Cationic supramolecules consisting of oligoethylenimine-grafted α-cyclodextrins threaded on poly(ethylene oxide) for gene delivery

Cationic supramolecules consisting of oligoethylenimine-grafted a-cyclodextrins threaded on poly(ethylene oxide)for gene delivery

Chuan Yang,1 Hongzhe Li,2 Xin Wang,2 Jun Li1,21Division of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1,Singapore 117574, Singapore2Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research),3 Research Link, Singapore 117602, Singapore

Received 19 September 2007; revised 30 November 2007; accepted 5 December 2007Published online 10 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31976

Abstract: In this study, three cationic polyrotaxanescomposed of multiple oligoethylenimine-grafted a-cyclo-dextrin rings threaded on a poly(ethylene oxide) chainhave been synthesized and characterized, and investi-gated for gene delivery. All three cationic polyrotaxanescould efficiently compact pDNA into small nanoparticles,with diameters ranging from 100 to 200 nm. In bothBHK-21 and MES-SA cell lines, the transfection efficiencymediated by the cationic polyrotaxanes were comparableor even higher than that of branched polyethylenimine

(PEI) with a molecular weight of 25 kDa, which is one ofthe most efficient gene-delivery vectors to date. Moreover,the cationic polyrotaxanes showed much lower cytotoxic-ity than branched PEI (25 kDa). Hence, these cationic polyrotaxanes have high potentials as new carriers for genedelivery. � 2008 Wiley Periodicals, Inc. J Biomed MaterRes 89A: 13–23, 2009

Key words: supramolecule; cationic polymer; cyclodextrin;polyethylenimine; gene delivery

INTRODUCTION

The safe and efficient DNA delivery remains acentral challenge to the application of gene therapyin the clinic. Recently, nonviral gene delivery vectorshave gained significant interest because of theincreasing concern of the severe immunogenicityand toxicity of viral vectors.1–4 A great number ofpolycations have been reported to be able to effectgene transfection, including homopolymers orcopolymers of polyethylenimine (PEI),5 poly(L-ly-sine),6 polyamidoamine7 poly(L-glutamic acid),8 polyphosphoester,9 and chitosan.10,11 Among these poly-mers, PEI homopolymers with a molecular weight(MW) higher than 25 kDa have been one of the mostpopular polymers used as gene carriers. They are

considered one of the gold standards for polymericnonviral gene delivery because of their high transfec-tion efficiency. However, the rather high toxicity ofthese PEI homopolymers strictly limits their applica-tion in gene therapy.12

Cyclodextrins (CDs) are a series of cyclic oligosac-charides composed of 6, 7, or 8 D(þ)-glucose unitslinked by a-1,4-linkages and named a-, b-, or g-CD,respectively. They are biocompatible, and do notelicit immune responses, and have low toxicities inanimal and human bodies.13 Since 1999, a class oflinear and CD-based polymers was introduced forgene delivery.14–16 Most of these polymers containedamines and CDs in the polymer backbone. Cationicpolymers modified with CDs were also reportedwith lower cytotoxicity and improved gene transfec-tion.17–20 Our group has been working on CD-CD-based supramolecular structures for drug and gene-delivery applications.21 Recently, we reported thesynthesis of novel a-CD-based cationic star polymersand supramolecules composed of multiple oligoethy-lenimine (OEI)-grafted b-CDs that are threaded andcapped on a triblock copolymer chain as new nonvi-ral gene delivery vectors, which showed good trans-fection efficiency and low cytotoxicities.22,23 We fur-ther synthesized cationic polyrotaxanes consisting of

Correspondence to: J. Li; e-mail: [email protected] grant sponsor: Academic Research Fund,

Ministry of Education, Singapore; contract grant number:R-397-000-031-112Contract grant sponsor: Institute of Materials Research

and Engineering, A*STAR, Singapore; contract grant number:IMRE/03-1R0516

� 2008 Wiley Periodicals, Inc.

multiple OEI-grafted a-CDs threaded and capped ona random copolymer of poly(ethylene oxide) (PEO)and poly(propylene oxide) as gene vectors.24

In this study, we have synthesized and character-ized a series of cationic polyrotaxanes consisting ofmultiple OEI-grafted a-CDs threaded on a homopoly(ethylene glycol) (PEG) chain, as a simple exam-ple of the cationic supramolecules as gene carriers.As a result, these cationic polyrotaxanes could effec-tively condense plasmid DNA (pDNA) into smallnanoparticles and deliver gene into cell with highgene-transfection efficiency.

