Journal of Controlled Release 210 (2015) 125–133

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Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre l

Facile construction of dual-bioresponsive biodegradable micelles withsuperior extracellular stability and activated intracellular drug release

Wei Chen a,b, Fenghua Meng a, Ru Cheng a, Chao Deng a, Jan Feijen a,b,⁎, Zhiyuan Zhong a,⁎⁎a Biomedical Polymers Laboratory, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry,Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, PR Chinab Department of Polymer Chemistry and Biomaterials, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, P.O. Box 217,7500 AE Enschede, The Netherlands

⁎ Correspondence to:W. Chen, Biomedical Polymers Labof Advanced Functional Polymer Design and Application,and Engineering, College of Chemistry, Chemical EnginSoochow University, Suzhou 215123, PR China.⁎⁎ Corresponding author.

E-mail addresses: j.feijen@utwente.nl (J. Feijen), zyzho

http://dx.doi.org/10.1016/j.jconrel.2015.05.2730168-3659/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 February 2015Received in revised form 13 May 2015Accepted 13 May 2015Available online 15 May 2015

Keywords:pH-sensitiveBiodegradable micellesReversible crosslinkingAnticancer drugsControlled release

It is still a major challenge for targeted cancer chemotherapy to design stable biodegradable micellar drugdelivery systems which show a rapid and complete intracellular drug release. Here, reversibly core-crosslinked pH-responsive biodegradable micelles were developed based on poly(ethylene glycol)-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate-co-pyridyl disulfide carbonate) [PEG-P(TMBPEC-co-PDSC)]copolymers and investigated for intracellular doxorubicin (DOX) release. PEG-P(TMBPEC-co-PDSC) copolymersformedmicelleswith a small size of 58.6 nmwere readily crosslinked by the addition of dithiothreitol (DTT). No-tably, in vitro release studies showed that under physiological conditions only ca. 19.9% ofDOXwas released fromthe reversibly crosslinked micelles in 24 h at a low micelle concentration of 40 μg/mL. The release of DOX wasaccelerated at pH 5.0 or in the presence of 10 mM glutathione (GSH) at pH 7.4, in which 64.2% and 44.1% ofDOX was released, respectively, in 24 h. The drug release was further boosted at pH 5.0 and 10 mM GSH, with98.8% of DOX released in 12 h. Moreover, DOX release was also facilitated by a 4 h incubation at pH 5.0 followedby incubation at pH 7.4 with 10 mM GSH. Confocal microscopy indicated that DOX was delivered and releasedinto the nuclei of RAW 264.7 cells following a 12 h incubation with DOX-loaded reversibly crosslinked micelles.MTT assays revealed that DOX-loaded reversibly crosslinked micelles had much higher antitumor activity thanirreversibly crosslinked controls, with low IC50 values of 1.65 and 1.14 μg/mL for HeLa and RAW 264.7 cells, re-spectively, following a 48 h incubation. The blank crosslinked micelles had a low cytotoxicity of up to a concen-tration of 0.8 mg/mL. These reversibly crosslinked pH-sensitive biodegradable micelles with superiorextracellular stability but activated intracellular drug release provide a novel platform for tumor-targetingdrug delivery.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Biodegradable polymeric micelles have been explored as one of themost promising drug delivery systems (DDSs) for targeted cancer che-motherapy, since they have overcome several problems that are associ-ated with traditional hydrophobic anticancer drugs, such as low watersolubility, nonspecific distribution and inefficient bioavailability in thebody [1,2,3,4,5,6]. Furthermore, as drug carriers, polymeric micellesshould be stable at low concentrations therefore achieving long in vivocirculation times and inhibiting premature drug release, and shouldhave specific tumor targetability as well as fast and maximum drug

oratory, Jiangsu Key LaboratoryDepartment of Polymer Scienceeering and Materials Science,

ng@suda.edu.cn (Z. Zhong).

release inside the target tumor cells [7,8,9,10]. The therapeutic efficacyof micellar drug-formulations would be drastically improved if thesepoints could successfully be met.

In recent years, stimulus-responsive nanosystems that releasepayloads in response to an intrinsic biological signal have been de-signed and explored for enhanced cancer therapy [11,12,13,14]. Forexample, pH-sensitive polymers containing acid-labile groups suchas ortho ester, hydrazone, cis-aconityl, and acetal have been widelystudied for drug delivery applications, since the pH in the environmentof tumor tissue is often 0.5–1.0 pH units lower than in normal tissue,whereas the pH in the intracellular endosomal/lysosomal compart-ments is as low as 4.0–6.5 [15,16]. Fréchet et al. reported thattrimethoxybenzylidene acetals are rapidly hydrolysed at slightly acidicpH and that these entities can be incorporated in drug delivery systemsfor pH-triggered drug release [17,18]. We have designed poly(ethyleneglycol)-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate)(PEG-PTMBPEC) block copolymers and prepared pH-responsive biode-gradable micelles and polymersomes [19,20]. As compared to the

126 W. Chen et al. / Journal of Controlled Release 210 (2015) 125–133

traditional aliphatic polyesters such as poly(ε-caprolactone) (PCL),polylactide (PLA), and lactide-glycolide copolymers (PLGA), PTMBPECcan be applied in nano-systems to induce fast swelling or evendissociation by hydrolysis of the acetal pendants at mildly acidicconditions. Furthermore, it has been demonstrated that hydroxypolycarbonates derived from pentaerythritol are prone to rapid degra-dation in vitro due to their highly hydrophilic nature [21]. Recently,we introduced disulfide bonds (S–S) into PEG-PTMBPEC copolymers(PEG-SS-PTMBPEC) to develop pH and reduction-sensitive micelles,which exhibit dually activated intracellular release behavior [22]. Wealso prepared pH-sensitive degradable chimeric polymersomes basedon trimethoxybenzylidene acetals for high loading and triggered re-lease of doxorubicin hydrochloride [23]. It should be noted, however,that these self-assembled polymeric nanocarriers, are often plaguedby inadequate in vivo stability, which leads to premature burstdrug release following i.v. injection, resulting in drug loss not onlyduring storage, but also in the blood circulation causing increasedside effects [24,25].

