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Synthesis and ch

aDepartment of Medicinal & Applied ChemisbDepartment of Biotechnology, College of Li

100 Shih-Chuan 1st Road, Kaohsiung 8070

Fax: +886-7-312-5339; Tel: +886-7-312-1101

† Electronic supplementary informa10.1039/c4ra04308a

Cite this: RSC Adv., 2014, 4, 31552

Received 9th May 2014Accepted 15th July 2014

DOI: 10.1039/c4ra04308a

www.rsc.org/advances

31552 | RSC Adv., 2014, 4, 31552–3156

aracterization of pluronic-block-poly(N,N-dimethylamino-2-ethyl methacrylate)pentablock copolymers for drug/gene co-deliverysystems†

Shih-Jer Huang,a Zhi-Rong Hsua and Li-Fang Wang*ab

We synthesized three pluronic-based cationic pentablock copolymers with different hydrophilic/lipophilic

balance (HLB) values using atom transfer radical polymerization (ATRP), including PF127-block-poly(N,N-

dimethylamino-2-ethyl methacrylate) (PF127-b-pDMAEMA), pluronic P123-block-poly(N,N-

dimethylamino-2-ethyl methacrylate) (PP123-b-pDMAEMA), and PL121-block-poly(N,N-dimethylamino-

2-ethyl methacrylate) (PL121-b-pDMAEMA). The copolymers self-assembled into core–shell structures,

which could be used to co-deliver a plasmid DNA (pEGFP) and a hydrophobic drug (epirubicin, EPI). The

physicochemical properties of the copolymers and drug-loaded micelles were thoroughly characterized.

The micelles had a high EPI encapsulation efficiency, �6%, and the EPI-loaded micelles exhibited a

similarly cytotoxic effect to free EPI. Among the three copolymers, the gene transfection efficiency of

PL121-b-pDMAEMA was the highest, indicating that the greater the hydrophobic effect the greater

cellular internalization was. The co-delivery effect of pEGFP and EPI was directly visualized using a

confocal laser scanning microscope. Thus, the pluronic-b-pDMAEMA micelles are a promising co-

delivery system for therapeutic pDNA and hydrophobic anticancer drugs.

Introduction

The rapid development of amphiphilic copolymers is mainlydue to their promising characteristics in drug delivery systems,including micelles,1 nanogels,2 and polymersomes.3 Amongthem, micelles have smaller hydrodynamic dimensions, usuallyspherical core–shell construction, in which the hydrophobiccore provides a depot for accommodation of hydrophobic drugsand the hydrophilic shell maintains water solubility. Althoughthe micelles have many advantages for biomedical applica-tions,4 the advancement of micellar drug formulations for theclinic has been challenging. Major barriers include difficulty intransport through the cell membrane and optimum drugdelivery necessary for therapeutic effect.5 Therefore, novelamphiphilic copolymers were designed with not only varioushydrophobic and hydrophilic segments, but also multiplefunctions.

Recently, amphiphilic copolymers containing cationicsegments have been extensively used as non-viral gene carriersincluding polyethylenimine (PEI),6,7 poly(N,N-dimethylamino-2-

try, Taiwan

fe Science, Kaohsiung Medical University,

8, Taiwan. E-mail: lfwang@kmu.edu.tw;

ext. 2217

tion (ESI) available. See DOI:

3

ethyl methacrylate) (PDMAEMA),8,9 poly(lysine) (PLL),10 andpolyamidoamine (PAAM).11 The cationic amphiphilic copoly-mers self-assembled into dispersive and stable micelles with acationic shell and a hydrophobic core. These novel carrierscondensed gene drugs via electrostatic interactions and loadedhydrophobic drugs in the hydrophobic core.

Due to the molecular complexity of cancer, the combinationtherapy of chemo and gene drugs becomes important for betterlong-term prognosis with fewer side effects.12 To furtherincrease therapeutic effects, advanced drug delivery systems(DDSs), capable of simultaneously delivering drugs and genes tothe site of action with specic time-programmed releaseproles, are important for drug development. Nanocarriersbased on cationic amphiphilic micelles for the simultaneous co-delivery of drugs and genes in combination therapy have beenstudied. Biodegradable cationic micelles were prepared fromPDMAEMA–PCL–PDMAEMA triblock copolymers and appliedfor the delivery of siRNA and paclitaxel (PTX) into cancer cells.The PTX-loaded PDMAEMA–PCL–PDMAEMA displayed higherdrug efficacy than free PTX and the co-delivery of VEGF siRNAand PTX showed an efficient knockdown of VEGF expression inPC3 cells.13 A co-delivery system was prepared from pluronic85-PEI/D-a-Tocopheryl polyethylene glycol1000 succinate (TPGS)/PTX/survivin shRNA (shSur) complex nanoparticles (PTPNs).The experimental results showed PTPNs could facilitate drugentry into cells and induce shSur into nuclei in both A549 and

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A549/T cells.14 Three amphiphilic star-branched copolymerscomprising polylactic acid (PLA) and PDMAEMA with AB3,(AB3)2, and (AB3)3 molecular architectures were synthesized toco-deliver microRNA and doxorubicin (DOX). The (AB3)3 archi-tecture micelles exhibited the highest transfection efficiency.8

When delivering DOX and miR-21 inhibitor (miR-21i) intoLN229 glioma cells, the micelles could mediate escaping miR-21i from lysosome degradation and the release of DOX intothe nucleus.

