Redox-triggered intracellular dePEGylation based on diselenide-linked polycations for DNA delivery

Journal ofMaterials Chemistry B

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View Article OnlineView Journal | View Issue

MOE key Laboratory of Macromolecular Syn

of Polymer Science and Engineering, Zheji

China. E-mail: [email protected]; Fax: +

† Electronic supplementary information (mPEG–SS–PEI, mPEG–PEI, and transfecpolyplexes are provided. See DOI: 10.1039

Cite this: J. Mater. Chem. B, 2013, 1,6418

Received 7th September 2013Accepted 25th September 2013

DOI: 10.1039/c3tb21241f

www.rsc.org/MaterialsB

6418 | J. Mater. Chem. B, 2013, 1, 64

Redox-triggered intracellular dePEGylation based ondiselenide-linked polycations for DNA delivery†

Wenyu Li, Peng Zhang, Kun Zheng, Qiaoling Hu and Youxiang Wang*

Extracellular stability to protect DNA against nucleases and stimulus-triggered intracellular DNA release are

key factors in designing non-viral gene vectors. In this study, the diselenide-linked polycation mPEG–SeSe–

PEI was developed as a new type of PEG-detachable gene vector for redox-responsive gene delivery. The

corresponding stable analog mPEG–PEI and the disulfide-linked polycation mPEG–SS–PEI were

synthesized as controls. The results showed that all the PEGylated polycations could condense DNA into

tightly packed spherical nanoparticles about 80 nm in size, which showed excellent stability under

physiological conditions. The results of zeta-potential measurements, stability tests and DNA release

ability assay indicated that at a GSH concentration of 0.3 mM, the diselenide bonds were more easily

cleaved than disulfide bonds, which facilitated dePEGylation and DNA release. Meanwhile, it was

interestingly found that mPEG–SeSe–PEI/DNA polyplexes showed higher gene expression than mPEG–

SS–PEI/DNA polyplexes in both HEK293T and HepG2 cells. Confocal laser scanning microscope (CLSM)

images revealed that mPEG–SeSe–PEI/DNA polyplexes showed more efficient endosomal escape ability

than mPEG–SS–PEI/DNA polyplexes. Based on these results, the diselenide bonds as a novel strategy are

more suitable to address the challenging problem of extracellular stability and intracellular DNA release.

Introduction

Non-viral vectors have attracted much attention due to their lackof immunogenicity, ease of manufacturing, and large nucleicacid loading capacity.1–3 A typical example is polyethylenimine(PEI), which has been studied widely due to its proton spongeeffect for relatively high transfection efficiency in vitro.4–6

However, PEI-based polyplexes, like most polyplexes, tend torapidly aggregate under physiological salt conditions.7–9 Thesepolyplexes are rapidly cleared from the bloodstream owing tononspecic interaction with proteins in physiological uids,resulting in poor transfection in vivo.8 PEGylation has been apreferred strategy to prolong the in vivo circulation time as aconsequence of the reduction of nonspecic interactions.10–13

However, hydrophilic shielding layers of PEGmay reduce cellularuptake and impede intracellular DNA release, leading to lowtransfection efficiency.14,15 Therefore, the ideal gene polyplexesshould have a subtle balance between extracellular stability toprotect DNA against nucleases and intracellular instability topermit DNA dissociation inside target cells.

It has been well documented that intracellular and extra-cellular environments differ in their pH, enzymatic activity, and

thesis and Functionalization, Department

ang University, Hangzhou 310027, P. R.

86 571 87953729; Tel: +86 571 87953729

ESI) available: 1H NMR spectra of SePA,tion efficiency of all the PEGylated/c3tb21241f

