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Soft Matter
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aDepartment of Chemistry, Northeast Norm
China. E-mail: [email protected] Laboratory of Polymer Ecomaterials, C
Chinese Academy of Sciences, Changchun 1
ciac.jl.cn
† Electronic supplementary informa10.1039/c2sm27189c
Cite this: Soft Matter, 2013, 9, 2224
Received 23rd September 2012Accepted 5th December 2012
DOI: 10.1039/c2sm27189c
www.rsc.org/softmatter
2224 | Soft Matter, 2013, 9, 2224–22
Disulfide crosslinked PEGylated starch micelles asefficient intracellular drug delivery platforms†
Aiping Zhang,a Zhe Zhang,a Fenghua Shi,a Jianxun Ding,b Chunsheng Xiao,b
Xiuli Zhuang,*b Chaoliang He,b Li Chen*a and Xuesi Chenb
Novel reduction-responsive disulfide core-crosslinked micelles based on amphiphilic starch-graft-
poly(ethylene glycol) (starch-g-PEG) were prepared and used for efficient intracellular drug delivery. The
starch-g-PEG copolymers can be conveniently prepared by grafting starch with carboxyl group
terminated PEG, and subsequently conjugated with lipoic acid for disulfide crosslinking. The self-
assembled starch-g-PEG micelles and the corresponding disulfide core-crosslinked micelles were then
characterized by transmission electron microscopy, dynamic laser scattering and fluorescence
techniques. It is interesting to observe that the hydrodynamic radii of disulfide core-crosslinked micelles
would increase gradually in phosphate buffered saline (PBS) due to the cleavage of the disulfide bond
in the micellar core, caused by the presence of reductive glutathione (GSH). The glutathione-responsive
behaviors of the disulfide core-crosslinked micelles should be attractive for intracellular drug delivery.
Thus, a model anticancer drug doxorubicin (DOX) was loaded into micelles and the in vitro drug release
in response to GSH was also studied. The results showed that only a small amount of loaded DOX was
released from the core-crosslinked starch-g-PEG micelles in PBS solution without GSH, while quick
release occurred in the presence of 10.0 mM GSH. Confocal laser scanning microscopy and flow
cytometry analyses further demonstrate that the disulfide crosslinked micelles exhibited a faster drug
release behavior in glutathione monoester (GSH-OEt) pretreated HeLa cells than that in the
nonpretreated and buthionine sulfoximine (BSO) pretreated cells. In addition, the DOX-loaded
crosslinked micelles show higher cellular proliferation inhibition against GSH-OEt pretreated HeLa and
HepG2 than against the nonpretreated and BSO pretreated ones. These results suggest that such
disulfide crosslinked starch-g-PEG micelles, which can efficiently release the loading drug in response to
intracellular GSH concentration, may provide favorable platforms for cancer therapy.
1 Introduction
Nowadays, there is a continuously growing interest in drugdelivery, especially for the treatment of cancer, which is one ofthe major causes of morbidity and mortality in the world.1
Conventional chemotherapy has proved partially successful intreatment and prolonging the lives of patients. The limitedclinical success is mostly due to the lack of tumor-selectivity ofanticancer drugs, which results in severe side effects to normaltissues and low efficacy against multi-drug resistant cancercells.2 Aiming to improve chemotherapy, tremendous effort hasbeen centered on the development of various nanocarriers that
al University, Changchun 130024, P. R.
hangchun Institute of Applied Chemistry,
30022, P. R. China. E-mail: zhuangxl@
tion (ESI) available. See DOI:
33
are capable of targeted controlled delivery of anticancer drugs,including polymeric micelles,3 vesicles,4 liposomes5 and nano-gels6 etc. These nanocarriers can not only enhance the aqueoussolubility and bioavailability of the drug but also improvedpharmacokinetics and biodistribution proles via the enhancedpermeability and retention (EPR) effect.