Co-delivery of chemo and gene drugs has a potential toefficaciously treat human diseases because of their synergeticeffects. Construction of a highly efficient multifunctional drugcarrier combining chemotherapy and gene therapy attractsmany researchers' attention. Pluronic copolymers consist ofhydrophobic poly(propylene) (PPO) segments and hydrophilicpoly(ethylene oxide) (PEO) segments. They self-assemble intomicelles in water with a hydrophobic core by PPO and ahydrophilic shell by PEO.15 Pluronic copolymers were used tomodify cationic polymers like PEI,16 PLL,17 and PDMAEMA18 toimprove gene transfection as well as to reduce their intrinsiccytotoxicity. The presence of the hydrophobic PPO block in thepluronic-modied PDMAEMA enhanced the association withDNA.18 Moreover, pluronic polymers promote cellular uptake ofpolyplexes because they inhibit P-glycoprotein (Pgp) to over-come multidrug resistance (MDR), subsequently enhancingtransgene expression.19

PDMAEMA is a well-known cationic polymer, easilycondensing nucleic acids and efficiently transfecting them. Thehigh transfection efficiency is attributable to the positive chargeof PDMAEMA forming a complex with nucleic acids and facili-tating a proton sponge effect. Compared with other cationicpolymers, PDMAEMA's macromolecular architecture is theeasiest to manipulate, including star, liner, gra, and block.20

In our previous work, we found PF127 modied PDMAEMA(PF127-b-pDMAEMA) reduced the cytotoxicity of PDMAEMA as agene vector and its transfection efficiency mainly depended onthe PDMAEMA chain length.21 The higher block length ofPDMAEMA indeed showed higher transfection efficiency butresulted in higher cytotoxicity as well. Since no one has usedpluronic-b-pDMAEMA to co-deliver chemo and gene drugs andcompared their performance using various pluronic polymerswith different hydrophilic/lipophilic balance (HBL) values. Inthis study, we synthesized a series of pentablock copolymersconsisting of pluronic polymers and a small length ofPDMAEMA (�35) to minimize the cytotoxic problem. Weselected three pluronic polymers, PF127, PP123, and PL121,with different lengths of PEO and similar lengths of PPO. Theeffect of the pluronic polymers used in preparation of penta-block copolymers on particle size, drug encapsulation, releasebehavior, cell viability, and gene expression were thoroughlystudied.

The pentablock copolymers were characterized using Fouriertransform infrared (FTIR) spectroscopy and 1H-nuclearmagnetic resonance (1H-NMR) spectroscopy. Hydrodynamicdiameters and zeta potentials were measured using dynamiclight scattering (DLS). Morphologies were obtained using atransmission electron microscope (TEM). To test the potency of

This journal is © The Royal Society of Chemistry 2014

pentablock copolymers as gene carriers, the gene transfectionefficiencies of polyplexes were studied using 293T cells. Toexamine the potency of pentablock copolymers as drug carriers,the cell viabilities of free epirubicin (EPI) and EPI-loadedmicelles were studied using 293T and KB cells. The co-deliveryeffect of EPI and pDNA was visualized using a confocal laserscanning microscope (CLSM).

ExperimentalMaterials

Pluronic F127, copper(I) bromide (CuBr), 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), deute-rium oxide (D2O) and deuterium chloroform (CDCl3) werepurchased from Sigma (St. Louis, MO). Epirubicin hydrochlo-ride was purchased from Zhejiang Hisun Pharmaceutical(Zhejiang, China). Pluronic L121 and P123 were purchased fromBASF (Ludwigshafen, Germany). 2,20-Bipyridine (Bpy), 2-bromo-2-methylpropionyl bromide, Amberlite® IR120, N,N-dimethylamino-2-ethyl methacrylate (DMAEMA) and pyrenewere purchased from Acros (Morris Plains, NJ). Aluminum oxideneutral (Al2O3) was obtained from Seedchem Company PTY.LTD (Melbourne, Australia). Fetal bovine serum (FBS) waspurchased from Biological Industries (Beit Haemek, Israel).Roswell park memorial institute medium (RPMI-1640) waspurchased from Invitrogen (Carlsbad, CA).

Preparation of macroinitiator (pluronic-Br)

In a two-neck round-bottom ask, each pluronic polymer (1mmol) was dissolved in 20 mL dichloromethane at roomtemperature. The solution was cooled to 0 �C and triethylamine(5 mmol) was added with stirring. Aer 20 min, 2-bromoiso-butryl bromide (5 mmol) was slowly added with a syringe thathad been purged with argon. Following 48 h reaction at roomtemperature, the product was obtained by precipitation inexcess n-hexane and dried under vacuum. The yield of PF127-Br,PP123-Br and PL121-Br were approximately 70.3, 42.8 and25.6%, respectively.

Preparation of pentablock copolymers

Pentablock copolymers (pluronic-b-pDMAEMA) were synthesizedusing a molar feed ratio of [DMAEMA] : [macroinitiator] :[CuBr] : [Bpy] at 40 : 1 : 2 : 2. Briey, macroinitiator (1 mmol),DMAEMA (40 mmol), 2-propanol (1.6 mL), and double-deionized(DD) water (0.4 mL) were added to a two-neck round-bottomask. The reaction was degassed by ve consecutive standardfreeze–pump–thaw cycles. CuBr (2 mmol) and Bpy (2 mmol) werequickly added to the mixture under an argon atmosphere.Following 4 h reaction at room temperature, the reaction wasstopped by diluting with DD water. The product was puried bydialysis against DD water using Mw cut-off 3500 membrane(Spectrum Labs, Rancho Dominguez, CA) for 2 days. The freeze-dried product was dissolved in toluene and passed throughaluminum oxide column and Amberlite® IR120 to remove catalystcomplexes. The puried solution was further precipitated inexcess n-hexane and dried under vacuum.

RSC Adv., 2014, 4, 31552–31563 | 31553

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Characterization of copolymers

The chemical structure of copolymers was determined usingproton nuclear magnetic resonance (1H-NMR) and Fourier-transform infrared (FTIR) spectroscopy. 1H-NMR spectra wereobtained from a Varian Mercury plus-200 spectrometer (PaloAlto, CA), using D2O and CDCl3 as a solvent. FTIR spectra wereacquired using a Perkin-Elmer System 2000 (Norwalk, CT). Themolecular weights of copolymers were measured by gelpermeation chromatography (GPC) using an Agilent 1100 series(Santa Clara, CA) equipped with a Shodex-KF804 column.Samples were dissolved in tetrahydrofuran (THF) at a concen-tration of 5 mg mL�1 and ltered through a 0.45 mm lter priorto injection. THF was used as an eluent at a ow rate of 1 mLmin�1. Polystyrene standards were used to generate a calibra-tion curve.