To improve the stability and to inhibit premature drug release, eitherthe micellar core or the shell could be crosslinked. It has been demon-strated that crosslinked biodegradable micelles have advantageousproperties like high drug loading efficiency, superior stability upon dilu-tion, prolonged circulation time, and enhanced drug accumulation atthe tumor site [26,27,28]. Very recently, we prepared photo-crosslinked pH-sensitive biodegradable micelles based on poly(eth-ylene glycol)-poly(2,4,6-trimethoxybenzylidene-pentaerythritolcarbonate-co-acryloyl carbonate) [PEG-P(TMBPEC-co-AC)] copoly-mers, which had superior extracellular stability by UV crosslinkingof AC units, and fast intracellular drug release through the acid-labile TMBPEC components [29]. These photo-crosslinked micellesprovided with galactose units enhanced drug accumulation in thehuman hepatoma SMMC-7721 tumor and exerted more efficient an-titumor activity, as compared to non-crosslinked micelles as well asnon-targeted micelles [30]. It should be noted; however, that irrevers-ible photo-crosslinkingmay induce incomplete drug release, and gener-ate non-degradable polyacrylate components. In recent years, muchattention has been given to the development of reversibly crosslinkedpolymeric micelles in that complete drug release can be obtained by

Scheme 1. Preparation of and activated intracellular drug release fr

de-crosslinking of the micelles [9,31]. In particular, crosslinking via di-sulfide bonds, which are prone to cleavage in the intracellular environ-ment due to the presence of a high reduction potential in the cytoplasmas well as in the cell nucleus, is an attractive approach to construct re-versibly crosslinked nanocarriers for triggered drug release [32,33]. Asubstantial number of reversibly crosslinked micelle systems using di-sulfide bonds have been prepared using cystamine and its derivativesas the reducible crosslinkers [34,35,36,37,38]. The reduction-sensitivereversibly crosslinked nanosystems can also be produced by oxidizingfree thiol groups in the shell- or core-forming blocks [39,40]. We havedeveloped disulfide-crosslinked micelles by conjugation of lipoic acidwhich can be self-crosslinked by ring-opening with a catalytic amountof DTT [41,42,43]. The self-crosslinking of pyridyl disulfide (PDS) unitsin polymers has emerged as another promising approach to develop re-versibly crosslinked nanosystems. The PDS unit is highly reactive, butspecific to thiols and provides disulfide-crosslinked structures by thethiol-disulfide exchange reaction under mild conditions [44,45,46,47].For instance, Thayumanavan et al. developed reduction-sensitive self-crosslinked nanogels based on a random copolymer containing pendantoligoethyleneglycol and PDS side chains. The disulfide-crosslinkednanogels are of particular interest for intracellular protein release inthat they on one hand possess superior colloidal stability and on theother hand are prone to rapid de-crosslinking and dissociation insidecells [48,49].

In this study, we report on the facile construction of reversiblycrosslinked pH-responsive micelles from poly(ethylene glycol)-b-poly(mono-2,4,6-trimethoxy benzylidene-pentaerythritol carbonate-co-pyridyl disulfide carbonate) [PEG-P(TMBPEC-co-PDSC)] block copol-ymer, in which the core-forming TMBPEC units are acid-labile and PDSfunctionalities allow formation of reduction-sensitive crosslinks(Scheme1). PEG-P(TMBPEC-co-PDSC)micelleswere designed to be sta-bilized by disulfide crosslinking with minimal drug leakage during cir-culation while quickly and completely releasing payloads upon arrivalinside the tumor cells. The synthesis, stability, in vitro drug release andtumor cell killing activity of DOX-loaded reversibly crosslinked PEG-(PTMBPEC-co-PDSC) micellar nanoparticles were investigated and theresults were compared with those obtained using DOX-loaded irrevers-ibly crosslinked PEG-P(TMBPEC-co-AC) micelles.

om reversibly crosslinked pH-sensitive biodegradable micelles.

127W. Chen et al. / Journal of Controlled Release 210 (2015) 125–133

2. Experimental section

2.1. Materials

Glutathione (GSH, 99%, Roche), zinc bis[bis(trimethylsilyl)amide](97%, Aldrich), doxorubicin hydrochloride (DOX·HCl, 99%, BeijingZhong Shuo Pharmaceutical Technology Development Co. Ltd.),1,4-dithio-D,L-threitol (DTT, 99%, Merck), and potassium persulfate(KPS, 99.5%, Aladdin) were used as received. Methoxy poly(ethyl-ene glycol) (PEG, Mn = 5.0 kg/mol) was purchased from Fluka anddried by azeotropic distillation from anhydrous toluene. Dichloro-methane (DCM) was dried by refluxing over CaH2 under an argonatmosphere. Mono-2,4,6-trimethoxybenzylidene-pentaerythritolcarbonate (TMBPEC), pyridyl disulfide cyclic carbonate (PDSC),and PEG-P(TMBPEC-co-AC) copolymer were synthesized accordingto our previous reports [19,29,50].