Polymeric micelles, as one kind of antitumor drug nano-carrier based on amphiphilic block/gra copolymers, havereceived considerable attention.7,8 The hydrophobic inner coreacts as a depot for drugs and the hydrophilic outer shell as aprotective interface between the hydrophobic core and theexternal aqueous milieu.9,10 Polymeric micelles offer severaldistinct advantages for anticancer drug delivery, such asimproved solubility, prolonged in vivo circulation time andpreferential accumulation at the tumor site via the EPR effect.However, one practical challenge is their low stability in vivo,because of the large dilution volume and/or interactions withcells and biomolecules presented in the blood, which oen leadto premature drug release, aggregation, and a diminishedability of the drug to reach its target. In order to overcome this
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limitation, different shell or core crosslinking approaches havebeen adopted, which can not only improve the stability ofmicelles but also control the drug release through the envi-ronment responsive linkages.8,11
As alternatives to traditional micelle systems, smart nano-carriers are actively pursued that can stably encapsulate thera-peutics and release them at a desired site in response to externalstimuli such as pH,12 redox,13–15 or enzyme16 conditions. Stimuli-responsive crosslinked micelles could minimize its prematurerelease in the circulating blood pool and lead to site-specicrelease of the drug modulated by the specic intracellularmicroenvironments, which can lead to aggressive anticanceractivity and maximal chemotherapeutic efficacy with fewer sideeffects.17–19 It is well known that the intracellular concentrationof GSH, a thiol-containing tripeptide that cleaves disuldebonds by a redox reaction,20,21 is substantially higher than thelevel in the extracellular environment.22 Taking advantage ofthis dramatic difference in the GSH concentrations, the smartreversible disulde crosslinked micelles may hold vast potentialfor targeting intracellular release of anticancer drugs.23,24
For any drug delivery systems including the crosslinkedmicelles to be practically useful, the nevertheless fundamentalconsideration is to tailor a safe biocompatible and biodegradablematerial. Starch, as a major dietary source of carbohydrates, is anattractive substitute for other chemically synthesized polymersdue to its non-toxicity, non-immunogenicity, stability in the airand compatibility with most drugs.25,26 Starch and its derivativesas non-polluting renewable resources for sustainable supply areused extensively in the pharmaceutical sector as tablets, capsules,liquids, suspensions, gels, inhalation products and strips tofacilitate the packaging and delivery of drugs. These advantagesalso make them an ideal substrate for the preparation of nano-carriers.27 For example, hydrophobic starch derivatives, such aspalmitoylated starch acetate28 and propyl starch,29 have beensynthesized in order to explore their application in formulatingnanoparticulate delivery to encapsulate anticancer drugs. Inaddition, nanosized self-assembled micelles of hydrophobicallymodied starch, which might be used as a potential drug carrier,were also reported.30
As we know, most researchers used starch and starch deriv-atives as the hydrophobic substrate. In order to increase thehydrophilicity of starch, they oen gra the hydrophilic sidechains to the starch backbone. In our work, we selected thenatural starch and PEG to formulate a micelle for drug delivery.The starch acted as the hydrophobic inner core and the PEGacted as the hydrophilic outer shell. The gra copolymers wereprepared through graing the PEG chains to the starch back-bone. The lipoic acid (LA) was also graed onto the starchbackbone to introduce the disulde crosslinker into themicellar core. The disulde crosslinked micelles show goodstability in phosphate buffered saline (PBS), but are prone todissociate under a reductive environment mimicking theintracellular conditions. The micellar characteristics wereinvestigated using transmission electron microscopy (TEM),dynamic light scattering (DLS) and uorescence techniques.Furthermore, DOX, as a model anticancer drug, was loaded intothe micelles by a simple dialysis technique. In vitro DOX release
This journal is ª The Royal Society of Chemistry 2013
from DOX loaded crosslinked micelles was accelerated in thepresence of GSH. The GSH mediated intracellular drug deliverywas also investigated against HeLa cells by pretreating the cellswith GSH-OEt or BSO, which could increase or decrease theconcentration of GSH in the cytoplasm, respectively. Thebiocompatibilities of micelles and cellular proliferation inhi-bition of DOX-loaded micelles were also investigated.
2 Experimental2.1 Materials
Monomethoxy poly(ethylene glycol) (PEG, Mn ¼ 2000 and 5000)was purchased from Sigma-Aldrich and used without furtherpurication. The carboxyl group terminated PEG (PEG–COOH)was synthesized according to the literature procedure.31 Waxymaize starch was purchased from Changchun Dacheng MaizeDevelopment Co., Ltd with 10% (w/w) moisture content and amacromolecular weight of 120 000. 4-Dimethylaminopyridine(DMAP), N-(3-dimethylaminopropyl)-N0-ethylcarbodiimidehydrochloride (EDC$HCl), lipoic acid (LA), D,L-1,4-dithiothreitol(DTT), glutathione (GSH), glutathione monoester (GSH-OEt),buthionine sulfoximine (BSO) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased fromSigma-Aldrich. Doxorubicin hydrochloride (DOX$HCl) waspurchased from Zhejiang Hisun Pharmaceutical Co., Ltd. All theother reagents and solvents were purchased from SinopharmChemical Reagent Co., Ltd and used as obtained.
2.2 Preparation of disulde crosslinked starch-g-PEGmicelles
2.2.1 Preparation of starch-g-PEG copolymers. The starch-g-PEG5K copolymers were conveniently prepared by conjugatingstarch with PEG5K–COOH. Typically, 1.0 g starch, 9.4 g PEG5K–
COOH, 0.72 g EDC$HCl and 0.023 g DMAP were dissolved in60.0 mL DMSO in a ame-dried ask. The conjugation wasperformed at 25 �C for 48 h. Then, the solvent and unreactedsubstances were removed by dialysis against deionized water for72 h. The solution was ltered and lyophilized to give theproduct starch-g-PEG5K as a white solid (yield: 81.5%). Similarly,starch-g-PEG2K copolymers were prepared using the samemethod with a yield of 79.8%.