MATERIALS AND METHODS

Materials

O,O0-bis(2-aminoethyl)poly(ethylene oxide) (PEO-bis(amine); the MW of PEO is 3350 Da) and 2,4-dinitro-1-fluo-robenzene (DNFB) were supplied by Aldrich. Pentaethyle-nehexamine was obtained from Fluka. a-Cyclodextrin waspurchased from Tokyo Kasei. Ethylenediamine, linear oli-goethylenimine (OEI-9; polyethylenimine with an averageMW of 423) and branched PEI (25 kDa) were also suppliedby Aldrich. DMSO-d6 and D2O used as solvent in theNMR measurements were also obtained from Aldrich.Qiagen kit and Luciferase kit were purchased from Qiagenand Promega (Cergy Pontoise, France), respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazodium bromide(MTT), penicillin, and streptomycin were obtained fromSigma.

Synthesis

The procedures for preparation of 4b from PEO-bis(-amine) 1 are given later as a typical example.

PEO-bis(amine) 1 (0.344 g) was added to 33 mL a-CDsaturated solution (0.145 g a-CD/mL H2O), and 0.5 gNaHCO3 was added to adjust the pH value of the solu-tion. The solution was ultrasonicated for 20 min and itgradually became turbid, producing the inclusion complex(IC) as precipitate. The reaction mixture was further stirredovernight at 48C. The precipitated IC was isolated by cen-trifugation and freeze-dried in vacuum. Yield, 3.677 g.

Then, DNFB (11.429 g, 61.45 mmol) was dissolved in 10mL of DMF. The solution was added dropwise to 3.677 gof the aforementioned IC 2 with stirring in a flask. Thestirring was continued overnight after the addition wascompleted. And then, the reaction mixture was centri-fuged, and the resulting wet solid was dissolved in 20 mLDMSO and poured into 300 mL MeOH to precipitate theproduct. The precipitate was centrifuged and washedthrice with MeOH. The resulting wet solid was dissolvedin 20 mL DMSO again and poured into 300 mL H2O toprecipitate the product. The resulting precipitate was cen-trifuged and washed thrice with H2O. Finally, the resultingwet solid was dried by freezing (liquid nitrogen) in vacuo,and 1.64 g of pure polyrotaxane 3 was yielded (87%).

The resulting polyrotaxane 3 (0.21 g, 0.011 mmol) wasdried at 408C overnight in vacuum. When the flask wascooled, 40 mL dry DMSO was injected under nitrogen.After all of 3 was dissolved, the DMSO solution of 3 wasadded dropwise during a period of 6 h under nitrogen to40 mL of anhydrous DMSO solution in which 1,1’-carbonyldiimidazole (CDI) (2.56 g, 15.75 mmol) was dissolved, andthe mixture was stirred overnight under nitrogen at roomtemperature. Then, the mixture of 300 mL tetrahydrofuran(THF) and 600 mL Et2O was poured in the resulting solu-tion to precipitate the product. The precipitate was centri-fuged and washed thrice with THF. Then, the resultingwet solid was dissolved in 40 mL DMSO, and this solutionwas slowly added dropwise during a period of 3 h into5.49 mL (18.9 mmol) of pentaethylenehexamine, whichwas dissolved in 40 mL of DMSO with stirring at roomtemperature, followed by stirring the mixture overnight.Nine hundred milliliters of THF was poured in the reac-tion mixture to precipitate the product. The precipitatewas centrifuged and washed thrice with THF, and theresulting crude product was purified by size exclusionchromatography (SEC) on a Sephadex G-50 column usingdeionized (DI) water as eluant. Finally, 0.111 g of brownsolid 4b was yielded (26%).

The yields and analytical data for all three cationic poly-rotaxanes are given later.

Cationic polyrotaxane 4a

Yield, 51%. 1H NMR (400 MHz, D2O, 228C): d 8.35 (s,4H, meta H of phenyl), 7.93 (s, 2H, ortho H of phenyl),4.95 (s, broad, 93H, H (1) of CD), 2.86–4.53 (m, broad,558H, H (3), H (6), H (5), H (2), and H (4) of CD, 303H,��CH2CH2O�� of PEO, 159H, CONCH2 of ethylenedia-mine), 2.67 (s, 159H, NCH2 of ethylenediamine).