2.2. Synthesis of PEG-P(TMBPEC-co-PDSC) and PEG-P(TMBPEC-co-AC)copolymer

The ring-opening copolymerization of TMBPEC and PDSC wascarried out in DCM at 50 °C, using methoxy PEG as an initiator andzinc bis[bis(trimethylsilyl)amide] as a catalyst. Briefly, in the glove-box under a nitrogen atmosphere, to a stirred solution of PEG(250 mg, 50 μmol), TMBPEC (200 mg, 0.588 mmol) and PDSC(200 mg, 0.738 mmol) in DCM (3.0 mL) were quickly added withzinc bis[bis(trimethylsilyl)amide] (10 mg, 26 μmol). The reactionvessel was sealed and placed in an oil-bath thermostated at 50 °C.The polymerization was allowed to proceed with magnetic stirringfor 4 days. The resulting polymer was isolated twice by precipitationfrom cold diethyl ether and dried at r.t. in vacuo. Yield: 78.2%. 1HNMR (400 MHz, CDCl3): PEG: δ 3.38, 3.64; TMBPEC moieties: δ3.82,3.99, 4.75, 5.97, 6.08; PDSC moieties: δ 1.08, 2.92, 4.09, 7.64, 7.07,8.45. Mn (1H NMR) = 10.9 kg/mol, Mn (GPC) = 10.2 kg/mol,PDI = 1.34. In a similar way, PEG-P(TMBPEC-co-AC) copolymerwas prepared to be used for the preparation of the irreversiblycrosslinked micelles. The Mn of the P(TMBPEC-co-AC) block wasevaluated to be 5.0 kg/mol with a molar ratio of TMBPEC and AC ap-proaching 1.25 as determined by 1H NMR.

2.3. Characterization

1H NMR spectra were recorded on a Unity Inova 400 spectrometeroperating at 400MHzusing deuterated chloroform (CDCl3) as a solvent.The chemical shifts were calibrated against residual solvent signals ofCDCl3. The average molecular weight and polydispersity of the copoly-mers were determined by a Waters 1515 gel permeation chromato-graph (GPC) instrument equipped with two linear PLgel columns(500 Ǻ and Mixed-C) following a guard column and a differentialrefractive-index detector. The measurements were performed using

PEG-OH O

O O

O

OMe

O

MeO OMe

TMBPEC

O O

O

+OMeO H +

SS

N

MeO

n

PDSC

CH2Cl2

Scheme 2. Synthesis of PEG-P(TMBPEC-co-PDSC) block copolymer by ring-opening copolymbis[bis(trimethylsilyl)amide] as a catalyst in CH2Cl2 at 50 °C.

CHCl3 as the eluent at a flow rate of 0.5 mL/min at 30 °C and a seriesof narrow polystyrene standards for the calibration of the columns.

2.4. Preparation of crosslinked micelles

Reversibly crosslinked PEG-P(TMBPEC-co-PDSC) micelles (RCM)were prepared by the solvent exchange method. Typically, understirring 2.0 mL of phosphate buffer (PB, 10 mM, pH 7.4) was addeddropwise into 0.20 mL of block copolymer solution in DMF(0.5 wt.%) at room temperature. The resulting suspension was dia-lyzed against PB (10 mM, pH 7.4) for 8 h to thoroughly removeDMF. The critical micelle concentration of PEG-P(TMBPEC-co-PDSC)was determined using pyrene as a fluorescence probe according toa previous report [22]. PEG-P(TMBPEC-co-PDSC) micelles with aconcentration of 0.50 mg/mL were readily crosslinked by the thiol-disulfide exchange reaction. In brief, to 2 mL of this micellar solution(0.50 mg/mL, 0.92 μmol of pyridyl disulfide groups) was added DTT(0.46 μmol) solution in ethanol. The mixture was perfused with ni-trogen for 10 min and stirred magnetically at 50 °C for 8 h. Thecrosslinking process was monitored by determining the amount ofpyridinethione byproduct using UV analysis [48]. PEG-P(TMBPEC-co-PDSC) micelles were isolated by dialysis against PB (10 mM,pH 7.4). Irreversibly crosslinked micelles (IRCMs) were preparedusing PEG-P(TMBPEC-co-AC) with a concentration of 0.5 mg/mLand potassium persulfate (KPS, 5 wt.% of the polymer) as a thermalinitiator at 50 °C for 8 h under N2 protection. The IRCMs were alsoisolated by dialysis against PB (10 mM, pH 7.4) and used as areduction-insensitive control. The size of the micelles was deter-mined by dynamic light scattering (DLS) at 25 °C using a ZetasizerNano-ZS (Malvern Instruments) equipped with a 633 nm He–Nelaser. Transmission electron microscopy (TEM) was performedusing a Tecnai G220 TEM operated at an accelerating voltage of200 kV. The samples were prepared by dropping 10 μL of a0.2 mg/mL suspension of the micelles on a copper grid followed bystaining with phosphotungstic acid. The stability of crosslinked mi-celles against extensive dilution and addition of THF was studiedusing DLS as described previously.