The uncrosslinked starch-g-PEG micelles were prepared bythe solvent exchange method. Briey, 5.0 mL deionized waterwas added dropwise to 1.0 mL starch-g-PEG copolymer solutionin DMSO (1.0 mg mL�1) under moderate stirring at 25 �C. Theresultant starch-g-PEG suspension was extensively dialyzedagainst deionized water to remove the DMSO solvent.
2.2.2 Synthesis of starch-g-PEG–LA copolymers. Starch-g-PEG5K–LA copolymers were prepared by conjugation of LA ontostarch-g-PEG5K. Typically, 1.0 g starch-g-PEG5K copolymers,0.036 g LA, 0.086 g EDC$HCl and 0.006 g DMAP were dissolvedin 40 mL DMSO in a ame-dried ask. The conjugation wasperformed at 25 �C for 48 h. Aer that, the solvent andunreacted substances were removed by dialysis againstdeionized water for 72 h. Then, the solution was ltered andlyophilized (yield: 73.5%). Similarly, starch-g-PEG2K–LA
Soft Matter, 2013, 9, 2224–2233 | 2225
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copolymers were prepared using the same method with atypical yield of 70.6%.
2.2.3 Preparation of disulde crosslinked starch-g-PEG–LAmicelles. The crosslinking mechanism is based on the thiol–disulde exchange reaction. Typically, the pH of starch-g-PEG–LA solution was adjusted to 8.5 using PBS, and the dispersionwas purged with nitrogen for 20 min. The micelles wereconveniently crosslinked in solution by treating with 10 mol%DTT relative to the amount of lipoyl units. Aer stirring for 24 hat room temperature, the solution was dialyzed against deion-ized water for 48 h. Then, the solution was ltered and lyophi-lized (yield: 90.6%).
2.3 Characterizations
The carbon and hydrogen content of copolymers was estimatedby elemental analysis (Vario EL III, Germany). 1H NMR spectrawere recorded on a Bruker AV 400 NMR spectrometer in dimethylsulfoxide-d6 (DMSO-d6). FT-IR spectra were recorded on a Bio-RadWin-IR instrument using the potassium bromide (KBr) method.DLS measurements were performed on a WyattQELS instrumentwith a vertically polarized He-Ne laser (DAWN EOS, Wyatt Tech-nology). The scattering angle was xed at 90�. TEMmeasurementwas performed on a JEOL JEM-1011 transmission electronmicroscope with an accelerating voltage of 100 kV. A drop of themicelle solution (0.1 g L�1) was deposited onto a 230mesh coppergrid coated with carbon and allowed to dry in air at 25 �C beforemeasurement. The excitation spectra of Nile Red were measuredon a PTI Fluorescence Master System with soware Felix 4.1.0 atan excitation wavelength of 550 nm and the emission wasmonitored from 580 to 720 nm.
2.4 In vitro drug loading and release
DOX was used as a model drug for drug loading. DOX loadeduncrosslinked and crosslinked micelles were prepared by asimple dialysis technique. Typically, micelles (20.0 mg), drug(4.0 mg) and triethylamine (0.7 mg) were rst mixed in 3.0 mLDMF. 1.0 mL deionized water was then added gradually understirring. The mixture was stirred at room temperature for 24 hand DMF was removed by dialysis against deionized water for24 h. The dialysis medium was changed four times and thewhole procedure was performed in the dark. Finally, the solu-tion was ltered and lyophilized. For determination of the drugloading content (DLC) and drug loading efficiency (DLE), thedrug loaded micelles were dissolved in DMF and analyzed byuorescence measurement using a standard curve method (PTIFluorescence Master System, lex ¼ 480 nm). The DLC and DLEof the drug loaded micelles were calculated according to eqn (1)and (2), respectively:
DLC ðwt %Þ ¼ amount of drug in micelles
amount of drug loaded micelles� 100 (1)
DLE ðwt%Þ ¼ amount of drug in micelles
total amount of feeding drug� 100 (2)
In vitro drug release proles of DOX-loaded crosslinkedmicelles were investigated in PBS (pH 7.4) with or without
2226 | Soft Matter, 2013, 9, 2224–2233
10mMGSH. The weighed freeze-dried DOX loadedmicelles wassuspended in 8.0 mL of the release medium and transferredinto a dialysis bag (MWCO 3500 Da). The release experimentwas initiated by placing the end-sealed dialysis bag into 50.0 mLof the release medium at 37 �C with continuous shaking at 80rpm. At specied time intervals, 2.0 mL dialysate was withdrawnand replaced with the fresh release medium. The amount ofreleased DOX was determined by uorescence measurement(lex ¼ 480 nm). The release experiments were conducted intriplicate.