Cationic polyrotaxane 4b

Yield, 26%. 1H NMR (400 MHz, D2O, 228C): d 8.39 (s, 4H,meta H of phenyl), 8.28 (s, 2H, ortho H of phenyl), 4.99 (s,broad, 93H, H (1)H of CD), 3.03–4.51 (m, broad, 558H, H (3),H (6), H (5), H (2), and H (4) of CD, 303H, ��CH2CH2O�� ofPEO, 150H, CONCH2 of pentaethylenehexamine), 2.68 (m,1350H, NCH2 of pentaethylenehexamine).

Cationic polyrotaxane 4c

Yield, 35%. 1H NMR (400 MHz, D2O, 228C): d 8.39 (s,4H, meta H of phenyl), 8.11 (s, 2H, ortho H of phenyl),5.03 (d, broad, 93H, H (1)H of CD), 2.96–4.54 (m, broad,558H, H (3), H (6), H (5), H (2), and H (4) of CD, 303H,��CH2CH2O�� of PEO, 70H, CONCH2 of OEI-9), 2.68 (m,1249H, NCH2 of OEI-9).

Gel permeation chromatography

Gel permeation chromatography (GPC) analysis for thecationic polyrotaxanes was carried out with a Shimadzu

14 YANG ET AL.

Journal of Biomedical Materials Research Part A

SCL-10A and LC-10AT system equipped with a SephadexG-75 column (size: 2.5 cm 3 32 cm), a Shimadzu RID-10Arefractive index (RI) detector. PBS buffer (13) solution wasused as the eluant. Fractions were collected per 1 mL andwere detected with a HORIBA SEPA-300 high-speed accu-rate polarimeter at a wavelength of 589 nm, with a celllength of 10 cm and a response of 2 s.

1H NMR

1H NMR spectra were recorded on a Bruker AV-400NMR spectrometer at 400 MHz at room temperature. The1H NMR measurements were carried out with an acquisi-tion time of 3.2 s, a pulse repetition time of 2.0 s, a 308pulse width, 5208-Hz spectral width, and 32 K datapoints. Chemical shifts were referenced to the solventpeaks (d 5 2.50 ppm for DMSO-d6 and 4.70 ppm for D2O).

13C NMR

13C NMR spectra were recorded on a Bruker AV-400NMR spectrometer at 100 MHz at room temperature. The13C NMR measurements were carried out using compositepulse decoupling with an acquisition time of 0.82 s, apulse repetition time of 5.0 s, a 308 pulse width, 20,080-Hzspectral width, and 32 K data points.

Plasmid

The plasmid used was pRL-CMV (Promega), encodingRenilla luciferase, which was originally cloned from themarine organism Renilla reniformis. All plasmid DNAswere amplified in Escherichia coli and purified according tothe supplier’s protocol (Qiagen, Hilden, Germany). Thepurity and concentration of the purified pDNA were deter-mined by absorption at 260 and 280 nm and by agarosegel electrophoresis. The purified pDNA was resuspendedin TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) andkept in aliquots at a concentration of 0.5 mg/mL.

Cells and media

All cell lines were purchased from ATCC (Rockville,MD). BHK-21 and MES-SA cells were maintained in Dul-becco’s modified eagle’s medium (DMEM) and McCoy’s5a medium supplemented with 10% heat-inactivated fetalbovine serum (FBS), 100 U/mg penicillin, and 100 lg/mLstreptomycin at 378C and 5% CO2, respectively. Opti-MEMreduced serum medium, and DMEM medium was pur-chased from Gibco BRL (Gaithersburg, MD). McCoy’s 5amedium was purchased from BCH-Biochrome (Germany).