18–6426

redox potential. These factors can be used as triggers fordisassociation of polyplexes. To overcome the limitation ofPEGylation, a great variety of PEG-conjugated polymers withstimulus-responsive chemical linkers, for example, acid-cleavable linkers,16–20 esterase cleavable lipid linkers,21,22 anddisulde bonds,23–25 have been proposed to enhance trans-fection efficiency. Intracellular glutathione (GSH) levels areapproximately 1000-fold higher than the concentration in theextracellular environment.26 Thus, disulde bonds have beenwidely used for reductive design in gene and drug delivery.Selenium and chalcogen sulfur are similar in many respects,including electronegativity, atom size, and accessible oxidationstates.27 The diselenide bonds possess reductive biodegradableability similar to that of disulde bonds.27–29 Specically, thebond lengths of disulde and diselenide bonds are 206 and232 pm, respectively.30 That is to say, diselenide bonds are moreeasily cleaved than disulde bonds. Zhang et al.31 synthesizednovel reduction-sensitive micelles based on a diselenide-containing PEG–PUSeSe–PEG triblock polymer. These micelleswere quite stable under a non-reductive environment, whereasthey could completely release Rhodamine B even at a relativelylow GSH concentration of 0.01 mg mL�1 within 8 h. Meanwhile,Gu et al.31 reported cross-linked oligoethylenimine using dis-elenide bonds to achieve efficient gene transfer with minimaltoxicity. However, few studies have focused on diselenide-linkedpolycations for DNA delivery to address the problematic chal-lenge of excellent extracellular stability and good intracellulargene release capability.

This journal is ª The Royal Society of Chemistry 2013

http://dx.doi.org/10.1039/c3tb21241f

http://pubs.rsc.org/en/journals/journal/TB

http://pubs.rsc.org/en/journals/journal/TB?issueid=TB001046

Scheme 1 Schematic illustration of DNA condensation and intracellular PEGdetachment induced by the cleavage of diselenide bonds.

Fig. 1 Synthetic route toward the preparation of various polycations (a) and the1H NMR spectrum of mPEG–SeSe–PEI (b).

Paper Journal of Materials Chemistry B

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. View Article Online

In this research, a diselenide bond was introduced betweenPEG and the PEI segment leading to a new type of PEG-detachable polycation (mPEG–SeSe–PEI) to address the abovedilemma of PEGylation. The corresponding stable analogmPEG–PEI and the disulde-linked polycation mPEG–SS–PEI wereseparately prepared and used as controls. The introduction ofPEG was expected to improve the stability and prolong the in vivocirculation time. The diselenide bonds were cleavable onceinside the cells and the redox-sensitive dePEGylation mightfacilitate pDNA release and gene expression (Scheme 1). Thechemo-physical properties of PEGylated polyplexes includingDNA binding ability, particle size, zeta-potential, and stabilityunder physiological conditions were characterized. Furthermore,their redox-sensitivity was tested and compared by monitoringtime-dependent particle sizes and by DNA release ability assay.The ability to mediate the gene expression of all the PEGylatedpolycations was further evaluated and compared.

ExperimentalMaterials

Branched polyethylenimine (PEI, 25 kDa) was purchased fromSigma-Aldrich. Selenium powder, sodium borohydride (NaBH4), 3-chloropropionic acid, hexanedioic acid, 2,20-dithiodipropionicacid, methoxypolyoxy-ethylene amine (mPEG–NH2, 5 kDa), N-hydroxy succinimide (NHS), and N,N0-dicyclohexylcarbodiimide(DCC) were obtained from Aladdin (Shanghai, China). Deoxy-ribonucleic acid (DNA, sh sperm, sodium salt), Cy3-DNA,glutathione (GSH), and N-[2-hydroxyethyl] piperazine-N0-[2-etha-nesulfonic acid] (HEPES, free acid, highpurity grade)wereobtainedfrom Sangon Biotech (Shanghai, China). Plasmid pEGFP (4733 bp)as plasmid DNA in the transfection experiment was purchasedfrom Clontech. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) was obtained from Bio Basic Inc. Lyso-tracker was purchased from J&K Chemical. Loading buffer waspurchased from TakaRa Biotechnology Co. Ltd (Dalian, China).

