Acta Biomaterialia 2 (2006) 207–212www.actamat-journals.com

Brief communication

Novel block copolymer (PPDO/PLLA-b-PEG): Enhancementof DNA uptake and cell transfection

Shanta Raj Bhattarai a, Ho-Keun Yi b, Narayan Bhattarai c, Pyoung Han Hwang d,Hak Yong Kim e,¤

a Department of Bionanosystem Engineering, Chonbuk National University, Chonju, South Koreab Department of Biochemistry, School of Dentistry, Chonbuk National University, Chonju, South Korea

c Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USAd Department of Pediatrics, School of Medicine, Chonbuk National University, Chonju, South Korea

e Department of Textile Engineering, Chonbuk National University, 664-14, 1-Ga Dukjin-Dong, Dukjin-Gu, Chonju, Chhalabucdo 56-756, South Korea

Received 24 June 2005; received in revised form 19 September 2005; accepted 6 October 2005

Abstract

The cationic lipid mediated uptake of plasmid DNA by cells in monolayer culture was signiWcantly enhanced with an aqueous solutionof the block copolymer poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol) (PPDO/PLLA-b-PEG). Plasmid uptake studies withDNA encoding the �-galactosidase gene and cytotoxicity evaluations were performed on MCF-7, NIH 3T3 and CT-26 cell lines. Trans-fection yields and time courses for maximum release of FITC labeled DNA in MCF-7 cells were observed and quantiWed by �-galactosi-dase assay and spectroXuorometry, respectively. The reported results suggest that the studied block copolymer might be useful for theenhancement of polycation mediated transfection and could Wnd application in gene therapy. 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol); Biodegradable DNA; Cell transfection; Cell proliferation

1. Introduction

Polyelectrolyte complexes formed between DNA andpolycations have recently been used for gene transfer inmammalian and bacterial cells [1,2]. Due to electrostaticbinding of DNA and polycations, these complexes arespontaneously formed in aqueous media. Polycations likepoly(L-lysine) (PLL), poly(N-ethyl-4-vinyl pyridinium bro-mide) (PEVP), and others permit uptake of the complexand can achieve cell transfection in the absence of bindingwith receptor [3]. Transfection reagents (Lipofectin andcalcium phosphate) are commercially available for thispurpose, but one problem encountered using these tech-niques is the relatively low eYcacy of DNA (or complex)release from endocytic compartments in the cytoplasm and

* Corresponding author. Tel.: +82 063 270 2351; fax: +82 063 270 2348.E-mail address: khy@moak.chonbuk.ac.kr (H.Y. Kim).

1742-7061/$ - see front matter 2005 Acta Materialia Inc. Published by Elsedoi:10.1016/j.actbio.2005.10.007

nucleus of the cell [1,2], resulting in low eYciency of geneexpression. Further, due to charge neutralization, thesecomplexes are often unstable in aqueous solutions and pre-cipitate, thereby hindering their application in gene deliv-ery [4].

Recently, nonionic micelles or unimers of surfactantblock copolymers have been used to transfer water insolu-ble drugs and polypeptides [4,5]. Work on these systemssuggested that they enhance the transport of chargedmolecules across cell membranes [6]. To the best of ourknowledge, there has been no previous report on applyingthe block copolymer poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol) (PPDO/PLLA-b-PEG) as anenhancer to improve polycation mediated cell transfection.Focusing on this application, we prepared an aqueous solu-tion of the triblock copolymer which was synthesized byour research group previously [7], and report here on itsability to enhance DNA uptake and cell transfection.

vier Ltd. All rights reserved.

208 S.R. Bhattarai et al. / Acta Biomaterialia 2 (2006) 207–212

2. Materials and methods

2.1. Preparation and characterization of copolymer particles in aqueous solutions

The preparation and characterization of the blockcopolymer was performed as previously published [7,8].(The weight average molecular weight of the copolymerwas 19,000, copolymer composition was PPDO/PLLA/PEG: 10/20/70, with the molecular weight of PEG fractionat 10,000.) A solution of triblock copolymer with speciWedconcentration in acetonitrile was added dropwise intodeionized water at room temperature. Then, the acetonitrilewas evaporated from the suspension under reduced pres-sure and the aqueous suspension was Wltered through a0.45�m pore disposable Wlter. The block copolymer con-centration in the Wnal suspension was 0.3%.

