦C/0.37 mmHg) in thepresence of methylene blue as inhibitor to obtain DMDMA asa colorless liquid in 63% yield (99% purity). 1H NMR (400 MHz,CDCl3), δ (ppm): 5.85–6.44 (m, 3H, CH2 CH), 3.76–4.38 (m, 5H,CH2CHCH2), 1.41 (d, 6H, C(CH3)2). 13C NMR (100 MHz, CDCl3), δ

(ppm): 165.9, 131.4, 127.9, 109.8, 73.6, 66.3, 64.7, 26.6, 25.4.

Synthesis of MPEG-Br macroinitiatorThe macroinitiator was prepared according to a reportedmethod.33 The stepwise addition of 2-bromopropionyl bromide(1.1 g, 5.0 mmol) solution in CH2Cl2 (10 mL) to a 100 mL three-neck flask containing a mixture of MPEG2000 (number-average

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molecular weight Mn = 2000 g mol−1; 4.0 g, 2.0 mmol) andtriethylamine (0.5 g, 5.0 mmol) in anhydrous CH2Cl2 (40 mL) inan ice bath was carried out with stirring for 48 h. After filtration,the filtrate was sequentially washed with 0.1 N HCl, 0.1 N NaOH anddistilled water followed by extraction with CH2Cl2 twice. The com-bined organic phase was dried over anhydrous MgSO4 and con-centrated. The crude product was redissolved in CHCl3 (3 mL) andprecipitated in diethyl ether (100 mL) twice to obtain MPEG-Br in83% yield. 1H NMR (400 MHz, CDCl3), δ (ppm): 4.40 (t, 1H, CH3CHBr),4.31 (t, 2H, CH2CH2OOC), 3.45–3.82 (m, repeating unit, OCH2CH2),3.37 (s, 3H, CH3OCH2), 1.82 (d, 3H, CH3CHBr). 13C NMR (100 MHz,CDCl3), δ (ppm): 170.0, 71.8, 70.5, 68.6, 64.8, 58.8, 39.8, 21.5.

Synthesis of MPEG-block-PDMDMAIn a typical atom transfer radical polymerization (ATRP) procedure,a Schlenk flask with a magnetic stir bar was charged with CuBr(14.4 mg, 0.1 mmol), MPEG-Br macroinitiator (213.5 mg, 0.1 mmol)and CuBr2 (1.1 mg, 0.005 mmol). The flask was degassed usingthree vacuum–nitrogen cycles. The liquid materials, PMDETAligand (17.0 mg, 0.1 mmol), DMDMA monomer (1.28 g, 7.0 mmol)and toluene (1.0 mL), which were degassed by bubbling withnitrogen for 20 min prior to use, were introduced into the reactionflask using syringes under nitrogen atmosphere. The reactionsystem was further degassed using three freeze–pump–thawcycles, and then immersed in an oil bath at 60 ◦C underthermostat control. After a predetermined polymerization time,the cooled reaction solution was diluted with THF (10 mL) andpassed through a neutral alumina column to remove the catalyst.The concentrated reaction solution was added dropwise intoa mixed solvent of hexane/diethyl ether (5/1 v/v) to obtainviscous polymer. The polymer was dried in vacuum at ambienttemperature for 4 h. 1H NMR (400 MHz, CDCl3), δ (ppm) for thePDMDMA hydrophobic block: 4.26–4.27 (m, nH, n(CH2CHCH2)),4.04–4.09 (m, n(2 + 1)H, n(COOCH2CHCH2)), 3.69–3.73 (m, nH,n(COOCH2CHCH2)), 2.28–2.39 (m, nH, CH on backbone), 1.51–1.93(m, 2nH, CH2 on backbone), 1.25–1.36 (m, 6nH, (CH3)2C); for theMPEG hydrophilic block: 3.54–3.64 (m, 4nH, n(OCH2CH2)), 3.36(s, 3H, CH3OCH2CH2), 1.12 (d, 3H, COCHCH3). 13C NMR (100 MHz,CDCl3), δ (ppm): 174.0, 109.6, 73.2, 71.9, 70.5, 69.0, 66.5, 65.0, 59.0,41.2, 35.6, 26.8, 25.3.