Synthesis of PEGylated polycations

The PEGylated polycations were prepared as illustrated inFig. 1a. Taking mPEG–SeSe–PEI for instance, 3,30-


diselenodipropionic acid (SePA) as a cross linker was rstlysynthesized according to the previous study with some modi-cations.32 Secondly, under a nitrogen atmosphere, 1.1 equiva-lent of SePA was activated by NHS (1.5 eq.) and DCC (1.5 eq.) inanhydrous dimethyl sulfoxide (DMSO) for 1 h. Then 1 equiva-lent of mPEG–NH2 dissolved in DMSO was added. The reactionmixture was maintained at room temperature for another 24 h.The resulting product was isolated by centrifugation to removeinsoluble by-products, precipitated in cold diethyl ether andthen puried by dialysis using a cellulose membrane (MWCO2000 Da) for 3 days followed by freeze-drying. Finally, mPEG–SePA (60 mmol), NHS (90 mmol), and DCC (90 mmol) dissolved in5 mL of anhydrous DMSO were added into a three-necked askand stirred under nitrogen for 1 h. PEI (10 mmol) dissolved in5mL of anhydrous DMSO was added into the abovemixture andstirred for another 24 h. The product was isolated as above anddialyzed against distilled water using a membrane (MWCO8000–14 000 Da) followed by freeze-drying. Similarly, mPEG–SS–PEI and mPEG–PEI were synthesized by the above processesusing 2,20-dithiodipropionic acid and hexanedioic acid as crosslinkers, respectively. The products were characterized by 1HNMR (300 MHz, Varian Spectrometer, USA) and FT-IR (Vector22, Bruker, Germany).

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Preparation of PEGylated polyplexes

Different polycations (mPEG–SeSe–PEI, mPEG–SS–PEI, andmPEG–PEI) were separately dissolved in 20 mM HEPES buffersolution containing 20 mM NaCl (pH 7.4), and PEGylated poly-plexes were prepared by vortexing an equal volume of polycationsolution with DNA solution (100 mg mL�1 in the above HEPESbuffer solution) at various N/P ratios. All the polyplexes wereprepared freshly and incubated for 30 min before analysis.

Characterization of PEGylated polyplexes

Agarose gel retardation assay. The DNA condensation capa-bility was examined by gel retardation assay. The polyplexescontaining 300 ng pDNA were prepared as above, mixed withloading buffer (5 : 1 by volume), loaded into each well of anagarose gel (1% by weight in 0.5 � TBE buffer) and subjected toelectrophoresis at 100 V for 50 min. Then the gel was immersedin ethidium bromide solution (0.5 mg mL�1) for 30 min andobserved using an UV illuminator (Gel Doc, Bio-Rad, USA).

Particle size and zeta (z-) potential measurements. Theparticle sizes and zeta potentials of the polyplexes weremeasured using a Malvern Zetasizer (Malvern Inst. Ltd., UK)equipped with either a four-side clear cuvette for particle sizeanalysis or a DTS 1060C cell for zeta-potential measurements.For particle size analysis, 4 serial measurements were carriedout at 25 �C (scattering angle 173�).

Transmission Electron Microscopy (TEM). TEM images wereobtained on a transmission electron microscope (JEM-1200EX,NEC, Tokyo, Japan) operated at 80 kV. A drop of the polyplexeswas deposited onto a 200-mesh carbon-coated copper grid for10 min. In order to obtain enough particles on the grid, theabove process was repeated three times.

Redox-sensitivity of diselenide- or disulde-linked polyplexes

The inuence of GSH on the zeta potentials of polyplexes.mPEG–SeSe–PEI/DNA and mPEG–SS–PEI/DNA polyplexes at anN/P ratio of 10 were prepared as described above and incubatedwith 0.3 mM GSH for 1 h. The GSH was prepared in the 20 mMHEPES buffer solution (pH 7.4, containing 20 mM NaCl). Thenthe zeta potentials of polyplexes treated with or without 0.3 mMGSH were evaluated by zeta potential measurements.

The inuence of GSH on the stability under physiologicalsalt conditions. The polyplexes at an N/P ratio of 10 wereprepared as mentioned above. The polyplexes were incubated inthe absence or presence of 0.3 mM GSH for 1 h. Then the saltconcentration was adjusted to 150 mM salt, and the time-dependent particle sizes were monitored using a Zetasizer(Malvern Inst. Ltd. UK).