2.5. pH and GSH-triggered size change of crosslinked micelles

The change of micellar size in response to pH and/or GSH wasfollowed by DLS measurements at 37 °C. RCM and IRCM suspensions(0.50 mg/mL) were diluted to a micellar concentration of 40 μg/mLwith PB (10 mM, pH 7.4), and then divided into four aliquots of 1 mL,which were adjusted to four different conditions: i.e., (i) PB (100 mM,pH 7.4) containing 10 μM GSH, (ii) acetate buffer (100 mM, pH 5.0),(iii) PB (100mM, pH 7.4) containing 10mMGSH, and (iv) acetate buffer(100mM, pH 5.0) containing 10mMGSH. The suspensions were gentlystirred at 37 °C and micelle sizes were monitored in time by DLS.

O O

O O

OMe

x

O

n

MeO OMe

O O

O

SS

N

Oy m

PEG-P(TMBPEC-co-PDSC)

erization of TMBPEC and PDSC using PEG-OH (Mn = 5.0 kg/mol) as an initiator and zinc

9 8 7 6 5 4 3 2 1

mk,ln

ji

g

f

e

d,h

c

b

ppm

CDCl3

a

O O

O O

OMe

x

O

n

MeO OMe

OMeO

O

O

SS

N

Oy m

a

i

hh

g

g

gff

e

dd

ccb

l

k

j

nm

Fig. 1. 1H NMR spectrum (400MHz, CDCl3) of PEG-P(TMBPEC-co-PDSC) block copolymer.

128 W. Chen et al. / Journal of Controlled Release 210 (2015) 125–133

2.6. Loading and release of DOX

DOX-loaded micelles were prepared by adding dropwise 2.0 mL ofPB (10 mM, pH 7.4) into a mixture of 200 μL of copolymer solution inDMF (5.0 mg/mL) and various volumes of DOX solution in DMSO(5.0 mg/mL, theoretical drug loading content (DLC) was 5, 10, and20 wt.%, respectively) under stirring at room temperature, followed bystirring for 0.5 h and adding DTT solution (50 mol% of PDS) in ethanol.The dispersion was perfused with nitrogen for 10 min and then stirredmagnetically at 50 °C for 8 h. The dispersion was finally dialyzed exten-sively against PB (10 mM, pH 7.4) with a MWCO of 3500 at room tem-perature in the dark for another 8 h.

The in vitro release of DOX from crosslinked PEG-P(TMBPEC-co-PDSC) micelles with a low micelle concentration of 40 μg/mL (loadedwith 6 μg/mL of DOX)was investigated at 37 °C under five different con-ditions, i.e., (i) PB (100 mM, pH 7.4), (ii) acetate buffer (100 mM,pH 5.0), (iii) PB (100 mM, pH 7.4) containing 10 mM GSH, and (iv) ac-etate buffer (100mM, pH 5.0) containing 10mMGSH, and (v) 4 h in ac-etate buffer (100 mM, pH 5.0) followed by PB (100 mM, pH 7.4)containing 10mMGSH. TheDOX-loadedmicellar suspensionwas divid-ed into five aliquots and immediately transferred to a dialysis tube witha MWCO of 12,000–14,000. The dialysis tube was immersed into 20 mLof appropriate buffer (100mM) and shaken at 37 °C. At desired time in-tervals, 5.0 mL of the release medium was taken out and replenishedwith an equal volume of fresh medium. To avoid oxidation of GSH, the

0

4

8

12

16

20

1 10 100 1000Size (nm)

Reversibly crosslinked micelles

Vol

ume

(%)

50 nm

A

Fig. 2. Size distribution of reversibly crosslinked PEG-P(TMBPEC-co-PDSC) micelles (A) and ir

release media were perfused with nitrogen gas. DOX release from thethermally-crosslinked IRCMs was used as a reduction-insensitive con-trol study. The concentration of DOX was determined by fluorescence(FLS920) measurements (excitation at 480 nm, emission at 600 nm).

To determine theDLC, DOX-loadedmicelle suspensionswere freeze-dried, dissolved in DMSO and analyzed with fluorescence spectroscopy.A calibration curve was obtained using DOX/DMSO solutions with dif-ferent DOX concentrations. To determine the amount of DOX released,calibration curves were run with DOX/corresponding buffer solutionswith different DOX concentrations at pH 5.0 and 7.4, respectively. Theemission at 600 nm was recorded. The release experiments were con-ducted in triplicate. The results are presented as the average± standarddeviation. The DLC and drug loading efficiency (DLE) were calculatedaccording to the following formula:

DLC wt:%ð Þ ¼ weight of loaded drug=total weight of polymer and loaded drugð Þ�100 %

DLE %ð Þ ¼ weight of loaded drug=weight of drug in feedð Þ � 100 %:

2.7. Confocal microscopy studies

RAW 264.7 cells were plated onmicroscope slides in a 24-well plate(5 × 104 cells/well) using DMEMmedium containing 10% FBS. The cellswere incubated with prescribed amounts of DOX-loaded micelles orfree DOXat 37 °C and 5% CO2. After incubation for 6 and 12h, the culturemedium was removed and the cells on the microscope plates werewashed three times with PBS. The cells were fixed with 4% paraformal-dehyde and the cell nuclei were stained with Hoechst 33342. Fluores-cence images of cells were obtained with a Confocal Laser ScanningMicroscope (Leica, TCS-SP2).