Acid-base titration was carried out using a PC-controlledsystem assembled with a 702 SM Titroprocessor, a 728 stirrer,and a PT-100 combination pH electrode (Metrohm, Herisau,Switzerland). Approximately 20 mg of each copolymer was dis-solved in 20 mL of 150 mM NaCl solution. The pH value of thesolution was adjusted to 2 using 0.1023 N HCl followed by back-titration to pH 11 using 0.0998 N NaOH.

Preparation and characterization of micelles

Micelles were prepared using a precipitation/solvent evapora-tion technique. Each pentablock copolymer (5 mg) in THF (200mL) was dropwise added into DD water (5 mL). The solution wassonicated for 3 min. The organic THF solvent was removed byrotary vacuum evaporation and the rest was freeze-dried. Fluo-rescence spectra were recorded on a Cary Eclipse uorescencespectrophotometer (Cary, Varian, CA). Pyrene was used as auorescence probe.22 Pyrene excitation spectra were recordedusing an emission wavelength at 390 nm. Emission and exci-tation slit widths were set at 2.5 and 2.5 nm, respectively. Acritical micelle concentration (CMC) value was determinedfrom the ratios of pyrene intensities at 339 and 334 nm andcalculated from the intersection of two tangent plots of I339/I334versus the concentrations of copolymers.

Epirubicin encapsulation (EPI-loaded micelle) and in vitrorelease

In this preparation, EPI HCl was solubilized in THF at aconcentration of 1 mg mL�1, containing 3 molar ratio of tri-ethylamine relative to EPI.23 To encapsulate epirubicin (EPI),THF (500 mL) containing a pentablock copolymer (5 mg) and EPI(0.5 mg) was dropwise added to DD water (5 mL). The mixturewas sonicated for 3 min. THF was removed by rotary vacuumevaporation and a 0.45 mm microporous lter was used toeliminate any unencapsulated EPI. The obtained micellarsolution was lyophilized and properly stored before furthercharacterization. To evaluate EPI loading efficiency, a driedsample was dissolved in THF and the concentration of EPI wasmeasured using a uorescence spectrometer at 590 nm. The EPIamount was calculated based on a standard calibration curve inEPI concentrations ranging of 0.8–12.5 mg mL�1. The loading

31554 | RSC Adv., 2014, 4, 31552–31563

efficiency (LE) and encapsulation efficiency (EE) were calculatedusing the following equations:

EE (%) ¼ (amount of drug in micelle/amount of drug in feed)

� 100 (1)

LE (%) ¼ {amount of drug in micelle/(amount of polymer

+ amount of drug in micelle)} � 100 (2)

The in vitro release of EPI was carried out in 0.1 Mphosphate-buffered saline (PBS) buffers at pH 7.4. The EPI-loaded micelle (30 mg) was placed in a dialysis membranewith MWCO of 3500 Da. The tube was then immersed in abeaker containing 30 mL of PBS, which was shaken at a speed of150 rpm and incubated at 37 �C. At predetermined time inter-vals, 2 mL of solution was withdrawn and the EPI content wasdetermined using a UV-vis spectrophotometer at 480 nm. Thesame amount of fresh PBS was replaced back to the beaker. Thereleased EPI amount was calculated from a standard calibrationcurve of pure EPI in the range of 2–40 mg mL�1.

Preparing plasmid DNA

pEGFP-C1 plasmid driven by a cytomegalovirus (CMV) promoterand pGL3-control plasmid with a Hind III/Xba I rey luciferasecDNA fragment cloned into the pCDNA vector were introducedinto the E. coli strain DH5a (Gibco-BRL, Gaithersbury, MD) andpuried using a kit (Maxi-V500 plasmid kit; Viogene, Sunnyvale,CA). The purity of the pDNA was certied by the absorbanceratio at OD260/OD280 and by distinctive bands of DNA fragmentsat corresponding base pairs in gel electrophoresis aerrestriction enzyme treatment of DNA. The pDNA was stored at�20 �C until used.

Preparing copolymer/pDNA polyplexes

All copolymer stock solutions were prepared at 2 mg mL�1 inDD water and the pH was adjusted to 5. The pDNA concentra-tion was xed at 3 mg/100 mL in DD water to measure pDNAbinding assay and 1 mg/100 mL for other studies. Equal volumesof pDNA and copolymer solution with different N/P ratiosranging of 1–20 were immediately vortexed at a high speed for60 s. Polyplexes were kept at room temperature for 10 min forcomplete complexation before analysis.

Characterization of nanoparticles

In this experiment, micelles were solubilized in DD water at aconcentration of 1 mg mL�1. The hydrodynamic diameters andzeta potentials of nanoparticles were measured using laserDoppler anemometry with a Zetasizer Nano ZS instrument(Marlvern Instruments, Worcestershire, UK). Light scatteringmeasurements were done with a laser at 633 nm and a 90�

scattering angle. Particle sizes and zeta potentials weremeasured three times. The morphologies of micelles (or poly-plexes) were observed using TEM (JEM-2000 EXII, JEOL, Japan).A carbon coated 200 mesh copper specimen grid (Agar ScienticLtd. Essex, UK) was glow-discharged for 1.5 min. The solution ofmicelles or polyplexes at N/P ¼ 9 was placed on the copper grid

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and allowed to dry for 5 days at room temperature. The imagesof samples were analyzed using Image J soware (NIH,Bethesda, MD).

Gel retardation assay

The DNA binding ability of polyplexes was evaluated using anagarose gel electrophoresis. The stability of copolymer/pDNApolyplexes with and without 10% FBS was evaluated using agel electrophoresis with 0.8% agarose in Tris acetate–EDTA(TAE) with EtBr (1 mg mL�1). A current of 100 V was applied tothe gels for 35 min, and DNA retention was visualized underultraviolet illumination at 365 nm.