DNA release ability assay

The protocol used to assess the DNA release ability of thePEGylated polyplexes in response to GSH was according to theliterature.33 The polyplexes containing 300 ng pDNA at an N/Pratio of 10 were incubated with or without GSH at a nalconcentration of 0.3 mM for 1 h, and mixed separately withheparin at various nal concentrations of 0, 10, 20, 30, 40, 60

6420 | J. Mater. Chem. B, 2013, 1, 6418–6426

and 80 mg mL�1. The mixture was incubated for 30 min andthen agarose gel electrophoresis was performed according tothe above method.

Cell cytotoxicity assay

A human embryonic kidney cell line (HEK293T) and a humanhepatoblastoma cell line (HepG2) were cultured in DMEM with10% FBS and 1% penicillin-streptomycin. Cells were main-tained under humidied air containing 5% CO2 at 37 �C. Cellcytotoxicity of different polycations was evaluated by MTT assay.Briey, the cells were seeded into 96-well plates at a density of8 � 103 cells per well. Following 24 h incubation, various pol-ycations were added into the cells at nal concentrations of 5,10, and 25 mg mL�1. Aer incubation for 48 h, 20 mL of MTT(5 mg mL�1, dissolved in PBS) was added. The cells were incu-bated for 4 h at 37 �C. Then the medium was removed, 200 mL ofDMSO was added and incubated for an additional 15 min at37 �C. The absorbance of 100 mL of the above mixture at 570 nmwasmeasured using a microplate reader (550, Bio-Rad, USA). Allexperiments were performed in sextuplicate.

In vitro transfection efficiency

HEK293T and HepG2 cells were seeded in 24-well plates at adensity of 5 � 104 cells per well. Before transfection, themedium was replaced by fresh DMEM with 10% FBS. Differentpolyplexes with 2 mg pEGFP at an N/P ratio of 10 were added andincubated for 6 h. PEI/DNA polyplexes at an N/P ratio of 10 wereused as control. Aer the cells were transfected for a total of48 h, they were observed using a uorescence microscope andtransfection efficiency was detected by ow cytometry (BDFACSCALIBUR, BD Bioscience, USA). All transfection experi-ments were performed in triplicate.

Confocal laser scanning microscope (CLSM) observation

HEK293T cells were seeded in glass base dishes at an initialdensity of 5 � 104 cells per dish and maintained overnight in1.5 mL of medium. Before the experiment, fresh culture mediumwas replaced. Then various polyplexes with 6 mg Cy3-DNA at anN/P ratio of 10 were added and incubated for 6 h in 1.5 mL ofmedium, followed by incubation in the absence of polyplexes foranother 6 h. Then, the endosome/lysosome was stained by lyso-tracker according to the manufacturer's protocol. The intracel-lular trafficking of Cy3-DNA was observed using a confocal laserscanning microscope (CLSM, Leica TS SP5, Germany).

Statistical analysis

Data are expressed as mean � standard deviation (SD). Statis-tical analysis was performed by ANOVA. The signicant levelwas set at p < 0.05.

Results and discussionSynthesis and characterization of PEGylated polycations

The synthetic route is shown in Fig. 1a. Briey, SePA as a cross-linker containing diselenide bonds was synthesized by reacting




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3-chloropropanoic acid with Na2Se2. The product was charac-terized by 1H NMR and FT-IR. As shown in Fig. S1 (see ESI†), thepeaks at d ¼ 2.65–2.75 and 3.00–3.10 were assigned to theprotons of the SeSeCH2CH2. In addition, the wavenumbers at1260 cm�1 and 927 cm�1 were associated with the stretchingand bending vibration of C–Se, respectively. These results sug-gested that SePA was successfully synthesized. To obtain anincreased number of carboxyl groups terminated on mPEG, themolar ratio of amine groups of mPEG–NH2 to carboxylategroups was controlled at 1 : 2.2. Subsequently, the carboxylgroup of mPEG–SePA reacted with amino groups of PEI to formmPEG–SeSe–PEI. As shown in Fig. 1b, the peak at 3.5 ppmreected the protons of the CH2 of PEG and the peaks at 2.0–2.8 ppm were assigned to the protons of the CH2CH2NH of PEI.Importantly, the PEG graing level was calculated by integra-tion of signals corresponding to the hydrogen atoms of the CH2