Preparation and characterization of polymeric micellesCritical micelle concentration (CMC) determination by fluorescencespectroscopyThe CMC of the amphiphilic block copolymer in phosphate buffersolution (PBS) was determined using a steady-state fluorescencespectrometer with pyrene as a fluorescent probe. A stock solutionof the copolymer was prepared in 0.1 mol L−1 PBS (pH = 7.4)by direct dissolution with vigorous stirring for 48 h. A knownamount of pyrene in CH2Cl2 was added to a series of vialsand CH2Cl2 was evaporated. To each vial was then added ameasured amount of stock solution, followed by the addition of0.1 mol L−1 PBS (pH = 7.4) affording solution concentrations from5.0 × 10−4 to 1.0 mg mL−1. The resulting solutions were stirred atroom temperature overnight to equilibrate the pyrene with themicelles. The final concentration of pyrene in each vial was kept at1.73 × 10−6 mol L−1.

The excitation spectra were scanned from 300 to 360 nm at anemission wavelength of 395 nm. The slit width for both excitationand emission was maintained at 2.5 nm. The intensity ratios atwavelengths of 338 and 333 nm (I338/I333) from the excitation

spectra were analyzed as a function of the logarithm of polymerconcentration.

Micelle formation and turbidity measurements

The preparation of polymeric micelles and turbidity measurementswere carried out according to published procedures.34 Deionizedwater was added at a rate of 5 µL min−1 to a polymer solution ofdioxane (1.0 wt%, 2 mL) under slight shaking. After each additionof water, the solution was left to equilibrate for a while until theoptical density was stable. The optical intensity (turbidity) wasmeasured at a wavelength of 600 nm using a quartz cell (pathlength of 1 cm) with a Hitachi U-3010 UV-visible spectrometer. Thecycle of water addition, equilibration and turbidity measurementwas continued until the desired amount of water was achieved(50–70 wt%). The solution was then dialyzed against distilledwater (500 mL) for 2 days during which the water was refreshedevery 4 h.

Micelle characterizationDynamic light scattering (DLS) measurements of polymer mi-celles were performed using a Zetaplus zeta potential analyzer(Brookhaven Instrument) equipped with ZetaPlus particle sizingsoftware and using a 35 mW solid-state laser operated at a laserlight wavelength of 660 nm. DLS measurements were carried outat 25 ◦C at a scattering angle of 90◦. All samples were filteredthrough Millipore membranes with pore sizes of 0.45 µm prior tomeasurement.

The polymeric micelles were imaged using a Hitachi H800transmission electron microscopy (TEM) instrument (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 100 kV. Forsample preparation, a drop of filtered sample solution was droppedon a carbon-coated copper grid, excess solution was wicked awaywith filter paper and the sample was left to dry in air.

Encapsulation of pyrene and pH-dependent pyrene fluorescencestudiesPyrene was loaded into the polymeric micelles by the addi-tion of deionized water (4 mL) via a syringe pump at a rateof 20 µL min−1 to dioxane solution (5 mL) containing pyrene(1.2 mg) and polymer (50 mg) with stirring at ambient tem-perature. For complete removal of dioxane and free pyrene,the pyrene-encapsulated micelle solution was transferred todialysis membranes (molecular weight cutoff of 3500) and di-alyzed against distilled water (500 mL) for 2 days. The dialyzedpyrene-loaded micelle solutions were filtered through a 0.45 µmfilter, and then divided into three 2 mL samples. The sampleswere adjusted to pH = 1.0 and 3.0 by the addition of variousvolumes of concentrated hydrochloric acid. All samples wereplaced in a laboratory shaker at 37 ◦C. At predetermined timepoints, 1 mL solution of each aliquot was taken for fluorescencemeasurements.

For measurement of the fluorescence intensity ratio of I380

to I384 at the moment of complete release of pyrene, 0.2 mLof concentrated hydrochloric acid was added to each aliquotafter 200 h hydrolysis detection. The percentage of releasedpyrene was expressed by the ratio of I380/I384 at determinedtime points to that at the moment of complete release ofpyrene.

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OO

O

O

n

Br

OO

O

O

m

pH-responsivefunctional group

OO

OHn2-bromopropionyl bromide

Et3N, CH2Cl2, 48h OO

O

O

Brn

OO+

OO

DMDMA

CuBr/PMDETA

toluene

MPEG-Br

MPEG-block-PDMDMA

Scheme 1. Synthesis of MPEG-block-PDMDMA amphiphilic copolymers by ATRP.