2.5 Intracellular drug release
The cellular uptake and intracellular release behaviors of DOX-loaded crosslinked micelles were observed by confocal laserscanning microscopy (CLSM) and ow cytometry using HeLacells.
For CLSM observation, cells were seeded onto glass cover-slips in six-well plates at 2 � 105 cells per well in 2.0 mL ofcomplete Dulbecco's modied Eagle's medium (DMEM) con-taining 10% fetal bovine serum, supplemented with 50 IU mL�1
penicillin and 50 IU mL�1 streptomycin, and cultured for 24 h,and then treated with BSO for 12 h or treated with GSH-OEt for 2h. Cells were washed by PBS and incubated at 37 �C for anadditional 3 h with DOX loaded crosslinked micelles at a nalDOX concentration of 10 mg L�1 in complete DMEM. Cellswithout GSH and BSO pretreatment were used as controls. Thenthe culture medium was removed and the cells were washedthrice with PBS. Thereaer, the cells were xed with 4% para-formaldehyde for 30 min at room temperature. And, the cellswere counterstained with 4,6-diamidino-2-phenylindole (DAPI)for cell nucleus and Alexa Fluor� 488 phalloidin for F-actinfollowing the manufacturer's instructions. CLSM images of thecells were obtained through confocal microscopy (OlympusFluoView 1000).
For ow cytometry studies, the cells were seeded in six-wellplates at 5 � 105 cells per well in 2.0 mL complete DMEM andcultured for 24 h, and then treated with BSO for 12 h or treatedwith GSH-OEt for 2 h. Cells were washed by PBS and incubatedat 37 �C for an additional 3 h with DOX loaded crosslinkedmicelles at a nal DOX concentration of 10 mg L�1 in completeDMEM. Cells without GSH and BSO treatment were used as acontrol. Thereaer, the culture medium was removed and thecells were washed with PBS thrice and treated with trypsin.Then, 2.0 mL PBS was added to each culture well, and thesolutions were centrifuged for 4 min at 3000 rpm. Aer removalof the supernatants, the cells were resuspended in 0.4 mL PBS.Data for 1 � 104 gated events were collected, and analysis wasperformed by ow cytometry (Beckman, California, USA).
2.6 Cell viability assays
The relative cytotoxicities of crosslinked micelles against HeLaand HepG2 cells were evaluated in vitro by a MTT assay. Thecells were seeded in 96-well plates at 1 � 104 cells per well in100 mL complete DMEM and incubated at 37 �C in a 5% CO2
atmosphere for 24 h, followed by removing the culture mediumand adding crosslinked micelle solutions at different
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Scheme 1 Synthetic pathway of starch-g-PEG–LA graft copolymers.
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concentrations (0–500 mg L�1). The cells were subjected to MTTassay aer being incubated for another 72 h. The absorbance ofthe solution was measured on a Bio-Rad 680 microplate readerat 490 nm. Cell viability (%) was calculated based on eqn (3):
Cell viability ð%Þ ¼ Asample
Acontrol
� 100 (3)
In eqn (3), Asample and Acontrol represented the absorbances ofthe sample and control wells, respectively.
The cytotoxicities of DOX-loaded crosslinked micellesagainst HeLa and HepG2 cells were also evaluated in vitro by aMTT assay. Similarly, cells were seeded into 96-well plates at 1�104 cells per well in 200 mL complete DMEM and further incu-bated for 24 h. Then, cells were treated with 0.5 mM BSO for12 h or 10.0 mM GSH-OEt for 2 h. Cells without pretreatmentwere used as a control. Aer washing cells with PBS, DOX-loaded crosslinked micelles were added with different DOXconcentrations (0–10 mg L�1 DOX). The cells were subjected toMTT assay aer being incubated for another 24, 48 and 72 h.The absorbance of the solution was measured on a Bio-Rad 680microplate reader at 490 nm. Cell viability (%) was calculatedbased on eqn (3).
2.7 Protein adsorption
Bovine serum albumin (BSA) was used as a model protein todetermine the protein adsorption of the crosslinked micelles.The micelles were incubated with a solution of BSA in PBS, withthe nal concentration of the micelles and BSA at 0.20 and0.25 mg mL�1, respectively. Aer incubation at 37 �C for adetermined time, 200 mL of each sample was withdrawn, thencentrifuged at 16 000 rpm for 16 min to precipitate the protein-adsorbed micelles.