2.8. MTT assay

The cytotoxicity of blank and DOX-loaded RCMs and IRCMs to HeLaand RAW 264.7 cells was studied using the MTT assay. Cells were seed-ed onto a 96-well plate at a density of 1 × 104 cells per well in 100 μL ofDulbecco'sModified Eaglemedium (DMEM) containing 10% FBS and in-cubated for 24 h (37 °C, 5% CO2). The medium was removed andreplenished by 90 μL of fresh DMEM medium containing 10% FBS.10 μL of micelle suspensions at different micelle concentrations in PB(10 mM, pH 7.4) was added. The cells were incubated for another48 h, the medium was aspirated and replaced by 100 μL of fresh medi-um, and 10 μL of MTT solution (5mg/mL) was added. The cells were in-cubated for another 4 h, and then 100 μL of DMSOwas added to dissolvethe resulting purple crystals. The optical densities at 570 nm were

1 10 100 10000

4

8

12

16

20

Vol

ume

(%)

Size (nm)

Irreversibly crosslinked micelles

50 nm

B

reversibly crosslinked PEG-P(TMBPEC-co-AC) micelles (B) determined by DLS and TEM.

10 100 10000

5

10

15

20

25

Vol

ume

(%)

Size (nm)

NCMs RCMs NCMs, 1000-fold dilution RCMs, 1000-fold dilution

A

1 10 100 10000

5

10

15

20

25

NCMs RCMs NCMs, 4-fold THF RCMs, 4-fold THF

Vol

ume

(%)

Size (nm)

B

Fig. 3. Stability of non-crosslinked PEG-P(TMBPEC-co-PDSC)micelles and reversibly crosslinked PEG-P(TMBPEC-co-PDSC)micelles after dilution (1000 times) (A) and addition of a 4-foldvolume of THF (B) as measured by DLS. The initial micelle concentration was 0.50 mg/mL.

129W. Chen et al. / Journal of Controlled Release 210 (2015) 125–133

measured using a BioTek microplate reader. Cells cultured in DMEMmedium containing 10% FBS (without micelles) were used as controls.

3. Results and discussion

3.1. Synthesis of amphiphilic PEG-P(TMBPEC-co-PDSC) copolymer

PEG-P(TMBPEC-co-PDSC) copolymer was prepared by ring-opening copolymerization of two cyclic carbonate monomers,TMBPEC and PDSC, using PEG (Mn = 5.0 kg/mol) as an initiatorand zinc bis[bis(trimethylsilyl)amide] as a catalyst in CH2Cl2 at 50 °C(Scheme 2). We have also developed block copolymers of PEG-

10 100 10000

4

8

12

16

20

Vol

ume

(%)

Size (nm)

0 h 4 h, pH 5.0, 10 mM GSH 20 h, pH 5.0, 10 mM GSH 30 h, pH 5.0

A

RCMs

Vol

ume

(%)

10 100 10000

4

8

12

16

20

Vol

ume

(%)

Size (nm)

0 h 30 h, pH 5.0, 10 mM GSH 30 h, pH 5.0

C

IRCMs

Fig. 4. pH and GSH-induced size change of reversibly crosslinked PEG-P(TMBPEC-co-PDSC)micC and D) in time followed by DLS. (A and C) pH 5.0; (B and D) pH 7.4. The micelle concentrati

PTMBPEC and PEG-P(TMBPEC-co-AC) using analogous methods ([19,20,29]). 1H NMR showed besides signals attributable to PEG (δ3.38,3.64) also peaks due to TMBPEC moieties (δ3.82, 3.99, 4.75, 5.97, 6.08)and PDSC moieties (δ 1.08, 2.92, 4.09, 7.64, 7.07, and 8.45) (Fig. 1).The average molecular weight (Mn) of the P(TMBPEC-co-PDSC) blockwas determined to be 5.9 kg/mol with a molar ratio of TMBPEC andPDSC close to 1, by comparing the integrals of signals at δ 5.98 (acetalprotons of TMBPECmoieties) and 2.92 (methylene protons neighboringto the disulfide bond of PDSC moieties) to 3.64 (methylene protons ofPEG), respectively. There were on average about ten PDSC units permolecule. GPC revealed that the resulting copolymer had a relativelylow polydispersity of 1.34 and an Mn of 10.8 kg/mol, close to that

10 100 10000

4

8

12

16

20

Size (nm)

0 h 4 h, pH 7.4, 10 mM GSH 30 h, pH 7.4, 10 mM GSH 30 h, pH 7.4, 10 uM GSH

B

RCMs

10 100 10000

4

8

12

16

20

0 h 30 h, pH 7.4, 10 mM GSH 30 h, pH 7.4, 10 uM GSH

Size (nm)

Vol

ume

(%)

D

IRCMs

elles (RCMs, A and B) and irreversibly crosslinked PEG-P(TMBPEC-co-AC)micelles (IRCMs,on was set as 40 μg/mL.

Table 1Characteristics of DOX-loaded non-crosslinked and reversibly crosslinked PEG-P(TMBPEC-co-PDSC) micelles.