Table 1. Molecular characteristics of MPEG-block-PDMDMA amphiphilic block copolymers

Block copolymeraMolar ratio of

macroinititor and monomer Temp. (◦C) Time (h) Mn,NMRb (g mol−1) Mn

c (g mol−1) Mw/Mnc

MPEG-block-PDMDMA (44/18) 1/80 60 14 5500 4700 1.17



MPEG-block-PDMDMA (44/65) 1/80 80 14 14 200 10 600 1.29

a The numbers in parentheses indicate the number of repeat units of each block. The number of repeat units of the hydrophobic block was determinedby 1H NMR analysis.b The number-average molecular weights of the block copolymers were calculated by 1H NMR analysis.c Determined using GPC.

RESULTS AND DISCUSSIONSynthesis and characterization of MPEG-block-PDMDMAThe synthesis of MPEG-block-PDMDMA block copolymer isoutlined in Scheme 1. MPEG-Br as macroinitiator was pre-pared by reacting monomethoxy-poly(ethylene glycol) with2-bromopropionyl bromide. Subsequently, a series of amphiphilicblock copolymers bearing hydrophobic blocks of various lengthswere fabricated via ATRP of DMDMA by adjusting the feed ratioof monomer to macroinitiator and polymerization conditions. Thepolymerization results are summarized in Table 1.

The chemical structures of MPEG-block-PDMDMA were con-firmed from the 1H NMR spectra; one typical 1H NMR spectrum ofMPEG-block-PDMDMA (44/18), (44 and 18 stand for the repeatingunit number of EG and DMDMA respectively) is shown in Fig. 1. Forthe MPEG block, the signals of the methylene protons (b) of therepeat units and methyl protons (a) at the end of the chain appearat 3.66 and 3.42 ppm, respectively. The characteristic signals ofthe PDMDMA block at 4.29, 4.09 and 3.78 ppm are assigned to theprotons of oxymethine (g), ester methylene (f) and oxymethylene(h), and the two single peaks at 1.35 and 1.42 ppm are assignedto the two methyl protons (i). The signal at 2.36 ppm (d) and thethree signals at 1.55, 1.70 and 1.96 ppm (e) corresponding to themethine and methylene protons of the PDMDMA backbone arealso observed.

The number-average molecular weight Mn,NMR and the numberof repeat units of the PDMDMA hydrophobic block were calculatedin accordance with Eqns (1) and (3), respectively; the results aresummarized in Table 1:

Mn,NMR = DPMPEG × 44 + DPDMDMA × 186 + 134 (1)

8 7 6 5 4 3 2 1 0

ee

OO

O

O

n

Br

OO

O

O

m

a

bc

de

f

ghi

i

Chemical Shift (ppm)

CDCl3

TMS

a

b

e

f, h

hg

i

d

c

Figure 1. 1H NMR spectrum of MPEG-block-PDMDMA (44/18) copolymer.

where

DPMPEG = Mn,MPEG

44(2)

DPDMDMA = 3 × I4.29

I3.42(3)

in which I4.29 and I3.42 are the proton integral areas for the signalsoccurring at δ = 4.29 and 3.42 ppm, 44 and 186 are the molar


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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Elution time (min)

a b

c

d

e

Figure 2. GPC traces of copolymers and MPEG-Br precursor:(a) MPEG-block-DMDMDA (44/65); (b) MPEG-block-DMDMDA (44/40); (c) MPEG-block-DMDMDA (44/25); (d) MPEG-block-DMDMDA (44/18);(e) MPEG-Br.

weights of the repeat units of the MPEG and PDMDMA blocksand 134 is the molar weight of the linkage moiety (–COCHBrCH3)between hydrophilic and hydrophobic blocks.

GPC traces of polymers obtained under various polymerizationconditions were measured. As shown in Fig. 2, the unimodalGPC traces of the resultant copolymers are shifted towardshigh molecular weight with increasing molecular weight ofDMDMA segments. No MPEG-Br macroinitiator and no PDMDMAhomopolymer are detected in the GPC traces. As shown inTable 1, the molecular weight distributions of the copolymersare in the range 1.10–1.29. The evidence of NMR spectraand GPC measurements substantiates the MPEG-block-PDMDMAdiblock structure, and not a physical mixture of MPEG andPDMDMA.