The BSA concentration of the supernatant was determinedusing UV-Vis spectroscopy by measuring the maximal absor-bance at 280 nm. Then, the amount of BSA adsorbed on themicelles was calculated with a standard calibration curve ofBSA. Poly(ethylene glycol) with a molecular weight of 5000(PEG5K) and branched polyethylenimine with a molecularweight of 25 000 (PEI25K) were used as controls.
3 Results and discussion3.1 Preparation and characterization of uncrosslinkedstarch-g-PEG micelles
The starch-g-PEG copolymers were conveniently prepared bygraing starch with PEG–COOH in DMSO, as shown in Scheme1. Based on the elemental analysis results of the obtainedcopolymers, the molar ratios of starch and PEG were calculatedand listed in Table 1.32 The chemical structures of representa-tive starch-g-PEG5K-2 copolymers were conrmed by 1H NMRand FT-IR. As shown in Fig. 1a, the 1H NMR resonances at 3.59–3.68 (proton 2, 3, 4, 5), 4.56 (proton 7) and 5.11–5.46 ppm(proton 1, 8, 9) were the characteristic signals of starch. Theresonances at 3.51 (proton 11, 12) and 3.23 ppm (proton 10)were attributed to the methylene protons and terminalmethoxyl protons of PEG, respectively. The FT-IR spectra(Fig. S1†) also conrmed the chemical structure of starch-g-PEG
This journal is ª The Royal Society of Chemistry 2013
copolymers, which showed the typical absorptions at 1108 cm�1
(yC–O–C) assigned to the ether bond of PEG.It is known that amphiphilic polymers can self-assemble into
micelles in selected solvents. In this study, we synthesized theamphiphilic starch-g-PEG copolymer, which contained ahydrophilic PEG side chain and a hydrophobic starch back-bone. Self-assemblies of starch-g-PEG were prepared by a dial-ysis method. Then the micelles were characterized by TEM andDLS. This revealed that the representative uncrosslinked starch-g-PEG5K-2 and starch-g-PEG2K-2 micelles took a sphericalmorphology with an average diameter of around 143 and 128nm (Fig. 2A and B), respectively. And, the hydrodynamic radii(Rh) measured by DLS were 164 � 5.6 and 131 � 4.9 nm,respectively (Fig. 2C).
The self-assembly of starch-g-PEG copolymers was alsocharacterized by uorescence spectra using Nile Red as theuorescence probe according to the literature.33,34 The criticalmicelle concentration (CMC) could be calculated by trackingthe uorescence intensity of Nile Red as a function of thesample concentration shown in Fig. S2.† As shown in Table 1,the PEG graing ratio only slightly affected the CMC values. TheCMC values were found to increase with the increase of PEGgraing ratio.35 In addition, an increase in the hydrophilic blockfrom PEG2K to PEG5K resulted in a twofold increase in the valuefor the CMC.36 For instance, the calculated CMC values ofstarch-g-PEG2K-2 and starch-g-PEG5K-2 were 3.5 mg L�1 and 8.3mg L�1, respectively.
Soft Matter, 2013, 9, 2224–2233 | 2227
Table 1 Characterizations of uncrosslinked starch-g-PEG micelles
Uncrosslinked micelles Feeding ratioa (g/g) Resultant ratiob (g/g) Rh (nm) CMC (mg L�1) DLC (wt%) DLE (wt%)
Starch-g-PEG5K-1 1/4.7 1/4.1 151 � 2.9 7.9 1.52 9.12Starch-g-PEG5K-2 1/9.4 1/8.5 164 � 5.6 8.3 1.31 7.86Starch-g-PEG5K-3 1/14.0 1/11.7 182 � 3.1 8.8 1.17 7.02Starch-g-PEG2K-1 1/1.9 1/1.7 119 � 3.1 3.1 1.34 8.04Starch-g-PEG2K-2 1/3.8 1/3.5 131 � 4.9 3.5 1.23 7.46Starch-g-PEG2K-3 1/5.6 1/5.1 146 � 4.8 4.2 1.02 6.12
a Feeding weight ratios of starch/PEG. b Resultant weight ratios of starch/PEG, calculated by elemental analysis.
Fig. 1 1H NMR spectra of starch-g-PEG5K-2 (a) and starch-g-PEG5K–LA-2 (b).
Fig. 2 TEM micrographs of uncrosslinked starch-g-PEG5K-2 (A), starch-g-PEG2K-2(B) micelles, and hydrodynamic radii (Rh) of uncrosslinked starch-g-PEG5K-2 (a)and starch-g-PEG2K-2 (b) micelles in PBS, pH 7.4 (C).