in PEG units and the hydrogen atoms of CH2CH2NH in PEIunits. The results suggested that each PEI backbone was graedwith about 4–5 PEG segments. According to the aboveprocesses, the corresponding stable analog mPEG–PEI and thedisulde-linked polycation mPEG–SS–PEI with a similar PEGgraing level to mPEG–SeSe–PEI were separately prepared ascontrols. Their 1H NMR spectra (Fig. S2, see ESI†) were similarto those of mPEG–SeSe–PEI.

Characterization of PEGylated polyplexes

For gene delivery, the ability of polycations to condense DNAinto the polyplexes is the primary requirement.34 Thus, the DNAbinding capability of polycations was examined by agarose gelelectrophoresis using PEI as a control. As shown in Fig. 2, allPEGylated polyplexes at N/P (nitrogen of PEI/phosphate of DNA)ratios ranging from 0.5 to 10 were electrophoresed separately inagarose gel. No uorescent DNA bands were observed for PEI(Fig. 2a) above the N/P ratio of 1, suggesting that the migrationof DNA in agarose gel was completely retarded. All PEGylatedpolycations including mPEG–SeSe–PEI, mPEG–SS–PEI, and

Fig. 2 Agarose gel electrophoresis assay of PEI/DNA (a), mPEG–SeSe–PEI/DNA(b), mPEG–SS–PEI/DNA (c), and mPEG–PEI/DNA (d) at various N/P ratios in 20 mMHEPES buffer solution (pH 7.4, 20 mM NaCl).


mPEG–PEI exhibited good DNA binding ability and the migra-tion of DNA in agarose gel was completely retarded above theN/P ratio of 2. These results suggested that PEGylation hadsome inuence on the DNA condensation ability, which wasconsistent with the previous study.35

It has been documented that the particle sizes and zeta-potentials are the major factors inuencing biodistribution andtransfection efficiency in gene delivery.36 All PEGylated poly-cations including mPEG–SeSe–PEI, mPEG–SS–PEI, and mPEG–PEI could condense DNA into small nanoparticles with adiameter of about 70–110 nm and the polydispersity index (PDI)of these polyplexes was in the range of 0.1–0.3. Above the N/Pratio of 10, the particle size of all the PEGylated polyplexesremained at around 80 nm, which was within the size require-ments for efficient cellular endocytosis.37 The well compactednanoparticles had a zeta-potential of about 10 mV (Fig. 3) and itwas signicantly decreased compared with that of PEI/DNApolyplexes (about 24 mV, not shown), due to the charge-shielding effect of PEG layers. The typical TEM image showedthat all the PEGylated polyplexes had a regular spherical shape(Fig. 4).

Redox-sensitivity of diselenide- or disulde-linked polyplexes

The intracellular GSH concentration (1–10 mM) is substantiallyhigher than extracellular levels (2 mM in plasma).38 Generally,normal cells have a lower concentration of cytosolic GSHcompared to the level of tumor cells.39 For gene delivery, poly-plexes inside cells were expected to quickly decondense DNA forefficient gene expression. Herein, 0.3 mM GSH, a relatively lowconcentration of GSH compared to the level of tumor cells, wasused to evaluate the redox-sensitivity of mPEG–SeSe–PEI/DNA

Fig. 3 Particle sizes (a) and zeta potentials (b) of various complexes composed ofmPEG–SeSe–PEI/DNA, mPEG–SS–PEI/DNA, and mPEG–PEI/DNA polyplexes in20 mM HEPES buffer solution (pH 7.4, 20 mM NaCl). The data are presented asmean � SD (n ¼ 3).

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Fig. 4 Typical TEM images of mPEG–SeSe–PEI/DNA (a), mPEG–SS–PEI/DNA (b),and mPEG–PEI/DNA (c) polyplexes at an N/P ratio of 10 in 20 mM HEPES buffersolution (pH 7.4, 20 mM NaCl). The scale bars represent 200 nm.