Characterization and morphology of polymeric micellesCMC determination and partitioning of pyrene in micellesThe amphiphilicity of the MPEG-block-PDMDMA copolymerprovides one good possibility for the copolymer to form assembledaggregates in aqueous solution. In this study, the CMC ofMPEG-block-PDMDMA in aqueous solution was characterizedusing the fluorescence technique with pyrene as a probe. Thismethod is based on the sensitivity of the pyrene probe to thehydrophobicity and polarity of its microenvironment.35 – 38 Thecharacteristic shift of the pyrene excitation spectral peak from333 to 338 nm following pyrene partition into the hydrophobiccore of micelles was utilized to determine the CMC of the blockcopolymers in aqueous solution. As an example, the excitationspectra of pyrene for various aqueous concentrations of theMPEG-block-PDMDMA (44/18) copolymer are shown in Fig. 3(a).With an increase in copolymer concentration, a red shift inthe absorption from 333 to 338 nm is observed. Figure 3(b)shows the intensity ratio of I338/I333 of the pyrene excitationspectra as a function of the logarithm of the copolymerconcentration for MPEG-block-PDMDMA (44/18). A negligiblechange in I338/I333 is detected at low concentration, while theintensity ratio substantially increases with increasing copolymerconcentration at higher concentrations. This indicates that the

300 310 320 330 340 350 360

0

500

1000

1500

2000

2500

3000

3500

Inte

nsity

Wavelength (nm)

IncreasingConcentration

(a)

-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.00.8

1.0

1.2

1.4

1.6

1.8

I 338

/I33

3

Log C (mg mL-1)

9.8 mg L–1

(b)

Figure 3. (a) Steady-state fluorescence excitation spectra monitored at390 nm for the pyrene probe in an aqueous solution of MPEG-block-PDMDMA (44/18) block copolymer at various concentrations at 25 ◦C.(b) Plot of I338/I333 of pyrene excitation spectra in water as a function ofthe concentration of MPEG-block-PDMDMA (44/18) at 25 ◦C.

pyrene molecules incorporate into the hydrophobic core regionof the micelles during assembled aggregation. The I338/I333 versuslog C plot presents a sigmoid curve; therefore, the CMC value wasdetermined from the crossover point in the low concentrationrange as shown in Fig. 3(b), with the results being summarizedin Table 2. Similar measurements were performed for the otherthree copolymers, and the corresponding plots are shown in Fig.S1 of the supporting information. The CMC values from 4.0 to10.0 mg L−1 for these copolymers are comparable to those ofsome polymeric amphiphiles previously reported.39,40 The effectof longer PDMDMA hydrophobic blocks is evident in the lowerCMC value, which indicates a strong tendency for the copolymersbearing longer hydrophobic blocks to be prone to formation ofmicelles in aqueous solution.

These micelles can be used to encapsulate hydrophobicmolecules such as drugs to improve their aqueous solubility,the encapsulation efficiency being affected by the hydrophobicityof the micelle core. The hydrophobicity of the PDMDMA micellarcore can be estimated by determining the partition equilibriumcoefficient, Kv, of pyrene in aqueous solutions of MPEG-block-PDMDMDA diblock copolymer. The calculation of the partitionequilibrium coefficient is based on the assumption of a simpleequilibrium distribution for pyrene between the micellar phase andthe water phase. The Kv values are calculated using a formulation


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Table 2. Solution properties of micelles formed from MPEG-block-PDMDMA copolymers

Block copolymerCMCa

(mg L−1)db

(nm) PDIcKv

(×10−5)

MPEG-block-PDMDMA (44/18) 9.8 80 0.35 1.61




a Measured at 25 ◦C.b Average dynamic dimension of the copolymer micelles determinedby DLS at 25 ◦C.c Polydispersity of particle dimension measured by DLS.

as previously described,36 which is expressed as

F − Fmin

Fmax − F= Kvχ (c − CMC)

1000ρ

where Fmin and Fmax are the average magnitude of the intensityratio (I338/I333) in the constant region in the low and highconcentration ranges, respectively, F is the intensity ratio in theintermediate concentration range of the copolymers, χ is theweight fraction of the PDMDMA block in the copolymer, c is theconcentration of the copolymer in aqueous solution, ρ is thedensity of the PDMDMA core in the micelles, which is assumedto be that of poly(2-hydroxylethyl acrylate) (ρ = 1.106 g cm−3) inthis study, and Kv is partition equilibrium coefficient of pyrene incopolymer aqueous solution.

The Kv values for pyrene were obtained by plotting a graphof (F − Fmin)/(Fmax − F) versus the concentration of MPEG-block-PDMDMA, as shown in Fig. S2 of the supporting information.The Kv values are summarized in Table 2. Kv values rangingfrom 1.61 × 105 to 4.86 × 105 indicate that these copolymerscan potentially be loaded with a hydrophobic drug with highencapsulation efficiency.41 As the hydrophobic block lengthincreases, Kv values increase, suggesting the hydrophobicitiesof the polymeric micellar core are enhanced.