2.2. Size and �-potential measurements

The size and �-potential measurements were also per-formed as previously published [8]. The particulate size inthe highly dispersed solution was determined by dynamiclight scattering using a Malvern System 4700 Instrument.All experiments were performed at 25 § 1 °C. The correla-tion decay function was analyzed by the cumulate methodto determine the average particle diameter. The �-potentialof the aqueous solution of the block copolymer was deter-mined by a laser zeta potential instrument (ELS-8000/6000,Otsuka Electronics). Measurements were performed at25 § 1 °C, on samples approximately diluted with 1 mMHEPES buVer.

2.3. Cell culture

Cells (NIH 3T3; mouse embryo cell, CT-26; colon cancercell and MCF-7; breast cancer cell) were purchased fromKorea Cell Line Bank (Seoul, Korea). These cell lines wereused for the transient transfection and the cytotoxicityexperiments, and grown at 37 °C under 5% CO2 atmo-sphere. The following media were used: (1) Dulbecco’smodiWed Eagle’s medium (DMEM) (Gibco) with 10% (v/v)fetal calf serum (Gibco) for CT-26 and MCF-7 cells, and (2)RPMI-1640 medium containing 10% (v/v) fetal bovineserum (FBS) (Gibco) for 3T3 cells. For all media, penicillin(100 U/mL) and streptomycin (100 �g/mL) was used.

2.4. Cytotoxicity evaluation

The toxicity of the aqueous solution of the block copoly-mer was evaluated by quantifying MCF-7, CT-26 and 3T3cell viability in vitro. BrieXy, three diVerent cell suspensionscontaining 1 £ 104 cells in their respective media (DMEMand REPI-1640) containing 10% FBS were distributed in a24-well plate and incubated in a humidiWed atmospherecontaining 5% CO2 at 37 °C for 24 h. After removing themedium, diVerent concentrations of the aqueous solution of

the block copolymer (1–10 mg/mL) were added to the 24-well plate, and were incubated for 5 h. The cell culture sur-face was washed with PBS solution, and fresh respectivemedia solution was added to the plates. The number of livecells was counted by the Trypan blue method after 24 h.The cytotoxicity of the aqueous solution of the blockcopolymer was expressed by the relative viability, whichwas deWned by the number of viable cells relative to the cellcontrol ( D 100%).

2.5. Preparation of DNA complexes

DNA or DNA/Lipofectin with aqueous solution of theblock copolymer was used for preparation of complexes inphosphate buVer (PBS, pH 7.5). The plasmid DNA (5 �g)and Lipofectin (15 �g) were mixed with or without diVerentconcentrations (0.2–3 mg/mL) of the block copolymer at aWnal volume of 1 mL PBS. Before further analysis, theresulting mixture was stored for 30 min at room tempera-ture.

2.6. Analysis of DNA complexes

DNA complexes were Wrst analyzed by gel electrophore-sis and further veriWed from sedimentation analysis withspectrophotometer. Samples were prepared as described inpreparation of DNA complexes. Resulting samples werestored in room temperature for 6 h and then centrifuged at1500 rpm (revolution per minute) at 4 °C for 20 min. Tenmicroliters of the supernatant from each sample was takenout and vortexed for 10 min before loading onto 1% aga-rose gel for gel electrophoresis. The remaining supernatantportion was discarded, and the sedimented portion of eachsample was again diluted with 20 �l autoclaved tripledistilled water and re-diluted in 1 mL autoclaved tripledistilled water for sedimentation analysis via spectrophoto-metric measurement of optical density (OD) at 260 nm.