Table 2 Characterizations of crosslinked starch-g-PEG micelles
Crosslinked micelles Rh (nm) DLC (wt%) DLE (wt%)
Crosslinked starch-g-PEG5K-1 97 � 6.1 9.52 57.12Crosslinked starch-g-PEG5K-2 108 � 2.3 8.87 53.22Crosslinked starch-g-PEG5K-3 114 � 6.0 8.62 51.72Crosslinked starch-g-PEG2K-1 77 � 1.5 9.18 55.08Crosslinked starch-g-PEG2K-2 86 � 6.2 9.01 54.06Crosslinked starch-g-PEG2K-3 93 � 5.5 8.48 50.88
Fig. 3 Change in the particle size (Rh) of crosslinked starch-g-PEG5K-2 micelles(A) and starch-g-PEG2K-2 micelles (B) in PBS, pH 7.4 containing 10 mM GSH.
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3.2 Preparation and characterization of disuldecrosslinked starch-g-PEG micelles
Starch-g-PEG–LA copolymers were prepared by conjugatingstarch-g-PEG with LA. The chemical structures of representativestarch-g-PEG5K-2–LA copolymers were conrmed by 1H NMR. As
2228 | Soft Matter, 2013, 9, 2224–2233
shown in Fig. 1b, the 1H NMR spectra showed resonances at1.39 (20), 1.56 (19, 21), 1.99 (23), 2.33 (18), 3.42 (22) and 3.61(22)ppm, which were the characteristic signals of LA. Then, thedisulde crosslinked micelles were prepared by thiol–disuldeexchange in the presence of DTT.20 Under the DTT reduction,the lipoyl rings were opened and the disulde bonds werereformed between different lipoyl units, leading to core
This journal is ª The Royal Society of Chemistry 2013
Fig. 4 In vitro DOX release from DOX-loaded crosslinked starch-g-PEG5K-1 (a), -2(b) and -3 (c) micelles without GSH, and starch-g-PEG5K-1 (d), -2 (e) and -3 (f)micelles with 10mMGSH (A); DOX release from DOX-loaded crosslinked starch-g-PEG2K-1 (a), -2 (b) and -3 (c) without GSH, and starch-g-PEG2K-1 (d), -2 (e) and -3(f) with 10 mM GSH (B) in PBS, pH 7.4, 37 �C. Data were presented as mean �standard deviation (n ¼ 3).
Fig. 5 CLSM images of HeLa cells incubated with DOX-loaded crosslinked starch-g-PEG5K-2 micelles (A–C) and starch-g-PEG2K-2 micelles (D–F): (A and D) cellspretreated with 0.5 mM BSO; (B and E) cells pretreated with 10 mM GSH-OEt; (Cand F) cells without pretreatment. For each panel, the images from left to rightshow cell nuclei stained with DAPI (blue), F-actin stained with Alexa Fluor�488phalloidin (green), DOX fluorescence in cells (red), and overlays of the threeimages.
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crosslinking of starch-g-PEG micelles. DLS measurementsshowed that the crosslinked micelles had smaller particle sizesthan the parent uncrosslinked micelles (Table 2).20
It is well-known that the disulde linkages are stable undernormal physiological conditions but respond to reductiveconditions (e.g. GSH) via reversible cleavage into free thiols. Toinvestigate whether the crosslinked micelles can decrosslinkunder a reductive intracellular environment, the change inmicelle size in response to 10.0 mM GSH was monitored overtime by DLS measurement (Fig. 3). Notably, the Rh values ofcrosslinked starch-g-PEG5K-2 and starch-g-PEG2K-2 micellesgradually increased from 108 � 2.3 to 200 � 4.3 nm and from88 � 6.2 to 196 � 6.1 nm with 10.0 mM GSH in PBS at pH 7.4 in24 h, respectively. It should be attributed to the decrease of thecore crosslinking density resulted from the cleavage of disuldecrosslinkers.20,37
Scheme 2 Illustration of reversible disulfide crosslinked starch-g-PEG micellesfor intracellular drug release triggered by GSH.
3.3 In vitro DOX loading and triggered release
DOX is a widely used antineoplastic drug in the treatment ofseveral adult and pediatric cancers, such as leukemia,lymphomas, breast cancer, and many other solid tumors. In the
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Fig. 6 Flow cytometric profiles of HeLa cells incubated with DOX-loadedcrosslinked starch-g-PEG5K-2 (A) and starch-g-PEG2K-2 (B) micelles: (a) cells pre-treated with 0.5 mM BSO; (b) cells without pretreatment; (c) cells pretreated with10 mM GSH-OEt.
Fig. 7 Cell viabilities of HeLa (A) and HepG2 (B) cells incubated with crosslinkedstarch-g-PEG5K-2 micelles (a), and crosslinked starch-g-PEG2K micelles (b) for 72 h.PEI25K was used as the positive control. Data are presented as mean � standarddeviation (n ¼ 6).