Gel-retardation experiments

All polymer stock solutions were prepared at a nitrogenconcentration of 1 mM in distilled water and the pH wasadjusted to 7.4. pRL-CMV (0.2 lg in 2 lL TE buffer) was

mixed with polymer at N/P ratios from 0 to 10. Each mix-ture was vortexed and incubated for �30 min at roomtemperature, and then analyzed on 1% agarose gel in TAErunning buffer (40 mM Tris-acetate, 1 mM EDTA) for 40min at 80 V in a Sub-Cell system (Bio-Rad Laboratories,CA). The gel was stained with ethidium bromide (0.5 lg/mL), and the DNA bands were visualized and photo-graphed by a UV transilluminator and BioDoc-It imagingsystem (UVP).

Cell viability assay

Two cell lines (BHK-21 and MES-SA) were cultured inthe DMEM medium (for BHK-21) and McCoy’s 5a me-dium (for MES-SA) supplemented with 10% FBS at 378C,5% CO2, and 95% relative humidity. For cell viabilityassay, the cells were seeded in a 96-well microtiter plate(Nunc, Wiesbaden, Germany) at a density of 10,000 cells/well. After 24 h, the culture media were replaced withserum-supplemented culture media containing serial dilu-tions of the polymers, and the cells were incubated for 24h. Then, 10 lL of sterile, filtered MTT stock solution inPBS (5 mg/mL) was added to each well, reaching a finalconcentration of 0.5 mg/mL. After 5 h, unreacted dye wasremoved by aspiration. The formazan crystals were dis-solved in DMSO (100 lL/well), and the absorbance wasmeasured using a microplate reader (Spectra Plus,TECAN) at the wavelength of 570 nm. The relative cellviability (%) was related to control cells cultured in mediawithout polymer. All experiments were conducted for sixsamples and averaged.

In vitro transfection and luciferase assay

Transfection studies were performed in BHK-21 andMES-SA cells using the plasmid pRL-CMV as reportergene. In brief, 24 h before transfection, 24-well plates wereseeded with cells at a density of 5 3 104/well. The poly-mer/DNA complexes at various N/P ratios were preparedby adding the polymer into DNA solutions dropwise, fol-lowed by vortexing and incubation for 30 min at roomtemperature before the transfection. At the time of trans-fection, the medium in each well was replaced withreduced-serum medium or normal medium. The com-plexes were added into the transfection medium and incu-bated with cells for 4 h under standard incubator condi-tions. After 4 h, the medium was replaced with 500 lL offresh medium supplemented with 10% FBS, and the cellswere further incubated for an additional 20 h under thesame conditions, resulting in a total transfection time of 24h. Cells were washed twice with PBS and lysed in 100 lLof cell culture lysis reagent (Promega). Luciferase geneexpression was quantified using a commercial kit(Promega) and a luminometer (Berthold Lumat LB 9507,Germany). Protein concentration in the samples was ana-lyzed using a bicinchoninic acid assay (Biorad, CA).Absorption was measured on a microplate reader (SpectraPlus, TECAN) at 570 nm and compared to a standardcurve calibrated with bovine serum albumin samples ofknown concentration. Results are expressed as relative

GENE DELIVERY OF CATIONIC SUPRAMOLECULES 15


light units per milligram of cell protein lysate (RLU/mgprotein).

Particle size and zeta-potential measurements

Both particle size and zeta potential of the polymer/pRL-CMV complexes were determined using a ZetasizerNano ZS (Malvern Instruments, Southborough, MA). Com-plex solutions (100 lL) containing 3 lg of DNA were pre-pared at various N/P ratios ranging from 2 to 30. Afterincubating for 30 min, the solutions of the complex werediluted to a final volume of 1 mL prior to measurements.Scattering light was detected at 1738, running at room tem-perature. The zeta potential measurements were carriedout using a capillary zeta-potential cell. For both particlesize and zeta potential measurements, the sampling timewas set to automatic mode, and the data obtained wereanalyzed in the cumulative analysis mode. In addition,each value was presented as the average of three runs.

Confocal microscopy

For confocal microscopy, the plasmid pEGFP-N1 (Clon-tech Laboratories, encoding a redshifted variant of wild-typegreen fluorescence protein (GFP), was used to examine theGFP expression in MES-SA cells. MES-SA cells were seededonto lab-Tek 4-chambered coverglass (Nalge-Nane interna-tional) at density of 5 3 104 cells/well in 500 lL of completeDMEM. After 24 h, transfection was undertaken with 2 lgEGFP plasmid. Each chamber was transfected in 0.3 mLreduced serum media. Twenty microliters of cationic polyro-

taxane 4b/DNA suspension was added per well. After 4 h,the transfection media were removed, and the cells werewashed. After 20 h of further incubation in serum-contain-ing media, the wells were washed with phosphate-bufferedsaline and imaged under a laser scanning confocal micro-scope (LSM 410, Carl Zeiss). GFP fluorescence was excitedat 488 nm, and emission was collected using a 515-nm filter.