Fig. 6 (a) Time-dependent change of the particle sizes of various PEGylatedpolyplexes under physiological salt (150 mM NaCl) conditions. The polyplexeswere treated with or without 0.3 mM GSH for 1 h at 37 �C (0 min means asprepared conditions); (b) the particle sizes of different polyplexes under physio-logical salt conditions in the absence or presence of 0.3 mM GSH for 24 h. Thedata are presented as mean � SD (n ¼ 3). * denotes statistically significantdifference at p < 0.05.


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and mPEG–SS–PEI/DNA polyplexes. Firstly, the zeta-potential ofthe polyplexes was measured respectively in the absence orpresence of 0.3 mM GSH. As shown in Fig. 5, aer treatment ofGSH for 1 h at 37 �C, the zeta-potentials of mPEG–SeSe–PEI/DNA polyplexes signicantly increased from around 8 mVto 18 mV and mPEG–SS–PEI/DNA polyplexes showed lessincrease, while no change was observed for mPEG–PEI/DNApolyplexes. These results indicated that in the experimentalreductive environment, the intermediate diselenide bondscan be cleaved quickly, resulting in effective dePEGylation.However, the bond energy of disulde bonds was larger thanthat of diselenide bonds. The cleavage of disulde bonds wasmore difficult than that of diselenide bonds.

On the other hand, during the DNA delivery process, thestability of polyplexes before endocytosis at the targeted siteis a prerequisite for efficient gene transfection.25 Time-dependent particle sizes were also investigated. As shown inFig. 6a, all PEGylated polyplexes showed excellent stabilityunder physiological salt conditions. Aer treatment with GSH,mPEG–SeSe–PEI/DNA polyplexes formed large aggregatesrapidly with sizes of nearly 1 mm under physiological condi-tions for 45 min, while the particle size of mPEG–SS–PEI/DNApolyplexes only increased from around 150 nm to 400 nm

Fig. 5 Zeta-potentials of different polyplexes in the absence or presence of 0.3mM GSH in 20 mM HEPES buffer solution (pH 7.4, 20 mM NaCl) for 1 h at 37 �C.The data are presented as mean � SD (n ¼ 3). * denotes statistically significantdifference at p < 0.05.

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within 45 min. The mPEG–PEI/DNA polyplexes still showedexcellent stability. Combined with the results of zeta-potentials,we speculated that due to the lower bond energy, the dis-elenide bonds were easily cleaved than disulde bonds. Therapid dePEGylation of mPEG–SeSe–PEI/DNA polyplexes led toaggregates under physiological salt conditions. However,when mPEG–SS–PEI/DNA polyplexes were incubated underphysiological salt conditions in the presence of GSH for 24 h(Fig. 6b), disulde bonds could also be cleaved and it resultedin large aggregates. The mPEG–SeSe–PEI/DNA polyplexesshowed quick redox-sensitivity compared with mPEG–SS–PEI/DNA polyplexes.

Many anionic proteins in the bloodstream would competewith the surface-covered cationic polymers and lead to DNArelease. Subsequently, in vitro pDNA decondensation experi-ments in the presence of 0.3 mM GSH were investigated tocompare the ability of DNA release caused by dePEGylation ofthe polyplexes. The concentration of heparin at which DNA isreleased from the polyplexes was used to evaluate the DNArelease ability of the PEGylated polyplexes.33 As shown in Fig. 7,for all the PEGylated polyplexes, a free pDNA band began toemerge at a heparin concentration of 60 mgmL�1, suggesting thatpDNA was decondensed and released partly from the polyplexes.However, aer treatment with 0.3 mM GSH, no detectablechanges were observed for mPEG–PEI/DNA polyplexes. ThemPEG–SeSe–PEI/DNA and mPEG–SS–PEI/DNA polyplexescompletely released their DNA at heparin concentrations of 40and 60 mg mL�1, respectively. These results further conrmed



Fig. 8 Relative cell viability of HEK293T (a) and HepG2 (b) cells exposed todifferent polycations at various concentrations. The data are presented as mean�SD (n ¼ 6). * denotes statistically significant difference at p < 0.05.