2.7. Gene transfection

MCF-7, NIH 3T3 and CT-26 cells were grown at 37 °Cunder 5% CO2 atmosphere. In the calcium phosphatemethod, 24 h before transfection, diVerent cell line mono-layers in 6 cm plates were supplemented with the serumcontaining media. Prior to cell transfection, 10 �g of theplasmid, 2 mM CaCl2, HEPES buVer and diVerent concen-trations of block copolymer (0.22–11 mg/mL) at a Wnal vol-ume of 1 mL serum and antibiotic-free media was made;and the resulting mixture was allowed to stand for 30 minat room temperature. During the transfection experiment,cells were supplemented with that mixture; and the plateswere slowly agitated for 2 min, and incubated for 4 h at37 °C, 5% CO2 atmosphere. After 4 h, media was replacedby fresh media containing 10% FBS, and again incubated inthe same conditions up to 48 h. For the Lipofectin method,complexes (DNA/Lipofectin) at a w/w ratio (1:3) weremixed with diVerent concentrations of block copolymer as

S.R. Bhattarai et al. / Acta Biomaterialia 2 (2006) 207–212 209

in the calcium phosphate method. The complexes were thenprepared in a Wnal volume of 1 mL serum-free media. Allother procedures were followed as in the commercial Lipo-fectin reagents protocol.

2.8. QuantiWcation of reporter gene expression

Reporter gene expression (�-galactosidase activity) wasmonitored using histochemical staining of cell monolayersaccording to a standard �-galactosidase protocol [9], andquantiWed with ortho-nitrophenyl-�-D-galactopyranoside(ONPG) essentially as described earlier [10].

2.9. Kinetics of FITC labeled DNA accumulation in cells

MCF-7 cells (2 £ 105) were seeded in 6 cm plates and cul-tured at 37 °C in DMEM media with 10% FBS. After trans-fection with FITC labeled DNA/Lipofectin at w/w ratio(1:3) and FITC labeled DNA/Lipofectin at w/w ratio (1:3)mixed with 1.4 mg/mL copolymer solution with a Wnal vol-ume of 1 mL in serum- and antibiotic-free DMEM media,samples were taken out periodically (0, 2, 4, 6 and 8 h), andwere washed and Wxed with 4% cold paraformaldehyde.Samples were observed with a UV microscope LSM 410(Zeiss, Oberkochen, Germany). To evaluate the Xuores-cence intensity of transfected cells, a spectroXuorometerwas used. After the cells were incubated with FITC labeledDNA/Lipofectin at w/w ratio (1:3) and FITC labeledDNA/Lipofectin at w/w ratio (1:3) mixed with 1.4 mg/mLcopolymer solution for 0, 2, 4, 6 and 8 h, they were washedand resuspended with PBS (phosphate buVer saline). TheXuorescence intensity of 1 £ 104 cells was measured with aFACScan spectroXuorometer (Becton Dickinson).

3. Results and discussion

The chemical structure of the block copolymer is shownin Fig. 1. Block copolymers were prepared from melt ringopening polymerization of the p-dioxanone, L-lactide withPEG in presence of stannous octoate catalyst. The amphi-philic nature of the copolymer and its characteristic of self-assembling in aqueous medium into nanoscopic structurehas been studied previously [8]. This paper reports a signiW-cant increase in cell uptake and transfection of mammaliancells using a combination of DNA/Lipofectin complex orDNA/calcium phosphate with an aqueous solution of theblock copolymer. The block copolymer applied for the

transfection study had the composition of PPDO/PLLA/PEG: 10/20/70 (weight ratio), reasonable particle size (aver-age diameter 70 nm) and with relatively low surface nega-tive charge (�-potential of ¡3 § 0.8 mV).

Fig. 2 shows the representative cytotoxicity data on thethree examined cell lines with increasing concentrations ofthe block copolymer aqueous solution. The aqueous solu-tion of the block copolymer showed no signiWcant toxicityon the cells. The cell viabilities in the presence of the solu-tion ranged between 78% and 120% of the control in allexperiments. At a maximum of the aqueous solution of theblock copolymer concentration of 10 mg/mL, the mean cellviabilities of the MCF-7 cell line showed about 95–120%viability compared with that of the control. The CT-26 cellsshowed a trend toward lower viability at higher polymersolution concentrations. This trend may be related to theaggregation and accumulation of the block copolymeraround the cell membrane at higher concentrations (>7 mg/mL) in aqueous solution, thus possibly interfering with nor-mal biological processes, and contributing to cytotoxicity.