AFM

A MultiMode-AFM Atomic Force Microscopy (DigitalInstruments) in a tapping mode was employed to image thenanoparticle samples. Briefly, silicon disks were soaked in50% acetone for a minimum period of 2 h, and then rinsedwith distilled water. After the silicon disks were dried com-pletely, 20 lL of cationic polyrotaxane 4c/DNA complexessolutions containing 1 lg of pRL-CMV and at the N/P ratioof 0, 1, and 10 were dropped on the silicon surface, respec-tively. The aforementioned solutions were volatilized to dry-ness at room temperature prior to measurements. All theAFM images were obtained with a scan rate of 0.5 or 1 Hzover a selected area of 2 lm 3 2 lm. The imaging analysiswas performed using Nanoscope software.

RESULTS AND DISCUSSION

Synthesis of cationic polyrotaxanes

Scheme 1 shows the synthesis procedures andstructures of the cationic polyrotaxanes (4a, 4b, and

Scheme 1. Synthesis procedures and structures of multiple OEI-grafted cationic polyrotaxanes (4a, 4b, and 4c). [Color fig-ure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

16 YANG ET AL.


4c). First, polypseudorotaxane 2 was formed betweenPEO-bis(amine) 1 and a-CD, and polyrotaxanes 3was synthesized by adding 2,4-dinitro-1-fluoro-ben-zene (DNFB) to the dry powder of 2, forming twobulky stoppers to block the two ends of the inclusioncomplex, in which about 15 a-CD rings are trappedon the PEG chain. Finally, linear OEIs with differentMW, ethylenediamine (k 5 1), pentaethylenehex-amine (k 5 5), and OEI with MW of 423 (OEI-9, k 59), were grafted to the a-CD molecules of polyrotax-anes 3 to give cationic polyrotaxanes 4a, 4b, and 4c,respectively.

Molecular characterization of cationicpolyrotaxanes

Figure 1 shows the SEC profiles of the cationic polyrotaxanes in comparison with pristine a-CD. The elu-tion curves for all three cationic polyrotaxanes wererecorded against RI, UV–vis absorption (Abs) at 358nm, and optical rotation (OR), while that for pristinea-CD was recorded against RI and OR, since it has noUV–vis absorption. As shown in Figure 1, a-CD hasrelative small molecular size, which was eluted out atthe low MW region of the column. In contrast, allthree cationic polyrotaxanes were eluted out at higherMW region of the column because of their larger mo-lecular sizes, and were detected by RI, Abs, and OR atthe same time, indicating that the cationic polyrotax-anes comprise the 2,4-dinitrophenyl (DNP) ends andcationic a-CD units. Compared to the other synthetic

Figure 1. GPC traces of a-CD and cationic polyrotaxane 4a,4b, and 4c detected by refractive index (RI), UV at 358 nm,and optical rotation (OR).

Figure 2. 1H NMR spectra of pristine a-CD (a), PEO-bis(amine) (b), and polyrotaxane 3 (c) in DMSO-d6.



polyrotaxanes, 4a was eluted out a bit later, whichcorresponds to its smaller molecular size than theother cationic polyrotaxanes, since 4a is grafted withthe shortest ethylenediamine chains. Each cationic poly-rotaxane showed a single peak in the SEC profiles,indicating that the cationic polyrotaxanes are pure,and there was no intra- or intermolecular crosslinking.

Figure 2 shows the 1H NMR spectra of polyrotax-ane 3 in comparison with pristine a-CD and PEO-bis(amine) 2 in DMSO-d6. In Figure 2(c), the peaksfor a-CD and EO segments of PEG, and the DNPend group were all observed, while they werebroadened as compared with the respective freecounterparts in Figure 2(a,b). This is due to the re-stricted molecular movement of the components inthe polyrotaxane. Quantitative comparisons betweenthe integral intensities of the peaks of a-CD andthose of threading copolymer segments gave thecompositions of the polyrotaxanes. In other words,the numbers of a-CD rings threaded in a single pol-yrotaxane chain could be determined. It was foundthat 15 molecules of a-CD, on average, werethreaded and blocked on a PEO chain.