Fig. 7 Agarose gel electrophoresis retardation assay of mPEG–SeSe–PEI/DNA (aand b), mPEG–SS–PEI/DNA (c and d), and mPEG–PEI/DNA (e and f) at an N/P ratioof 10 treated without or with 0.3 mM GSH and incubated in the presence ofvarious concentrations of heparin. The first lane on the left of the figures is pDNA.


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that in the experimental reductive environment, the intermediatediselenide bonds were more easily cleaved than disulde bonds,which facilitated dePEGylation and DNA release.

Cell cytotoxicity study

The cell cytotoxicity of mPEG–SeSe–PEI, mPEG–SS–PEI, andmPEG–PEI was investigated by MTT assay using the humanembryonic kidney cell line (HEK293T) and the human hep-atoblastoma cell line (HepG2) using PEI as a control. As shownin Fig. 8, the cell cytotoxicity of all polycations increased withthe polymer concentration. The PEGylated polycations showedlower cytotoxicity than PEI in both HEK293T and HepG2 cells(Fig. 8). For instance, at the same concentration of 25 mg mL�1,only around 30% viability was found aer treatment withPEI, while all the PEGylated polycations still had 80–90%viability. These results indicated that PEGylation signicantlydecreased the cell cytotoxicity of all the PEGylated polycations,which was due to the well-known biocompatibility of PEGand the reduced disturbance of the cell membrane caused bythe charge-shielding effect of PEG layers. A similar ndingindicated that reduced cytotoxicity of cationic polymer wasachieved by introducing the PEG segment into the cationicpolymer.40

Fig. 9 pEGFP expression mediated by polycations/DNA polyplexes at an N/Pratio of 10 exposed to HEK293T cells (a) and HepG2 cells (b). The data are pre-sented as mean � SD (n ¼ 3). * denotes statistically significant difference at p <0.05.

In vitro transfection of mPEG–SeSe–PEI/DNA polyplexes

The in vitro transfection efficiency of mPEG–SeSe–PEI/DNA,mPEG–SS–PEI/DNA, and mPEG–PEI/DNA polyplexes was


evaluated in HEK293T and HepG2 cells using pEGFP. PEI/DNApolyplexes as golden standard transfection vectors at anN/P ratio of 10 were used as a control. Herein, the mean

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uorescence intensity of transfected cells was used to evaluatethe gene expression.

As shown in Fig. 9, the mean uorescence of transfected cellswas apparently dependent on cell types, and higher uores-cence intensity was observed in the HEK293T cells. The mPEG–PEI/DNA polyplexes had the lowest gene expression among thePEGylated polyplexes, which was due to the hindered DNArelease ability caused by PEG shells.15,41 It was interestinglyfound that mPEG–SeSe–PEI/DNA polyplexes showed highergene expression than mPEG–SS–PEI/DNA in HEK293T cells(Fig. 9a), similar to the trend observed in HepG2 cells (Fig. 9b).For example, in HEK293T cells, the uorescence intensity ofmPEG–SeSe–PEI/DNA polyplexes at an N/P ratio of 10 wasalmost 1.8-fold higher than that of mPEG–SS–PEI/DNA poly-plexes. The percentage of transfected cells detected by owcytometry showed the same trend as the results for uorescenceintensity (Fig. S3, see ESI†). In our previous study, it was foundthat intracellular dePEGylation caused by light irradiation wasbenecial for DNA release and efficient transfection.41 For genedelivery, fast endosomal escape capability was needed to avoidthe degradation of DNA and polyplexes inside cells were

Fig. 10 Intracellular distribution of pDNA polyplexed with mPEG–PEI (a), mPEG–Slabeled pDNA (red) were incubated with HEK293T cells for 6 h, followed by incubperformed using a 63� objective. The acidic late endosomes and lysosomes were stathe yellow fluorescence overlapped by red and green fluorescence.

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expected to quickly decondense DNA for efficient gene expres-sion. Due to the quick redox-sensitivity of diselenide bonds, wespeculated that the higher transfection of mPEG–SeSe–PEI/DNApolyplexes might be due to fast endosomal escape capabilityand facilitated DNA release.