Cell experiments

HEK 293T cells (human embryonic kidney 293T cell line) andKB cells (human nasopharyngeal epidermoid carcinoma cellline) were cultivated at 37 �C under humidied 5% CO2 inDMEM and RPMI-1640, supplemented with 10% FBS and 100mg mL�1 penicillin-streptomycin. The medium was replenishedevery three days and the cells were sub-cultured aer they hadreached conuence.

Cytotoxicity

The cytotoxicities of copolymers were measured using the MTTassay in 293T and KB cells. 293T cells or KB cells (5 � 103 cellsper well) were seeded in 96-well tissue culture plates at 37 �Cunder 5% CO2 for 24 h in DMEM and RPMI-1640 mediumcontaining 10% FBS. The cytotoxicity was evaluated by deter-mining the viability aer 24 h incubation with variousconcentrations of copolymers (2.5–100 mg of copolymer permilliliter).

The cytotoxicities of free EPI and EPI-loaded micelles weremeasured using the MTT assay in KB cells. The cells wereseeded in 96-well tissue culture plates at a density of 5 � 103

cells per well in RPMI-1640 medium containing 10% FBS at 37�C under 5% CO2 for 24 h. The equivalent EPI concentrations ofEPI-loaded micelles were controlled within 0.43–9.28 mM. Aer24 h, the cells were washed trice with PBS and incubated foranother 48 h. The number of viable cells was measured by theestimation of their mitochondrial reductase activity using thetetrazolium-based colorimetric method. The half maximalinhibitory concentration (IC50) was analyzed using SigmaPlotsoware.

In vitro transfection

The transfection assay was evaluated using a pGL-3 plasmid in293T cells. The transfection efficiencies of polyplexes werecompared with those of naked DNA as a negative control, Lip-ofectamine 2000 (LIPO, Invitrogen), and branched PEI (25 kDa,N/P ¼ 10) as positive controls. The 293T cells were seeded at adensity of 1 � 105 cells per well in 12-well tissue culture platesand incubated in DMEM medium containing 10% FBS for 24 hbefore transfection. When the cells were at 50–70% conuence,the culture medium was replaced with 1 mL of DMEM with orwithout 10% FBS. Polyplexes with N/P ratios ranging of 1–20

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were prepared using different amounts of copolymers and axed pDNA amount of 1 mg to a nal volume of 100 mL. Aer leto stand for 10 min, polyplexes were added to each well con-taining the cells and incubated for 4 h. The medium wasreplaced with 1 mL of fresh DMEM and the cells were incubatedfor 44 h post-transfection. The transfected cells were rinsedgently with 1 mL of 0.1 M PBS (twice) and added to a 200 mL perwell of lysis buffer (0.1 M Tris–HCl, 2 mM EDTA, and 0.1%Triton X-100, pH 7.8). The luciferase activity was monitoredusing a microplate scintillation and luminescence counter aermixing the contents of a 50 mL per well of supernatant with thecontents of 50 mL per well of luciferase assay reagent (Promega).The total protein content of the cell lysate was examined using aBCA protein assay kit (Pierce, Rockford, IL).

Confocal laser scanning microscopy (CLSM)

The cells were seeded at a density of 1 � 105 cells per well in 12-well plates containing one glass coverslip per well in RPMI-1640supplemented with 10% FBS and incubated for 24 h. Subse-quently, the culture medium was replaced with 1 mL of RPMI-1640 without 10% FBS. The EPI-loaded micelle (PL121-b-pDMAEMA)/pDNA polyplex was added and incubated for 4 h.The medium was replaced with 1 mL of fresh RPMI-1640 andthe cells were incubated for 24 h post-transfection. The cover-slips were removed and washed three times with PBS. The cellnuclei were stained using Hoechst 33342 (5 mg mL�1) for 15min. Next, the cells were xed with 3.7% paraformaldehyde for30 min and the cells on the coverslips were washed 3 times withPBS and mounted with uorescent mounting medium on glassslides. A CLSM (Fv 1000; Olympus, Tokyo, Japan) was used forcell imaging.

Statistical analysis

Means and standard deviations (SD) of data were calculated.Comparison between groups was tested using Student's t-testand P < 0.05 was considered as signicant.

Results and discussionPreparing and characterizing copolymers

In this work, an ATRP technique was applied to obtain penta-block copolymers. The central triblock copolymers were plur-onic F127, P123 and L121, which were reacted with 2-bromoisobutyl bromide to form ATRP macroinitiators followedby copolymerization with DMAEMA. The chemical structure ofPF127-Br was conrmed from the 1H-NMR spectrum at the peakintensity of 1.92 ppm (b, C(CH3)2–Br of 2-bromo-2-methylpropionyl groups) and 1.18 ppm (a, methyl protons ofthe PPO block) (Fig. 1a). The degree of halogenation wasdetermined to be �95%, indicating the terminal hydroxylgroups were approximately substituted. The PF127-Br wasfurther characterized using GPC. The number-averaged molec-ular weight (Mn) and polydispersity index (PDI) of PF127-Br were�13 000 g mol�1 and 1.27 (data not shown). The synthesis of aseries of pluronic macroinitiators was well-controlled.

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Fig. 1 (a) 1H-NMR and (b) FTIR spectra of PF127, PF127-Br and PF127-b-pDMAEMA.

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The pentablock copolymers composed of DMAEMA werefurther prepared via ATRP using pluronic-Br as a central block(Scheme 1). Fig. 1b shows the 1H-NMR spectrum of PF127-b-

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pDMAEMA. The chemical shis in the region of 2.25–2.63 ppmwere mainly associated with the methyl (b, N–CH3) andmethylene (c, N–CH2) protons of the DMAEMA segments. The

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Scheme 1 Synthesis of pluronic-b-pDMAEMA pentablock copolymers.