Micelle DLC (wt %) DLE (%) Size b

(nm)PDI b

Theory Determined a

Non-crosslinked 5 3.4 66.8 62.4 ± 0.8 0.0810 6.5 62.6 90.3 ± 1.6 0.1220 12.1 55.1 167.2 ± 2.1 0.15

Crosslinked 5 3.3 64.8 63.6 ± 2.6 0.1610 6.3 60.5 77.4 ± 1.6 0.1320 12.0 54.5 84.6 ± 2.3 0.13

a Determined by UV measurement.b Determined by DLS.

130 W. Chen et al. / Journal of Controlled Release 210 (2015) 125–133

determined by 1H NMR (10.9 kg/mol), confirming the controlledsynthesis of PEG-P(TMBPEC-co-PDSC) diblock copolymer.

3.2. Micelle formation and crosslinking

DLS measurements showed that PEG-P(TMBPEC-co-PDSC) formedmicellar nanoparticles with an average diameter of 58.6 nm and a nar-row size distribution in PB (10 mM, pH 7.4), (Fig. S1A). TEM revealeda homogeneous distribution of spherical nanoparticles with sizes in ac-cordance with those determined by DLS. The critical micelle concentra-tion (CMC) of PEG-P(TMBPEC-co-PDSC) copolymers was ca. 1.46 mg/Las determined by fluorescence measurements using pyrene as aprobe. PEG-P(TMBPEC-co-PDSC) micelles were readily crosslinked bythe thiol-disulfide exchange reaction. DLS results and TEM images re-vealed that the size and PDI of PEG-P(TMBPEC-co-PDSC)micelles beforeand after crosslinking were almost the same (Figs. S1A and 2A). Theprogress of the crosslinking reaction was conveniently monitored byfollowing the release of 2-pyridothione (reaction byproduct) by UV ab-sorption at 343 nm[48]. It is assumed that formation of a single disulfidebond will require cleavage of two PDS units and produce two 2-pyridothione molecules. Considering the mechanism of the reaction,the addition of 50 mol% DTT (relative to the PDS groups) could resultin the complete formation of the disulfide bonds via cleavage of PDSunits and thiol-disulfide exchange, assuming 100% reaction efficiency.The final crosslinking efficiency was determined to be ca. 86% basedon the ratio of released amount of 2-pyridothione to the total theoreticalamount of 2-pyridothione (Fig. S1B). The PEG-P(TMBPEC-co-AC) mi-celles were crosslinked using KPS as a thermal initiator at 50 °C. DLS re-sults and TEM images showed that these irreversibly crosslinkedmicelles had an average size of 91.6 nm (Fig. 2B), which was similar tothe size of PEG-P(TMBPEC-co-AC) micelles using UV-crosslinking [29].

0 4 8 12 16 20 240

20

40

60

80

100

Cum

ulat

ive

rele

ase

(%)

Time (h)

pH 5.0, 10 mM GSH pH 5.0 pH 7.4, 10mM GSH pH 7.4A

Fig. 5. pH and/or redox-triggered release of DOX from reversibly crosslinked PEG-P(TMBPEC-cocrosslinked micelles at pH 5.0 for 4 h followed by pH 7.4 and 10 mM GSH (mimicking the intr

The colloidal stability of RCMs against extensive dilution and organicsolvent dilution was investigated using DLS measurements. Notably,RCMs following 1000-fold dilution showed only a slight increase in hy-drodynamic diameter and maintained a low PDI, while significant ag-gregation was observed for the non-crosslinked micelles (NCMs)under otherwise the same conditions (Fig. 3A). Moreover, unlikeNCMs that were completely dissociated into unimers upon addition of4-fold THF, RCMs just swelled (Fig. 3B). These results confirmed thatmi-celles were successfully crosslinked by the thiol-disulfide exchange re-action and that the colloidal stability of crosslinked micelles had beensignificantly improved. The colloidal stability of IRCMswas also remark-ably increased with respect to that of the non-crosslinked micelles(Fig. S2).

3.3. Redox and pH-induced size change of crosslinked PEG-P(TMBPEC-co-PDSC) micelles

The size change of crosslinked PEG-P(TMBPEC-co-PDSC) micelles inresponse to acidic pH and 10 mM GSH was studied by DLS. RCMsswelled from 58 nm to about 180 nm in 30 h at pH 5.0 (Fig. 4A),which was similar to that observed for IRCMs (Fig. 4C). The increasedsize was due to the increased hydrophilicity of themicellar core as a re-sult of acetal hydrolysis. Interestingly, a small increase in the size ofRCMs from 58 nm to 120 nmwas observed after incubation of thesemi-celles for 4 h at pH 7.4 in the presence of 10mMGSH due to cleavage ofthe disulfide bonds (Fig. 4B). However, no further significant changeswere observedwhen the RCMswere kept in this medium for prolongedtimes (i.e., 30 h). The results can be explained by the fact that althoughthemicelles were de-crosslinked, no acetal hydrolysis in the core of themicelles took place. Furthermore, the micelle concentration (40 μg/mL)was higher than the CMC (1.46 mg/L) of the non-crosslinked micelles.To mimic extracellular reductive conditions, the stability of RCMs wasalso assessed at pH 7.4 in the presence of 10 μMGSH. RCMs showed lit-tle size change over 30 h (Fig. 4B).Meanwhile, it was found that the sta-bility of IRCMswas not affected by the addition of 10 μMor even 10mMGSH at pH 7.4 in 30 h (Fig. 4D).