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current study, DOX was used as a model drug and loaded intothe uncrosslinked and crosslinked micelles by simply mixing ofdrug and micelles in DMF and subsequent dialysis againstdeionized water. As shown in Tables 1 and 2, the DLE ofuncrosslinked starch-g-PEG micelles were in the range of 6.12–9.12%, while the DLE of crosslinked starch-g-PEG micelles were50.88–57.12%. These data indicated that the crosslinkingstructure can improve the drug loading capacity and stability ofstarch-g-PEG copolymer micelles.
The DOX release behaviors of the DOX-loaded crosslinkedmicelles were investigated with or without 10.0mMGSH in PBS atpH 7.4. The cumulative release percentages of DOX loaded incrosslinked starch-g-PEG5K micelles versus time were plotted inFig. 4A. In the absence of GSH, the release of DOX from cross-linked starch-g-PEG5K micelles was slow without burst release;only about 35% of the loaded DOX was released over 59.5 h.However, DOX release was accelerated in PBS with 10.0 mMGSH,analogous to the intracellular reductive environment; about 70%of the loaded DOX was released in 12 h and 90% of the loadedDOX was released in 59.5 h. The fast DOX release from thecrosslinked micelles under the reductive conditions was mostlikely due to the decrease in core cross-linking density caused by
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the cleavage of disulde bonds (Fig. 3). Fig. 4B shows the releaseof DOX from crosslinked starch-g-PEG2Kmicelles in PBS with andwithout 10.0 mM GSH. Similarly, about 85% of the loaded DOXwas released in the reductive conditions, relative to 30% of theloaded DOX released in the media without GSH over 72.5 h. Theresults suggest that the disulde crosslinked starch-g-PEGmicelles could effectively hinder the release of the encapsulateddrug in normal physiological conditions, while releasing drugsrapidly in response to intracellular GSH.23,38 Included, we areconvinced that these reversibly crosslinked micelles will havetremendous potential for targeted cancer chemotherapy.
3.4 Intracellular DOX release and cellular proliferationinhibition
To test the feasibility of the micelles for intracellular anticancerdrug release, the cellular uptake and intracellular drug releasebehaviors of DOX-loaded crosslinked micelles were monitoredwith CLSM and ow cytometry in HeLa cells. HeLa cells wererst pretreated with 0.5 mM BSO for 12 h to inhibit the intra-cellular synthesis of GSH or with 10.0 mM GSH-OEt for 2 h toimprove the intracellular GSH concentration.39,40 HeLa cellswithout pretreatment were used as a control. Then, the DOX-loaded crosslinked starch-g-PEG5K-2 and starch-g-PEG2K-2
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Fig. 8 Proliferation inhibitions toward HeLa (A–C) and HepG2 cells (D–F) incubated with DOX-loaded crosslinked starch-g-PEG5K-2 micelles with various DOXconcentrations for 24 (A and D), 48 (B and E) and 72 (C and F) h. The cells were pretreated with 0.5 mM BSO or 10 mM GSH. The non-pretreated cells were used as acontrol. Data are presented as mean � standard deviation (n ¼ 6).
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micelles were incubated with the pretreated and non-pretreatedHeLa cells for 3 h. For both types of micelles, the strongestintracellular DOX uorescence intensity was observed in theGSH-OEt pretreated cells while the weakest DOX uorescenceintensity was observed in the cells pretreated with BSO (Fig. 5).These data indicated that the higher intracellular GSHconcentration should accelerate the degradation of the disul-de crosslinker, resulting in intracellular release of DOX fromcrosslinked starch-g-PEG micelles and subsequent localizationof DOX in the cell nucleus (shown in Scheme 2).41 The triggered
Fig. 9 Proliferation inhibitions toward HeLa (A–C) and HepG2 cells (D–F) incubconcentrations for 24 (A and D), 48 (B and E) and 72 (C and F) h. The cells were pretcontrol. Data are presented as mean � standard deviation (n ¼ 6).
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intracellular drug release was further quantitatively analyzed byow cytometry. As shown in Fig. 6, the ow cytometric histo-grams of the GSH-OEt pretreated cells incubated with DOX-loaded crosslinked micelles shi clearly in the direction of highuorescence intensity compared with that of the control cells.In contrast, the weakest uorescence intensity was shown in thecells incubated with BSO.42 The results conrm that the higherconcentration of GSH could promote the degradation of thedisulde bond and accelerate the intracellular release of DOXfrom the micelles.
ated with DOX-loaded crosslinked starch-g-PEG2K-2 micelles with various DOXreated with 0.5 mM BSO or 10 mM GSH. The non-pretreated cells were used as a
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Fig. 10 BSA adsorption on the crosslinked starch-g-PEG5K-2 (a), starch-g-PEG2K-2 micelles (b), PEG5K and PEI25K after incubation at 37 �C for different periodsof time.