Table 1 Molecular weights and properties of copolymers

SampleMn,theo

a

(g mol�1)Mn

b

(g mol�1)DPc

(DMAEMA)DPd

(DMAEMA)DPe

(EO)DPf

(PO) HLBd PDIe ConversiongYield(%)

PF127-b-pDMAEMA 17 800 14 370 33 23 200 65 22 1.22 0.83 53.2PP123-b-pDMAEMA 12 080 7150 34 22 40 70 8 1.20 0.85 39.2PL121-b-pDMAEMA 10 680 6000 38 23 10 68 1 1.27 0.95 12.4

a Theoretical Mn calculated as (target DPn � 157 g mol�1) � actual fractional conversion achieved and includes initiator residue. b The number-averaged molecular weight (Mn) of copolymers was done by GPC. c The number of DMAEMA per copolymer chain was estimated by 1H-NMR.d The number of DMAEMA per copolymer chain was estimated by GPC. e The number of EO and PO segments and Hydrophilic/LipophilicBalance (HLB) were obtained from ref. 15. f PDI ¼ Mw/Mn measured by GPC. g The conversion was estimated by 1H-NMR.

Fig. 2 Titration profiles of PF127-b-pDMAEMA, PP123-b-pDMAEMAand PL121-b-pDMAEMA.

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chemical shi at 4.12 ppm was attributed to the methyleneprotons adjacent to the oxygen moieties of ester linkages (d,H2C–O–C]O). The number of repeating units of DMAEMA incopolymers could be determined from 1H-NMR spectra. Theintegration areas of the methyl (–CH3) protons at 1.18 ppm(peak a) of the pluronic segments and the methyl (N–CH3)protons of the DMAEMA segments at 2.25 ppm (peak b) wereused to calculate the nal copolymer composition. The numberof the DMAEMA segment of the three copolymers was�35 units(Table 1). This value could be correlated to the monomer/macroinitiator ratio in feed. The 1H-NMR spectra of PP123-b-pDMAEMA and PL121-b-pDMAEMA were shown in ESI Fig. S1.†

The FTIR spectrum of PF127-Br peaked at 1726 cm�1, whichwas attributable to characteristic C]O stretching. In addition,the enhanced stretching bands of PDMAEMA at 1726 cm�1

(C]O), 2767 cm�1 and 2819 cm�1 (C]N), implied thesuccessful polymerization of the pentablock copolymer(Fig. 1c). The FTIR spectra of PP123-b-pDMAEMA and PL121-b-pDMAEMA were shown in ESI Fig. S2.† The molecular weightsand their corresponding MW distribution were measured byGPC and listed in Table 1. The number of the DMAEMAsegments estimated from the MW measurements was�23 units, which was lower than the value calculated by NMR.This might be due to the fact that the MWmeasurement by GPCis relative to polystyrene standards, where its chemical structureis quite different from that of the pentablock copolymers. The

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narrow polydispersity (PDI) of 1.20–1.30 suggested the poly-merization kinetics of DMAEMA with pluronic-Br was success-fully controlled by ATRP.24

Fig. 2 shows the titration proles of PF127-b-pDMAEMA,PP123-b-pDMAEMA, and PL121-b-pDMAEMA. The copolymersshowed a similar buffer capacity. The apparent pKa values of the

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Scheme 2 Preparation of pluronic-b-pDMAEMAmicelle to co-deliverhydrophobic drugs and gene drugs.

Fig. 3 (a) Critical micelle concentration measurements of pluronic-b-pDlight scattering (DLS) diagrams of PF127-b-pDMAEMA micelle (b) withou

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amino groups were 7.29, 7.33, and 7.30, for PF127-b-pDMAEMA,PP123-b-pDMAEMA, and PL121-b-pDMAEMA, respectively.These values were close to that reported on pluronic L92-pDMAEMA (pKa ¼ 7.1)18 and independent of the central plur-onic polymers used.

Micellar properties

The amphiphilic copolymers of pluronic-b-pDMAEMA, con-sisting of the hydrophilic PDMAEMA and PEO segments and thehydrophobic PPO segments, provided an opportunity toexamine their self-assembly behavior in aqueous solutions

MAEMA; transmission electron microscope (TEM) images and dynamict EPI and (c) with EPI.

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Table 2 Hydrodynamic diameters, zeta potentials, and EPI encapsulation of micellesa

Sample Dh (nm) PDI Zeta (mV) E.E (%) L.E (%)

PF127-b-pDMAEMA 206.9 � 9.5 0.29 � 0.02 19.1 � 0.5 68.1 � 11.3 6.2 � 1.0PP123-b-pDMAEMA 271.0 � 30.0 0.33 � 0.02 21.5 � 0.6 78.7 � 10.6 7.2 � 0.9PL121-b-pDMAEMA 350.9 � 24.9 0.32 � 0.03 18.7 � 0.7 71.8 � 5.7 6.8 � 0.5

a E.E: encapsulation efficiency; L.E: loading efficiency.

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(Scheme 2). As listed in Table 1, the numbers of the PPO blockwere 65, 70, and 68, and those of the PEO block were 200, 40,and 10 for PF127, PP123, and PL121, respectively. To measurethe self-assembled ability of copolymers, the CMC values ofmicelles were determined using pyrene as a uorescence probe(Fig. 3a). The CMC values were 4.99 � 10�2, 3.64 � 10�2, and3.54 � 10�2 mg mL�1 for PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA, respectively. Comparedthe values with PF127, PP123, and PL121 of 3.53 � 10�2, 2.53 �10�2, and 4.40 � 10�3 mg mL�1,15 the introduction ofPDMAEMA to the pluronic polymers increased water-solu-bility,25 leading to an increase in CMC value. In addition, theCMC values of the copolymers showed the same trend as theirnascent pluronic polymers, showing the higher the hydropho-bicity the smaller the CMC value was.