It should be noted that a pronounced size change of RCMs was ob-served at pH 5.0 in the presence of 10 mM GSH, in which large aggre-gates with a diameter over 1000 nm were formed in 20 h (Fig. 4A).Since the pK(SH) is about 9, the reduction ability of glutathione atpH 5.0 becomes weaker as compared to that at pH 7.4. Nevertheless,glutathione still has a relatively strong reduction ability at pH 5.0 [51].Therefor the size changes of the RCMs at acidic pH and 10 mM GSHare not only induced by acetal hydrolysis but also by the cleavage of di-sulfide bonds. The combination of de-crosslinking under reductive con-ditions and acetal hydrolysis for the RCMs will likely further induce

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Fig. 6. CLSM images of RAW264.7 cells following 6 or 12 h of incubationwith DOX-loadedmicelles and freeDOX (10 μg/mL). For eachpanel, the images from left to right showed cellnuclei stained byDAPI (blue), DOXfluorescence in cells (red), and overlays of both images.The scale bars correspond to 25 μm in all the images. A and B: DOX-loaded reversiblycrosslinked PEG-P(TMBPEC-co-PDSC) micelles (RCMs), incubation for and 6 and 12 h, re-spectively; C and D: DOX-loaded irreversibly crosslinked PEG-P(TMBPEC-co-AC) micelles(IRCMs), incubation for and 6 and 12 h, respectively; and (E) free DOX, 6 h.

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synergistic effects for intracellular drug release. In contrast, the sizechange of the IRCMs at pH5.0 in the presence of 10mMGSHwas similarto that observed at pH 5.0 in the absence of GSH (Fig. 4C). Therefore,RCMs while possessing superior colloidal stability under extracellularconditions are prone to rapid dissociation under acidic pH and redoxdual-stimuli mimicking the intracellular conditions.

3.4. Loading and in vitro release of DOX

DOX was loaded into non-crosslinked and crosslinked PEG-P(TMBPEC-co-PDSC) micelles at theoretical DLC's of 5, 10 and 20 wt.%.Drug loading levels for crosslinked and non-crosslinked micelles weresimilar, with drug loading efficiencies (DLE) in the range of 59.8 to66.8% (Table 1). The average sizes of non-crosslinked PEG-P(TMBPEC-co-PDSC) micelles increased from 62.4 to 167.2 nm while the sizes ofcrosslinked micelles increased from 63.6 to 84.6 nm with DOX loadingcontents increasing from 3.4 to 12.1 wt.% (Table 1). Analogous trendswere also observed for DOX-loaded irreversibly crosslinked PEG-P(TMBPEC-co-AC) micelles (Table S1).

The in vitro release of DOX from RCMs was investigated at 37 °Cunder different conditions, i.e., (i) pH 7.4, (ii) pH 5.0, (iii) pH 7.4 and10mMGSH, (iv) pH 5.0 and 10mMGSH, and (v) 4 h at pH 5.0 followedby pH7.4 and 10mMGSH (mimicking the intracellular trafficking path-way). The results showed that the release of DOX from both crosslinkedmicelles was largely inhibited, even at a low micelle concentration of40 μg/mL, in which only ca. 19.8% and 17.2% DOX was released at phys-iological pH in 24 h from reversibly (Fig. 5A) and irreversibly (Fig. S3)crosslinked micelles, respectively. It is interesting that DOX releasefrom the RCMs was boosted in the first two hours (22.4% of DOX) atpH 7.4 in the presence of 10 mM GSH, due to cleavage of the disulfidecrosslinks. However, only 44.1% of drugwas released in 24 h, as DOX re-lease from the de-crosslinked micelles was still inhibited by the stronghydrophobic interaction between DOX and TMBPEC units. The releaseresults are in accordance with the size changes observed for RCMs atpH 7.4 in the presence of 10 mM GSH (Fig. 4B). The release of DOXwas significantly accelerated at pH 5.0mainly due to pH-induced acetalhydrolysis. For example, 64.2% of DOX was released from the RCMs in24 h (Fig. 5A), which was also observed for the IRCMs under otherwisethe same conditions (Fig. S3). Notably, the fastest and almost completedrug release was observed at pH 5.0 in the presence of 10 mM GSHwherein 98.7% of DOX was released from RCMs within 12 h (Fig. 5A).This is in line with the observation that RCMs were rapidly destabilizedat pH 5.0 in the presence of 10 mMGSH (Fig. 4A). It was found that theaddition of 10mMGSHhad noeffect onDOX release from IRCMsboth atpH 5.0 and pH 7.4 (Fig. S3). In order to simulate the intracellular traffick-ing process, drug release studies were first performed at pH 5.0 (mim-icking the acidic endosomal compartments) for 4 h and subsequentlyat pH 7.4 and 10 mM GSH (mimicking the reductive environment ofthe cytosol), which has interestingly resulted in faster and more thor-ough DOX release from PEG-P(TMBPEC-co-PDSC) micelles (ca. 95.0%of DOX released in a total time of 24 h) than at either pH 5.0 or pH 7.4and 10 mM GSH conditions (Fig. 5B). DOX release from the IRCMs atpH 5.0 was similar as that for the RCMs during the first 4 h, but was sig-nificantly inhibited at pH 7.4 in the presence of 10 mMGSH (Fig. 5B). Itappears, therefore, that reversibly crosslinked pH-sensitive degradablemicelles on one hand possess superior stability at physiological condi-tions due to the core-crosslinked structure with S-S bonds and on theother hand are able to rapidly and thoroughly release drug by the pHand reduction dual-stimuli simulating the intracellular traffickingprocess.