Mechanism of the enhanced gene expression of mPEG–SeSe–PEI/DNA polyplexes

Next, to gain more insight into the relationship between theintracellular dePEGylation and transfection efficiency, intra-cellular trafficking of DNA was observed using a confocal laserscanning microscope (CLSM). mPEG–SeSe–PEI, mPEG–SS–PEI,and mPEG–PEI complexed with Cy3-DNA (red) at an N/P ratio of10 were prepared as above. HEK293T cells exposed to variouspolyplexes were incubated for 6 h followed by incubation in theabsence of polyplexes for another 6 h. Lyso Tracker (green) wasused as an endo/lysosomal marker. As shown in Fig. 10a, thegreen and red uorescence partly overlapped to generate yellowuorescence inside the cells (indicated by arrows), whichmeansthat many of the mPEG–PEI/DNA polyplexes were colocalized in

S–PEI (b), and mPEG–SeSe–PEI (c) at an N/P ratio of 10. The polyplexes with Cy3-ation in the absence of polyplexes for another 6 h. The CLSM observation wasined with lyso-tracker (green). The scale bar represents 10 mm. The arrows indicate




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endo/lysosomes even aer incubation for 12 h. In contrast, formPEG–SS–PEI/DNA polyplexes with disulde bonds (Fig. 10b), asmall amount of overlapped yellow uorescence was stillobserved. However, in the case of mPEG–SeSe–PEI/DNA poly-plexes, almost no yellow uorescence was observed, suggestingfast endosomal escape. The reason was probably due to theincreased osmotic pressure in the endosome induced bydetached PEG chains.42 These results conrmed our aboveinference. Combined with the DNA release ability assay, wespeculated that the fast endosomal escape capability of mPEG–SeSe–PEI/DNA polyplexes was attributed to the activity of thediselenide bonds, which were most easily destroyed by reducingagents. Therefore, compared with the disulde bonds, the quickredox-sensitivity of diselenide bonds endowedmPEG–SeSe–PEI/DNA polyplexes with fast endosomal escape ability and facili-tated DNA release capability, resulting in efficient transfection.

Conclusions

In summary, a diselenide-linked cationic polymer mPEG–SeSe–PEI was synthesized and developed with the aim of detachingthe PEG layers in response to the intracellular reducing envi-ronment. The corresponding stable analog mPEG–PEI andthe disulde-linked polymer mPEG–SS–PEI were separatelyprepared and compared. All PEGylated polycations composed ofmPEG–SeSe–PEI and mPEG–SS–PEI exhibited high DNAbinding ability above an N/P ratio of 2. They could condenseDNA into small spherical nanoparticles with a diameter ofabout 70–110 nm and showed excellent stability under physio-logical salt conditions. Aer treatment with 0.3 mM GSH,mPEG–SeSe–PEI/DNA polyplexes showed fast aggregation,while the particle size of mPEG–SS–PEI/DNA polyplexes onlyincreased from around 150 nm to 400 nm within 45 min. Theseresults indicated that in the experimental reductive environ-ment, diselenide bonds were more easily cleaved than disuldebonds. The DNA release ability assay indicated that in thepresence of GSH, mPEG–SeSe–PEI/DNA polyplexes completelyreleased DNA at a lower concentration of heparin than mPEG–SS–PEI/DNA polyplexes, which was benecial for the intracel-lular DNA release and efficient gene transfection. Meanwhile, itwas interestingly found that mPEG–SeSe–PEI/DNA polyplexesshowed higher gene expression than mPEG–SS–PEI/DNA poly-plexes in both HEK293T and HepG2 cells. CLSM imagesrevealed that mPEG–SeSe–PEI/DNA polyplexes showed moreefficient endosomal escape ability than mPEG–SS–PEI/DNApolyplexes. These results suggested that the diselenide-linkedcationic polymer had great potential as an effective bio-responsive gene vector for gene therapy.

Acknowledgements

This work was nancially supported by the National NaturalScience Foundation of China (21074110, 51273177).

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Redox-triggered intracellular dePEGylation based on diselenide-linked polycations for DNA delivery