The hydrodynamic diameters of micelles were measured byDLS and summarized in Table 2. The hydrodynamic sizes ofmicelles decreased as increasing the PEO block in a copolymer.The sizes were also estimated from TEM images, �175 nm, 180nm, and 250 nm for PF127-b-pDMAEMA, PP123-b-pDMAEMA,and PL121-b-pDMAEMA, respectively. These particle sizes weresmaller than the values measured by DLS because of the dryingeffect. The average core diameters of PF127-b-pDMAEMA(Fig. 3b), PP123-b-pDMAEMA (ESI Fig. S3a†), and PL121-b-pDMAEMA (ESI Fig. S3c†) were in the order of�80, 112, and 188nm, respectively. The sizes strongly depend on the ratio ofhydrophilic and hydrophobic segments in the backbone. Thelargest HLB value of 22 for PF127 forced the self-assembly of thehydrophobic PPO core the easiest, resulting in the smallestparticle size with the clearest corona layer.15 In contrast, thelowest HLB value of 1 for PL121 formed the biggest core area.The zeta potential values of the three micelles were similar,ranging from 18.7 to 21.5 mV, due to the analogous number ofcationic DMAEMA segments.

Fig. 4 In vitro EPI released profiles of EPI-loaded micelles done in 0.1M PBS at pH ¼ 7.4 at 37 �C (n ¼ 3).

Drug encapsulation and release

EPI was encapsulated via a precipitation/solvent evaporationmethod. All three micelles showed a similar loading capacity of�70% (Table 2). As seen in TEM images, all three drug-loadedmicelles showed a spherical shape with a nice size distribu-tion. The sizes of the EPI-loaded PF127-b-pDMAEMA (�330 nm,Fig. 3c) and EPI-loaded PP123-b-pDMAEMA micelles (�355 nm,ESI Fig. S3b†) increased dramatically as compared with thosewithout EPI (Fig. 3b and ESI Fig. S3a†). In the highest hydro-phobic PL121-b-pDMAEMA, both the DLS and TEM resultsshowed the size decreased aer the drug had been loaded(compared the image of ESI Fig. S3d† with that of S3c). The size

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of PL121-b-pDMAEMA decreased from 250 nm to 200 nm whenEPI was encapsulated. This might be explained by the enhancedhydrophobic effect in the drug and the core.26 In addition, thehydrophilic corona layer of the micelles faded when the drugwas loaded (see Fig. 3c, ESI Fig. S3b and S3d†) because of thehigh electron density of the drug.

Fig. 4 exhibits the in vitro drug release proles of EPI-loadedmicelles. The initial burst release of EPI might be attributed tothat EPI molecules located within the corona or at the interfacebetween the corona and core. Such EPI does not need to diffusethrough the large segments of the core and the drug release ratewas rapid. Following the burst release, EPI was continuallyreleased from the EPI-loaded micelles over 50 h without a lagtime. Interestingly, the release rate of PP123-b-pDMAEMA wasslightly faster than those of PF127-b-pDMAEMA and PL121-b-pDMAEMA. This result implied the PL121-b-pDMAEMA withthe lowest HLB enhanced the interactions between EPI and thehydrophobic PPO core and sustained the drug release. On theother hand, the PF127-b-pDMAEMA had the highest HLB,leading to a difficulty in drug diffusion because of a thickerhydrophilic layer on the surface.27 Hence, choosing a pluronicpolymer with a suitable HLB is possible to facilitate a control-lable drug release rate.

Cytotoxicity

The in vitro cytotoxicities of micelles were measured using anMTT method in KB (Fig. 5a) and 293T cells (Fig. 5b). The cell

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Fig. 5 Relative cell viabilities of (a) KB and (b) 293T cells exposed topluronic-b-pDMAEMA copolymers for 24 h (n ¼ 8); (c) relative cellviabilities of KB cells exposed to free EPI or EPI-loaded pluronic-b-pDMAEMA micelles at various EPI concentrations for 24 h followed by48 h post incubation (n ¼ 8).

Fig. 6 Gel electrophoresis to test pDNA retention in copolymers/pDNA polyplexes at variousN/P ratios (a) without 10% FBS, and (b) with10% FBS. Naked pDNA was used as a reference, and numerals of each

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viability was dose dependent in both the KB and 293T cells. Thecell viability dramatically dropped at a concentration of >30 mgmL�1 in KB cells but gradually decreased in 293T cells. Theimpact of different pluronic polymers on cell viability was trivialat a low concentration. However, at a high concentration of 100mg mL�1, it seems PF127-b-pDMAEMA had higher cell viabilitythan other two pentablock copolymers.

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To examine the cell viability of drug-loaded micelles, thecells were exposed to free EPI or the EPI-loaded micelles atvarious concentrations for 48 h (Fig. 5c). The IC50 values were0.20, 0.45, 0.79, and 0.55 mg mL�1 for free EPI, PF127-b-pDMAEMA/EPI, PP123-b-pDMAEMA/EPI, and PL121-b-pDMAEMA/EPI, respectively. Free EPI exhibited the best cyto-toxic effect as compared with the EPI-loadedmicelles because ofit having the most rapid diffusion of drug inside the cells toperform its cell-inhibiting effect. No signicance was observedamong the EPI-loadedmicelles with different pluronic polymersin the cytotoxic study.

Characterization of copolymer/pDNA polyplexes

The gel retardation assay was performed to examine the DNAbinding ability of copolymers and pDNA at various N/P ratios.Since no exposed pDNA was stained by EtBr at every N/P ratioranging of 1 �12, PF127-b-pDMAEMA, PP123-b-pDMAEMA, andPL121-b-pDMAEMA had excellent binding ability with pDNAboth with (Fig. 6b) and without 10% FBS (Fig. 6a).

Three copolymers/pDNA polyplexes showed stable hydrody-namic diameters at an N/P of $6 and their hydrodynamicdiameters were �100 nm (ESI Fig. S4a†). The polyplexes showedpositive charges with zeta potentials of 7–14 mV (ESI Fig. S4b†),slightly increasing with an increase in N/P ratio. The copolymers/pDNA polyplexes at N/P¼ 9 formed spheroid and their sizes wereapproximately 95 nm, 102 nm, and 108 nm for PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA/pDNA,respectively measured by TEM (ESI Fig. S5†). The strong elec-trostatic interactions between the positively-charged pluronic-b-pDMAEMA and the negatively-charged pDNA magnicently

graph indicate an N/P ratio.