Turbidity measurements and micelle morphological analysisThe turbidity of the copolymers was measured using a UV-visible spectrometer when water was added progressively todioxane solution of the diblock copolymers. As shown in Fig. 4,the turbidity value reaches a plateau after one or two jumpsupon addition of water, which is associated with the formationof polymeric micelles. For MPEG-block-PDMDMA (44/18) andMPEG-block-PDMDMA (44/25), one jump in the turbidity curvesis observed with the addition of deionized water, while forMPEG-block-PDMDMA (44/40) and MPEG-block-PDMDMA (44/65)bearing longer hydrophobic blocks, the appearance of two jumpsin the turbidity curves probably indicates the presence of variousassembly morphologies prior to the final stable morphology.42

The turbid mixtures at the end of the measurements were dialyzedagainst deionized water to remove dioxane, and the morphologyand size distributions of the copolymer micelles were investigatedusing TEM and DLS, respectively.

The average copolymer micelle sizes determined from DLS arepresented in Table 2. One typical DLS plot of the MPEG-block-PDMDMA (44/65) copolymer is shown in Fig. 5, and the othersare shown in Fig. S3 of the supporting information. The polymer

0 10 20 30 40 500.0

0.5

1.0

1.5

2.0

2.5

3.0

MPEG-block-PDMDMA (44/18)



OD

(T

urbi

dity

)

Water wt%


Figure 4. Turbidity curves of MPEG-block-PDMDMA copolymers in 2 mLof dioxane at a concentration of 1.0 wt% as a function of the amount ofdeionized water added to the polymer solution. The turbidity value forsmall amounts of added water is close to zero for all samples. The curves ofMPEG-block-PDMDMA (44/25), (44/40) and (44/65) are shifted for clarity.

0 100 200 300 400 500 600 7000

20

40

60

80

100

Rel

.Int

.

Diameter (nm)

Figure 5. DLS plot of micelle size distribution of MPEG-block-PDMDMA(44/65) block copolymer.

micelles have characteristic bimodal distributions with a smallersize component below 100 nm and a larger size component ofseveral hundred nanometers. Their overall effective hydrodynamicdiameters range from 80 to 400 nm, and the polydispersity arebetween 0.11 and 0.35. Micelles with size range of several hundrednanometers are probably due to the intermicellar aggregationof the amphiphilic block copolymers, which indicates most ofthe MPEG-block-PDMDMA copolymers form a multi-core micellestructure, rather than a simple core–shell structure.43,44

The morphology of the copolymer micelles was furtherexamined using TEM. A representative image of the micelle-forming MPEG-block-PDMDMA (44/65) is shown in Fig. 6. Themicelles are close to spherical and have a mean number-averagediameter of 350 nm. The diameters of the micelles determinedfrom the TEM data are less than the effective diameters measuredby DLS, probably because DLS determines hydrodynamic diameter


97

3


Figure 6. TEM image of micelle-forming MPEG-block-PDMDMA (44/65) block copolymer. The scale bar is 500 nm.

of the polymeric micelles in solution, while TEM analysis involvesthe micelle morphology in a dry state.

Encapsulation and pH-dependent release of pyreneThe ability of the micelles to release encapsulated pyrene from theirhydrophobic compartment in response to ketal removal at acidicpH was monitored using fluorescence spectroscopy. As shown inFig. 7, pyrene release from the copolymer micelles is both time-and pH-dependent. The percentage of pyrene released increasesgradually with incubation time and reaches a plateau at pH = 1.0and 3.0. For these micelles, release of 50% of the encapsulatedpyrene is achieved at pH = 1.0 in 20 h. In the case of pH = 3.0, thepyrene release rate from the micelles depends on the hydrophobicblock length of the copolymers. For MPEG-block-PDMDMA (44/18)and MPEG-block-PDMDMA (44/25), pyrene release is triggeredafter about 80 h incubation, and nearly 70 and 25% of pyrenerelease is completed in 260 h, respectively. The phenomenon ofrelease starting after a period of incubation indicates that themicelles likely remain stable until a critical number of hydrophobicgroups have been lost.26 In contrast, for MPEG-block-PDMDMA(44/40) and MPEG-block-PDMDMA (44/65) at pH = 3.0, no pyrenerelease is detected during 260 h incubation, possibly becausethese copolymers with longer hydrophobic blocks need longerincubation time to reach the critical number of hydrophobicgroups to destroy the multi-core structure. As a control, thefluorescence of the samples at pH = 7.4 remains constant over aperiod of 320 h.