To form the transfecting complex, we mixed the plasmidDNA and DNA/Lipofectin at w/w ratio (1:3) with diVerentconcentrations (0.22–5 mg/mL) of block copolymer. Cor-rect complex formation corresponding to the complete

Fig. 2. Cytotoxicity data from three diVerent cell lines as a function ofincreasing concentration of aqueous solution of the block copolymer.Data are presented as a percent of control cell number, where controlrefers to cells growing in normal conditions without block copolymeraddition. Data represent the means of three experiments, §SD.

Fig. 1. Chemical structure of the block copolymer. The subscripts m, n, o, p, and q represent the variable repeat numbers for the units.

210 S.R. Bhattarai et al. / Acta Biomaterialia 2 (2006) 207–212

immobilization or interaction of the DNA was determinedby sedimentation analysis and further conWrmed byagarose gel electrophoresis. Fig. 3 illustrates the complexforming capacity corresponding to the DNA/Lipofectininteraction with block copolymer solution. The bar dia-gram represents the spectrophotometer results of thesedimented portion of DNA to show that with increasingconcentration of the block copolymer from 0.2 to 2 mg/mL,the absorbance increased and was maximum at 1.4 mg/mLparticle concentration. Increased absorbance means anincrease in the plasmid DNA sediment portion which cor-responds to the binding or complexation with the aqueoussolution of block copolymer and subsequent settling assediment. These results were veriWed by sedimentation orsupernatant analysis using gel electrophoresis (results notpresented). From these data we concluded that the blockcopolymer of PPDO/PLLA-b-PEG might play an impor-tant role in enhancing complex formation between DNAand cationic lipid (Lipofectin).

It has been suggested that the eYcacy of transfectionwith complexes formed between DNA and cationic poly-mer strongly depends upon the complex composition[11,12]. This report examined the complexes having theoptimal composition with PPDO/PLLA-b-PEG blockcopolymer, which were shown to be the most eVectiveenhancer for transfection on three diVerent cell lines. TheeVect of internalization of the plasmid encoded �-galactosi-dase gene into MCF-7, CT-26 and NIH 3T3 cells at 37 °C isshown in Table 1. Particularly, no recovery of the �-galac-tosidase activity was observed when cells were incubatedwith free DNA, or DNA with block copolymer. Resultsalso showed the �-galactosidase activity varied with thethree diVerent cell lines under the same w/w ratio of DNA/Lipofectin or DNA/Lipofectin with block copolymer, with

Fig. 3. The eVect of diVerent concentrations of aqueous solution of theblock copolymer mixed with a constant amount of plasmid DNA (5 �g)/Lipofectin (15 �g) on complex formation. The control of 0 mg/mL repre-sents pure plasmid DNA/Lipofectin. Data are the means from threeexperiments, §SD.

the highest activity observed for MCF-7 cells. For thisreason the MCF-7 cells were selected for further experi-mentation. As we know, the DNA/Lipofectin complex iseVectively internalized by the cells [13]. However, adminis-tration of this complex in the presence of block copolymerof PPDO/PLLA-b-PEG further increased plasmid uptakewith levels in cells exceeding those observed during Lipo-fectin method. The substantial enhancement of plasmidinternalization was also observed on MCF-7, NIH 3T3 andCT-26 cell lines with calcium phosphate precipitationmethod (Table 2), but the eVect of the block copolymer onthe uptake of the DNA was not as pronounced comparedto the Lipofectin method.

Enhancement of transfection was also observed whenthe DNA/Lipofectin was administrated with diVeringconcentrations of the block copolymer solution on MCF-7cells (Fig. 4). Qualitatively, higher transfection eYciencycorresponded to an obvious increase in the number of darkblue cells observed with light microscopy (photographsnot shown). This enhancement gradually increased withincreasing concentration of block copolymer up to 1.2 mg/mL. There was no signiWcant diVerence between 1.2 and1.4 mg/mL. Although the optimal conditions of serum,media solution pH, and other parameters might also beimportant to achieve maximal eYciency, even in the limitedconditions examined, the transfection eYciency wasmarkedly higher at a w/w ratio of DNA/Lipofectin (1:3)with 1.4 mg/mL block copolymer concentration than that

Table 1�-Galactosidase activity ((U/mg of total protein in lysate) D [OD420/0.0045 £ assay volume (mL)] min¡1 mg¡1) for three diVerent cell lines withLipofectin transfection

a Concentrations of DNA, Lipofectin and block copolymer were 5 �g,15 �g and 1.2 mg with Wnal volume 1 mL serum and antibiotic-free media,respectively for all cell lines. Data represent the means of three experi-ments, §SD. �-Galactosidase activity was determined using ONPG assubstrate [10].