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Fig. 7 Relative cell viabilities of copolymers and their copolymers/pDNA polyplexes in 293T cells as a function of polyplexes at variousN/P ratios (n ¼ 3, *P < 0.05). The N/P ratio of PEI (25K)/pDNA was 10.

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reduced the particle size of the polyplexes. In addition, pluronicpolymers assisted the polyplexes to form a core–shell structure, inwhich the core of PPO segments could stabilize PDMAEMA/pDNApolyplexes.18 Thus, a micelle-based gene delivery system couldfacilitate cellular uptake and enhance stability, leading toimproved gene expression.28

Fig. 8 In vitro gene expression of copolymers/pDNA polyplexes in293T cells using various N/P ratios (a) without 10% FBS, and (b) with10% FBS (n ¼ 3, *P < 0.05). The N/P ratio of PEI (25K)/pDNA was 10.

Cytotoxicity and in vitro gene transfection of polyplexes

The gene transfection efficiencies and cytotoxicities ofcopolymers/pDNA polyplexes at various N/P ratios were testedand compared with those of LIPO/pDNA, PEI (25K)/pDNA at N/P¼ 10, and naked pDNA in 293T cells. In Fig. 7, polyplexesexhibited higher cell viability than LIPO/pDNA and PEI/pDNA.As the N/P ratio increased, cell viability gradually decreased.At an N/P ratio of $15, the highest hydrophilic PF127-b-pDMAEMA/pDNA showed the lowest cytotoxicity as comparedwith PP123-b-pDMAEMA/pDNA and PL121-b-pDMAEMA/pDNA.This result agreed with the previous study using the copolymersalone (Fig. 5). Thomas et al.29 studied the cytotoxicity of unal-kylated and alkylated PEIs, and found the alkylated PEIs dis-played no or reduced cytotoxicity, compared with the parentPEI. Tian et al.30 also reported reduced cytotoxicity of PEI aerintroducing a biocompatible hydrophobic poly(g-benzyl-L-glutamate) moiety. In contrast, some authors have reportednegative effects of hydrophobic modications in comparisonwith parent polymers.31,32 Thus, the hydrophobic effect ofpolycations on cytotoxicity remains controversial.

The transfection ability of copolymers was assayed by pGL3-control plasmid for luminescence measurement and conductedin with and without 10% FBS culture medium. Without 10%FBS (Fig. 8a), PP123-b-pDMAEMA/pDNA and PL121-b-pDMAEMA/pDNA exhibited higher gene expression than didPF127-b-pDMAEMA/pDNA at a high N/P ratio of $6 (p < 0.05).The higher hydrophobic characteristics of PP123-b-pDMAEMA/pDNA and PL121-b-pDMAEMA/pDNA facilitated higher cellular

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uptake of the polyplexes with increasing interactions betweenthe cell membrane and the polyplexes. On the other hand, theserum-containing environment caused the aggregation betweenthe protein and polyplexes, reducing the gene transfection. Atan N/P ratio of $15 with 10% FBS (Fig. 8b), the gene trans-fection efficiency of pluronic-b-pDMAEMA/pDNA decreased butwas still higher than that of PEI/pDNA. The PL121-b-pDMAEMA/pDNA showed signicantly higher gene expression than theother two polyplexes at the N/P ratio of $15. According to theprevious report,33 the more hydrophobic PL121 supplied lessprotection to the polyplex than the more hydrophilic-formedpolyplexes against serum aggregation. On the other hand, thehighest hydrophobic effect of PL121 provided the highestpossibility for cellular uptake because of hydrophobic interac-tions. Based on the best gene expression observed in the PL121-b-pDMAEMA/pDNA, it seems the impact of hydrophobic prop-erty on cellular uptake is a dominating factor in this study.

Co-delivery

To verify the co-delivery effect of PL121-b-pDMAEMA, EPI andEGFP plasmid were used as the model drug and gene. The

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Fig. 9 Confocal images of the EPI-loaded PL121-b-pDMAEMA/pDNApolyplex at an N/P ratio of 15.

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solvent evaporation method was used for EPI encapsulationfollowed by condensation with pDNA. The EPI loadingconcentration of the PL121-b-pDMAEMA in this study was 0.625mg mL�1 and the EPI-loaded micelle/pDNA polyplex wasprepared at an N/P ratio of 15. The combination effect of theEPI-loaded micelle/pDNA was observed using CLSM in KB cells(Fig. 9). Aer 24 h of incubation, the CLSM image showed EPIwas delivered into the cells and released in the cytoplasm. Thesimilar co-delivery result was observed using PL121-b-pDMAEMA as a delivery vector (ESI Fig. S6†). The presence ofEPI did not affect gene transfection. The GFP protein was highlyexpressed inside the cells. Thus, we concluded that the plur-onic-b-pDMAEMA micelles seem a potential carrier for drug/gene co-delivery.

Conclusions

This study successfully synthesized PF127-b-pDMAEMA, PP123-b-pDMAEMA, and PL121-b-pDMAEMA copolymers with similarDMAEMA chain lengths via ATRP. The copolymers could easilyself-assemble into core–shell micelles in aqueous solutions toco-deliver EPI and pDNA into cancer cells. The micelles hadhigh capacity for EPI encapsulation and the EPI-loaded micelleshad similar cell-killing effects to free EPI. The copolymers werecapable of carrying pDNA, showing high gene transfectionefficiencies. Among the three copolymers, the PL121-b-pDMAEMA showed the highest gene transfection efficiency ascompared with the PF127-b-pDMAEMA and PP123-b-pDMAEMAin both with and without 10% FBS culture medium. The CLSMresult demonstrated the PL121-b-pDMAEMA delivered the EPImolecules and pEGFP inside the cells. The PL121-b-pDMAEMAmicelles displayed a combinational property.

Acknowledgements

We are grateful for the nancial support from the Ministry ofScience and Technology of Taiwan under grant numbers ofNSC-98-2221-E037-001-MY3 and NSC-102-2325-B037-005. Thisstudy is also supported by “Aim for the Top Journals Grant”

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under grant number KMU-DT103007 from Kaohsiung MedicalUniversity.