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In order to investigate the biocompatibility of the cross-linked micelles, the cell cytotoxicity was estimated. The in vitrocytotoxicities of crosslinked micelles toward HeLa and HepG2cells were evaluated using a MTT assay. As shown in Fig. 7, theviabilities of cells treated with micelles for 72 h were around100% at all test concentrations up to 500 mg L�1. The resultsrevealed that the crosslinked starch-g-PEG micelles had lowcytotoxicity and could be safely used as biocompatible carriersfor efficient intracellular drug delivery.
Next, the ability to inhibit the cell proliferation of DOX-loadedcrosslinked starch-g-PEG5K-2 and starch-g-PEG2K-2 micelles wasalso evaluated in HeLa and HepG2 cells. Cells were pretreatedwith 0.5mMBSO or 10.0mMGSH-OEt for 12 h and 2 h separatelyand then incubated with the DOX-loaded crosslinked micelles.The cells without pretreatment were used as a control. It wasnoted that BSO and GSH-OEt at the tested concentrations did notshow any cytotoxicities to HeLa and HepG2 cells.42 In general, theeffects of DOX-loaded crosslinked starch-g-PEG micelles on thecell proliferation of cancer cells was found to be time-dependent(Fig. 8 and 9). The in vitro cell-proliferation inhibition study forDOX-loaded crosslinked starch-g-PEG5K-2 micelles is shown inFig. 9. In contrast to the control cells, the HeLa and HepG2 cellspretreated with GSH-OEt and BSO exhibited the highest andlowest inhibition efficiency, respectively. There was a sametendency when cells were incubated with the DOX-loaded cross-linked starch-g-PEG5K-2 micelles (Fig. 10). Meanwhile, theproliferation of cells incubated with free DOX was not affected bythe pretreatment of GSH and BSO.42 The results revealed that thefaster DOX release from DOX loaded micelles was triggered byhigher intracellular GSH concentration, which enhanced theinhibition of the cellular proliferation.
3.5 Protein adsorption
The characterization of in vitro hemocompatibility was alsoimportant, since the micelles are mostly designed to be admin-istrated via intravenous injection for drug delivery applications.Here, the protein adsorption behaviors of the crosslinked starch-g-PEG micelles were assessed using BSA as a model protein, and
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the results are shown in Fig. 10. PEG5K and PEI25K were used asthe negative and positive controls, respectively. Because of thePEG shell, the micelles showed very limited BSA adsorption,which was comparable to the PEG control. Thus, we can concludethat the crosslinked starch-g-PEG micelles are hemocompatible,allowing potential applications as drug delivery vehicles.43
4 Conclusions
In this study, a series of disulde crosslinked micelles based onstarch-g-PEG copolymers were prepared and used for stimuli-responsive intracellular drug delivery. The starch-g-PEG copoly-mers can be conveniently prepared by graing starch with PEG–COOH. The starch-g-PEG copolymers were then further modiedwith LA for producing disulde crosslinking micelles. The Rh ofcrosslinked starch-g-PEG micelles was smaller than that of theuncrosslinked ones; and the Rh of crosslinked micelles canincrease twofold aer 24 h incubation at pH 7.4 solution con-taining 10.0 mM GSH. Based on the GSH responsive Rh variationbehavior, it is expected that the disulde crosslinked starch-g-PEGmicelles should be interesting for intracellular drug delivery.Thus, DOX, as the model anticancer drug, was loaded into thedisulde crosslinked micelles and the redox sensitive release ofthe payload DOX was tested in vitro. A relatively high DLE wasclearly observed in crosslinked starch-g-PEG micelles, ascompared to that of the uncrosslinked ones, indicating theimproved stability of the micelles aer core crosslinking. Thein vitro drug release proles revealed that only a small amount ofthe loaded DOX was released at 59.5 h without GSH, while up toabout 90% of the loaded DOX can be quickly released in thepresence of 10.0 mM GSH. The disulde crosslinked micellesexhibit a faster drug release behavior in GSH-OEt pretreatedHeLacells than in the nonpretreated and BSO pretreated cells. More-over, higher cellular proliferation inhibition efficacy was achievedtoward GSH-OEt pretreated HeLa and HepG2 cells, as comparedto nonpretreated and BSO pretreated cells. Protein adsorptionresults indicated that the crosslinked micelles showed verylimited BSA adsorption. Therefore, with the good biocompati-bility and accelerated intracellular drug release, the disuldecrosslinked starch-g-PEGmicelles provide a favorable platform toconstruct smart drug delivery systems for cancer therapy.
Acknowledgements
This research was nancially supported by the National NaturalScience Foundation of China (Projects 50903012, 51273037,51003103 and 21174142), Jilin Science and Technology Bureau(International Cooperation Project 20120729), Jilin HumanResources and Social Security Bureau (201125020).
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