In order to confirm that pyrene-loaded release is triggeredby ketal removal, and not by hydrolysis of the ester bondin the DMDMA units under acidic conditions, concentratedhydrochloride acid was added to a polymer solution of THF/PBS(0.1 mol L−1; 1/1 v/v) maintaining the pH value of the solutionaround 1.0, followed by stirring for 14 h at ambient temperature,and the resultant polymer was investigated using 1H NMR analysis.As shown in Fig. 8, besides the presence of the characteristicsignals of the MPEG moiety at 3.66 ppm (b), the characteristicsignals after ketal deprotection of the PDMDMA block appear at

0 10 20 30 40

0

20

40

60

80

100 MPEG-block-PDMDMA (44/18)MPEG-block-PDMDMA (44/25)MPEG-block-PDMDMA (44/40)MPEG-block-PDMDMA (44/65)

% I

nitia

l Flu

ores

cenc

e

Time (h)

(a)

0 20 40 60 80 100 120 140 160 180 200 220 240 260

0

20

40

60

80

100

120

MPEG-block-PDMDMA (44/18)MPEG-block-PDMDMA (44/25)MPEG-block-PDMDMA (44/40)MPEG-block-PDMDMA (44/65)

% I

nitia

l Flu

ores

cenc

e

Time (h)

(b)

Figure 7. Time dependence of pyrene release from micelles of MPEG-block-PDMDMA at (a) pH = 1.0 and (b) pH = 3.0.

2.44 ppm (e) and 1.5–1.9 ppm (d) corresponding to the methineand methylene protons in the backbone. The methine protonsignal (g) shifts from 4.29 to 4.16 ppm, overlapping with theester methylene protons (f), and the characteristic signals ofthe two methyl protons in the 1,3-dioxolane ring of the originalPDMDMA block at 1.35 and 1.42 ppm almost disappear. Thisevidence suggests that the transition from amphiphiles to doublehydrophiles for the studied block copolymers is performed byketal removal from the PDMDMA hydrophobic block under acidicconditions. The ketal deprotection of the PDMDMA hydrophobicblock leading to the elimination of the driving force for themicellar aggregates triggers the escape of encapsulated pyrenefrom the micelle core. Acetone as hydrolyzate, a compound onthe FDA GRAS list, is benign in the physiological environment.Encouraged by these promising results, further studies exploringmore polymeric micelles made pH-sensitive by adjusting theirmolecular architecture are underway in our laboratory.

CONCLUSIONSA series of amphiphilic block copolymers with hydrophobicblocks bearing pH-sensitive ketal groups were fabricated by ATRP


97

4


8 6 5 4 3 2 0 -1

OO

O

O

n

Br

OO

HO

HO

m

a

bc

d

e

f

gh

Chemical Shift

H2O

CD3ODb

a

ce df, gh

7 1

Figure 8. 1H NMR spectrum of double hydrophilic polymer fabricated viaMPEG-block-PDMDMA (44/40) copolymer after ketal removal.

with MPEG-Br as macroinitiator under various polymerizationconditions. The CMC values of the copolymers in aqueoussolution range from 4.0 to 10.0 mg L−1, and the hydrophobicityof the micellar cores was estimated by measuring the partitionequilibrium constant (Kv) of pyrene in micellar solutions of thecopolymers. The values of Kv (1.61 × 105 to 4.86 × 105) indicatethe potential high encapsulation efficiency of hydrophobicdrugs. The micelle formation process was monitored using UV-visible spectroscopy, the average micellar dimensions in aqueoussolution characterized by DLS were from 80 to 400 nm and themicellar morphology was spherical as observed with TEM. ThepH-responsive release of molecules from the block copolymermicelles was studied at pH = 1.0 and 3.0. Ketal deprotectionfrom the PDMDMA block of the copolymers triggered release ofencapsulated molecules.

ACKNOWLEDGEMENTSThis work was supported by the National Natural ScienceFoundation of China (NSFC, grant no. 20804003) and startinggrants of Beijing University of Chemical and Technology.

SUPPORTING INFORMATIONSupporting information may be found in the online version of thisarticle.

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Synthesis and self-assembly behavior of pH-responsive amphiphilic copolymers containing ketal functional groups