3.5. Intracellular DOX release and antitumor activity

To ascertain that the crosslinkedmicelles have good cellular uptake,the intracellular drug release profiles of DOX-loaded crosslinked mi-celles were investigated with CLSM using RAW 264.7 cells. Notably,

comparable DOX fluorescence was observed in the cytoplasm of RAW264.7 cells following 6 h of incubation with either DOX-loaded RCM orIRCM (Fig. 6A and C), in which the micelles are encountering theendosomal pH, inducing a similar DOX release. After incubation of thecells with DOX-loaded RCMs for an incubation time of 12 h, the DOXfluorescence in the cell nuclei became even stronger (Fig. 6B), whichwas similar to that observed for RAW 264.7 cells following 6 h of incu-bation with free DOX (control experiments) (Fig. 6E). The uptake ofDOX by the cell nuclei is crucial because DOX has to intercalate withDNA to induce cell death [52]. In contrast, after 12 h of incubationwith DOX-loaded IRCMs, the fluorescence was not increased and stillmainly present in the cytosol as well as in the perinuclei regions of thecells (Fig. 6D). These different intracellular drug release behaviors ofRCMs and IRCMs are most likely due to the fast DOX release fromRCMs after their escape from the endosomes as a result of reduction-

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132 W. Chen et al. / Journal of Controlled Release 210 (2015) 125–133

triggered de-crosslinking in the cytosol. In comparison, drug releasefrom IRCMs would be much slower in the cytosol (neutral pH), as indi-cated in Fig. S3. These results have demonstrated that reversiblycrosslinked pH-sensitivemicelles couldmediatemore efficient intracel-lular anticancer drug release than the IRCMs.

MTT assays using HeLa and RAW 264.7 cells revealed that RCMsand IRCMs were practically non-toxic (cell viabilities ≥ 94%) up toa tested concentration of 0.8 mg/mL (Fig. 7), confirming that thesedegradable micellar particles have good biocompatibility. DOX-loaded RCMs and DOX-loaded IRCMs, however, displayed significantanti-tumor activity towards HeLa and RAW264.7 cells following 48 hof incubation (Fig. 8). It should be noted that DOX-loaded RCMs hadlow IC50 (half-maximal inhibitory concentration) values of 1.65 and1.14 μg DOX equiv./mL for HeLa and RAW 264.7 cells, respectively,which were close to those obtained with the pH and reductiondual-responsive non-crosslinked PEG-SS-PTMBPEC micelles [22].IRCMs displayed inferior antitumor activity under otherwise the sameconditions with IC50 values of 19.8 and 12.6 μg DOX equiv./mL forHeLa and RAW 264.7 cells, respectively, which was mainly due tothe incomplete and slow drug release from the irreversiblycrosslinked structures. The higher anti-tumor activity of DOX-loaded RCMs as compared to DOX-loaded IRCMs is in accordancewith the confocal microscopy results. Interestingly, the anti-tumoractivity of DOX-loaded RCMs approached that of free DOX (IC50 =0.41 and 0.38 μg/mL for HeLa and RAW 264.7 cells, respectively).It should further be noted that the anti-tumor activity of DOX-loaded RCMs might be further enhanced by using targeting ligandssuch as folic acid, aptamer, peptide, antibody or antibody fragments

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Fig. 8. The in vitro antitumor activity of DOX-loaded reversibly crosslinked PEG-P(TMBPEC-(IRCMs), and free DOX. (A) HeLa cells; (B) RAW 264.7 cells. The cells were incubated with DOviation (n = 4).

that facilitate efficient and specific cellular uptake of micelles. It isevident that these reversibly crosslinked pH-sensitive micelleswhile possessing superior extracellular stability can rapidly andcompletely deliver and release anticancer drugs following internal-ization by cancer cells.

4. Conclusions

Wehave demonstrated that pH-sensitive degradable RCMs based onPEG-P(TMBPEC-co-PDSC) copolymers while possessing superior stabil-ity in the extracellular environment undergo rapid de-crosslinking anddisassembly at endosomal pH followed by the exposure to cytoplasmicglutathione, resulting in enhanced intracellular anticancer drug release.In these intelligent micelles several features have been integrated:(i) they are degradable and have low cytotoxicity; (ii) they show gooddrug loading levels; (iii) they show superior stability at low concentra-tions with inhibited (premature) drug release; and (iv) they promotefast andmaximumdrug release inside the cancer cells inwhich drug re-lease is activated during the whole intracellular trafficking process, i.e.,not only in the mildly acidic endosomal compartments but also in thehighly reducing cytoplasm and cell nuclei. Further in vivo experimentsusing RCMs decoratedwith targeting ligands are currently being carriedout in our lab. We are convinced that these pH-sensitive degradableRCMs provide an interesting platform for targeted and efficient deliveryof potent chemotherapeutics.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2015.05.273.

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Acknowledgment

This work was supported by the National Natural Science Founda-tion of China (NSFC 51173126, 81261120557 and 51373113), the Na-tional Science Fund for Distinguished Young Scholars (51225302), theNatural Science Foundation of Jiangsu Province (BK20131166), and aProject Funded by the Priority Academic Program Development ofJiangsu Higher Education Institutions.

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