References

1 J. L. Markman, A. Rekechenetskiy, E. Holler andJ. Y. Ljubimova, Adv. Drug Delivery Rev., 2013, 65, 1866–1879.

2 M. D. Moya-Ortega, C. Alvarez-Lorenzo, A. Concheiro andT. Losson, Int. J. Pharm., 2012, 428, 152–163.

3 J. S. Lee and J. Feijen, J. Controlled Release, 2012, 161, 473–483.

4 D. E. Lee, H. Koo, I. C. Sun, J. H. Ryu, K. Kim and I. C. Kwon,Chem. Soc. Rev., 2012, 41, 2656–2672.

5 M. Elsabahy and K. L. Wooley, Chem. Soc. Rev., 2012, 41,2545–2561.

6 T. K. Endres, M. Beck-Broichsitter, O. Samsonova, T. Renetteand T. H. Kissel, Biomaterials, 2011, 32, 7721–7731.

7 J. Chen, H. Tian, X. Dong, Z. Guo, Z. Jiao, F. Li, A. Kano,A. Maruyama and X. Chen, Macromol. Biosci., 2013, 13,1438–1446.

8 X. Qian, L. Long, Z. Shi, C. Liu, M. Qiu, J. Sheng, P. Pu,X. Yuan, Y. Ren and C. Kang, Biomaterials, 2014, 35, 2322–2335.

9 Y. Wang, C. Y. Hong and C. Y. Pan, Biomacromolecules, 2013,14, 1444–1451.

10 J. Deng, N. Gao, Y. Wang, H. Yi, S. Fang, Y. Ma and L. Cai,Biomacromolecules, 2012, 13, 3795–3804.

11 S. Pan, C. Wang, X. Zeng, Y. Wen, H. Wu and M. Feng, Int. J.Pharm., 2011, 420, 206–215.

12 M. Khan, Z. Y. Ong, N. Wiradharma, A. B. Attia andY. Y. Yang, Adv. Healthcare Mater., 2012, 1, 373–392.

13 C. Zhu, S. Jung, S. Luo, F. Meng, X. Zhu, T. G. Park andZ. Zhong, Biomaterials, 2010, 31, 2408–2416.

14 J. Shen, Q. Yin, L. Chen, Z. Zhang and Y. Li, Biomaterials,2012, 33, 8613–8624.

15 A. V. Kabanov, E. V. Batrakova and V. Y. Alakhov, J.Controlled Release, 2002, 82, 189–212.

16 A. Albanese, P. S. Tang and W. C. Chan, Annu. Rev. Biomed.Eng., 2012, 14, 1–16.

17 E. Jeon, H. D. Kim and J. S. Kim, J. Biomed. Mater. Res., Part A,2003, 66, 854–859.

18 C. Alvarez-Lorenzo, R. Barreiro-Iglesias, A. Concheiro,L. Iourtchenko, V. Alakhov, L. Bromberg, M. Temchenko,S. Deshmukh and T. A. Hatton, Langmuir, 2005, 21, 5142–5148.

19 A. V. Kabanov, P. Lemieux, S. Vinogradov and V. Alakhov,Adv. Drug Delivery Rev., 2002, 54, 223–233.

20 S. Agarwal, Y. Zhang, S. Maji and A. Greiner, Mater. Today,2012, 15, 388–393.

21 S. J. Huang, T. P. Wang, S. I. Lue and L. F. Wang, Int. J.Nanomed., 2013, 8, 2011–2027.

22 T. Peng, Y. Li, D. Ahn, B. S. Kim and D. S. Lee, Macromol.Res., 2013, 21, 400–405.

23 K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano,Y. Sakurai, S. Fukushima, K. Okamoto and G. S. Kwon, J.Controlled Release, 2000, 64, 143–153.

24 K. Matyjaszewski, Macromolecules, 2012, 45, 4015–4039.

This journal is © The Royal Society of Chemistry 2014

Paper RSC Advances

Publ

ishe

d on

15

July

201

4. D

ownl

oade

d by

Chr

istia

n A

lbre

chts

Uni

vers

itat z

u K

iel o

n 27

/10/

2014

18:

34:1

8.

View Article Online

25 H. Y. Hong, Y. Y. Mai, Y. F. Zhou, D. Y. Yan and Y. Chen, J.Polym. Sci., Polym. Chem., 2008, 46, 668–681.

26 Q. He, W. Wu, K. Xiu, Q. Zhang, F. Xu and J. Li, Int. J. Pharm.,2013, 443, 110–119.

27 K. H. Jeong, S. H. Kwon and Y. J. Kim, Macromol. Res., 2008,16, 418–423.

28 D. J. Gary, H. Lee, R. Sharma, J. S. Lee, Y. Kim, Z. Y. Cui,D. Jia, V. D. Bowman, P. R. Chipman, L. Wan, Y. Zou,G. Mao, K. Park, B. S. Herbert, S. F. Konieczny andY. Y. Won, ACS Nano, 2011, 5, 3493–3505.

This journal is © The Royal Society of Chemistry 2014

29 M. Thomas and A. M. Klibanov, Proc. Natl. Acad. Sci. U. S. A.,2002, 99, 14640–14645.

30 H. Tian, W. Xiong, J. Wei, Y. Wang, X. Chen, X. Jing andQ. Zhu, Biomaterials, 2007, 28, 2899–2907.

31 A. Neamnark, O. Suwantong, R. K. Bahadur, C. Y. Hsu,P. Supaphol and H. Uludag,Mol. Pharm., 2009, 6, 1798–1815.

32 H. Eliyahu, A. Makovitzki, T. Azzam, A. Zlotkin, A. Joseph,D. Gazit, Y. Barenholz and A. J. Domb, Gene Ther., 2005,12, 494–503.

33 T. C. Lai, K. Kataoka and G. S. Kwon, Biomaterials, 2011, 32,4594–4603.

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