Figure 3 shows the 1H NMR spectra of the cationicpolyrotaxanes in comparison with pristine a-CD. Inthe spectra of (b–d) in Figure 3, the signals for a-CD,the grafting OEI chains, the threading copolymer, andthe DNP ends were observed, while the peaks weremuch broadened because of the restriction of the mo-lecular motion by molecular interlocking and thegrafting of the OEI units. From the 1H NMR spectra,

the average number of OEI chains grafted to each a-CD (y) was estimated. Corresponding to cationic pol-yrotaxane 4a, 4b, and 4c, the number of the OEIchains grafted to one a-CD molecule in these cationicpolyrotaxanes is about 5.2, 4.9, and 2.3, respectively.It indicates that, the longer the OEI chain, the lessnumber of OEI chains could be grafted to each a-CDring because of the influence of the steric hindranceof OEI chains on the grafting reaction.

Figure 4 shows the 13C NMR spectra of cationicpolyrotaxane 4b in comparison with pristine a-CDand pentaethylenehexamine. In Figure 4(c), all peaks

Figure 3. 1H NMR spectra of pristine a-CD (a), and cationic polyrotaxanes 4a (b), 4b (c), and 4c (d) in D2O.

Figure 4. 13C NMR spectra of pristine a-CD (a), pentae-thylenehexamine (b), and cationic polyrotaxanes 4b (c) inD2O.

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attributed to the a-CD, the grafting OEI, and thethreading copolymer were observed clearly; and thepeaks were broadened because all components ofthe cationic polyrotaxane formed an integrated mac-romolecular system that restricts their molecularmotion. The peak at d 158.3 ppm corresponds to thecarbon of carbonyl groups, which links the OEIchains to a-CD rings. Compared to pristine a-CD,the peak of C-6 of a-CD in 4b shifted from 60.8 to64.7 ppm. This is an evidence that the grafting ofOEI chains mainly happened at the 6-positionhydroxyl groups. In fact, of the three types ofhydroxyl groups of a-CD, those at the 6-position(primary hydroxyl groups) are the most nucleophilicand are thought to be modified under the weak ba-sic conditions.25

Formation of cationic polyrotaxane/DNA complexes

The ability of the cationic polyrotaxanes to condensepDNA into particulate structures was confirmed byagarose gel electrophoresis, particle size, and zetapotential measurements, as well as AFM images.

DNA condensation capability is a prerequisite forpolymeric gene vectors. In this study, the complexa-tion of cationic polyrotaxanes with DNA was ana-lyzed using agarose gel electrophoresis. Figure 5shows the gel retardation results of cationic polyro-taxane/DNA complexes with increasing N/P ratiosin comparison with branched PEI (25 K)/DNA com-plex. In comparison with PEI (25 K), cationic polyro-taxanes 4a, 4b, and 4c could compact pDNA entirelyat the higher N/P ratio of 3–4, indicating that thesepolyrotaxanes have slightly lower DNA condensa-tion ability than PEI (25 K).

Figure 6 shows the particle size and zeta potential ofcationic polyrotaxane/DNA complexes in comparisonwith PEI (25 K)/DNA complex at various N/P ratios.In Figure 6(a), all three cationic polyrotaxanes could effi-ciently compact pDNA into small nanoparticles. Withthe increase of N/P ratio, the diameters of the com-plexes formed by 4a, 4b, and 4c with DNA decreasedsharply. After N/P ratio reached 8, they stabilized andranged within 100–200 nm. These results are similar tothe case of branched PEI (25 K)/DNA complex.