DNA forma 3T3 CT-26 MCF-7

Free DNA – – –DNA/copolymer – – –DNA/Lipofectin 0.31 0.3 0.41DNA/Lipofectin/copolymer 0.72 0.9 1.36

Table 2�-Galactosidase activity ((U/mg of total protein in lysate) D [OD420/0.0045 £ assay volume (mL)] min¡1 mg¡1) for three diVerent cell lines withcalcium phosphate transfection

a Prior to cell transfection, 10 �g of the plasmid, 2 mM CaCl2, HEPESbuVer and 1.4 mg/mL block copolymer in a Wnal volume of 1 mL serumand antibiotic-free media was made. The resulting mixture stood for30 min at room temperature prior to cell contact. Data represent themeans of three experiments, §SD. �-Galactosidase activity was deter-mined using ONPG as substrate [10].

DNA forma 3T3 CT-26 MCF-7

Free DNA – – –DNA/copolymer – – –DNA/calcium phosphate 0.31 0.30 0.41DNA/calcium phosphate/copolymer 0.62 0.51 0.70

S.R. Bhattarai et al. / Acta Biomaterialia 2 (2006) 207–212 211

observed in DNA/Lipofectin only as a control (Fig. 4). Thiscomposition was anticipated to be near optimal based onthe study of DNA/Lipofectin interaction with the blockcopolymer in the sedimentation analysis.

The possible reason for the increased uptake and trans-fection activity of these complexes may be related to theability of the hydrophilic PEG segment in the block copoly-mer to reduce the precipitation of DNA/polycation (DNA/Lipofectin) complex, and lead to greater dispersion in theaqueous phase with less negative surface charge on thecomplex. The amphiphilic nature of the block copolymermay also aid in membrane interactions.

At the 1.4 mg/mL block copolymer concentration, thetime course for release of DNA into the MCF-7 cells andtheir nuclei was measured (Fig. 5). The Xuorescence inten-sity as measured by spectrophotometry was markedlyhigher in cells after 4 h of incubation with block copolymerthan at the initial time point and at 2, 6, and 8 h. Theseresults were qualitatively conWrmed with Xuorescencemicroscopy. We also speciWcally monitored the intensity inthe nucleus of the MCF-7 cell and only found visible Xuo-rescence in the cell nucleus at 4 h when incubated withblock copolymer.

In conclusion, the major Wnding of this paper was thatPPDO/PLLA-b-PEG block copolymer enhanced the inter-nalization and transfection capacity of the cationic lipidmediated (Lipofectin) DNA complexes into three diVerentcells (MCF-7, CT-26, and NIH 3T3). Results showed theblock copolymer to be nontoxic, enhance complex formingtendency, and increase cell transfection and maximumrelease of DNA into MCF-7 cells in a relatively shortperiod (4 h). Based on these results, the block copolymer(PPDO/PLLA-b-PEG) might be used as a novel pharma-ceutical block copolymer to improve DNA precipitation aswell as aggregation in current protocols for polycation-

Fig. 4. Transfection eYcacy using �-galactosidase assay on MCF-7 cellsincubated with diVering copolymer concentrations and constant concen-trations of DNA (5 �g) and Lipofectin (15 �g). Data represent the meansof three experiments, §SD.

mediated mammalian cell transfection where low expres-sion from therapeutic genes is problematic. These resultsalso warrant further investigation of the eVects of nonionicsurfactants, speciWcally PPDO/PLLA-b-PEG block copoly-mers, on transfection as more eYcient techniques for genedelivery may be developed. Detailed study of this blockcopolymer and its further applications are ongoing.

Acknowledgement

This research was supported by the Korean Ministry ofScience and Technology through the Center for HealthcareTechnology Development.

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