Zeta potential is an indicator of the surface chargeof polymer/DNA naoparticles, and a positive surfacecharge allows an electrostatic interaction betweennegatively charged cellular membranes and the posi-tively charged complexes.26 As shown in Figure 6(b),the surface net charge of the complexes of pDNAwith PEI (25 K) and cationic polyrotaxanes 4a, 4b, or4c increased dramatically as the N/P ratio increasedfrom 2 to 4 and stabilized at an N/P ratio of 10 andabove. After the N/P ratio reached 10, the zeta poten-tial of the complexes of pDNA with all three cationic

polyrotaxanes and PEI (25 K) was strongly positiveand varies within the same range (20–35 mV), whichresults in a similar affinity for cell surface.27

Figure 7 shows representative taping-mode AFMimages of naked pDNA (ND) and cationic polyrotax-ane 4c/DNA complexes at an N/P ratio of 1 and 10.The images obtained clearly demonstrate significantmorphological differences when different N/P ratioswere applied, as well as the formation of compactnanoparticles. In Figure 7(a), loose, supercoiledstructure of pDNA could be observed when pDNAwas not condensed by a cationic polymer. At an N/Pratio of 1, supercoiled plasmid pDNA could still beidentified under AFM while small and compact nano-

Figure 5. Electrophoretic mobility of plasmid DNA in thecomplexes between cationic polyrotaxanes and DNA incomparison with PEI (25 kDa)/DNA complex at variousN/P ratios.



particles were formed at the same time. Compared tothis partial condensation at an N/P ratio of 1, thesame amount of pDNA could be tightly packed andcompletely formed pDNA complexes in the form ofspherical nanoparticles at an N/P ratio of 10.

Cytotoxicity of cationic polyrotaxanes

Cytotoxicity is one of the most important factorsto be considered in selecting polymeric materials

as gene carriers. Figure 8 shows the results ofin vitro cytotoxicity studies of cationic polyrotax-anes in two cell lines (BHK-21 and MES-SA) usingMTT assay. As shown in Figure 8, all the cationicpolyrotaxanes showed a dose-dependent effect oncytotoxicity. It is worth noting that these cationicpolyrotaxanes exhibited less toxicity in both cul-tured BHK-21 and MES-SA cells than the PEI con-trol. The slopes of the dose–response cytotoxicitycurves were much steeper for PEI (25 K) thanthose for 4a, 4b, and 4c. One possible reason isthat the introduction of CD and copolymer resultsin the lower density of amino groups, and the highdensity of amino groups is always considered asan important factor leading to high cytotoxicity ofPEI.28 These results also appeared to be supportedby the calculated half maximal (50%) inhibitoryconcentration IC50 values: in BHK-21 cell lines, theIC50 value of PEI (25 K) was less than 14 lg/mL,while those of cationic polyrotaxanes 4a, 4b, and4c were 33, 40, and 43 lg/mL, respectively. Thesimilar trend was also observed in MES-SA celllines.

Gene transfection mediated by cationicpolyrotaxanes

In vitro gene transfection efficiency of cationic polyrotaxane/DNA complexes was assessed using lucif-erase as a marker gene in BHK-21 and MES-SA cells.Figure 9 shows the gene transfection efficiency ofcationic polyrotaxanes at various N/P ratios, com-pared with those of branched PEI (25 K) at an N/Pratio of 10 and ND in the absence and presence ofserum in BHK-21 and MES-SA cells.

In BHK-21cells [Fig. 9(a,b)], the transfection effi-ciencies mediated by cationic polyrotaxanesincreased with an increase of N/P ratio when con-ducted in the presence of serum. Particularly, 4bexhibited the highest transfection efficiencies among

Figure 7. Atomic force microscopy (AFM) images of the supercoiled pDNA (a) and cationic polyrotaxane 4c/DNA com-plexes at N/P ratios of 1 (b) and 10 (c). [Color figure can be viewed in the online issue, which is available at www.inter-science.wiley.com.]

Figure 6. Particle size (a) and zeta potential (b) of thecomplexes between cationic polyrotaxanes and DNA incomparison with PEI/DNA complex at various N/P ratios.[Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]

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the three cationic polyrotaxanes, even much higherthan those of branched PEI (25 K) at an N/P ratio of10. However, when conducted in the absence ofserum, all the three cationic polyrotaxanes showedmuch lower transfection efficiencies than PEI (25 K)at an N/P of 10.

In MES-SA cells [Fig. 9(c,d)], under both serumand serum free conditions, the transfection efficien-cies of cationic polyrotaxanes also increased with anincrease of N/P ratio. At higher N/P ratios, thetransfection efficiencies mediated by 4b were compa-rable to or even much higher than those of branchedPEI (25 K) at an N/P ratio of 10.

Confirmation of the gene-delivery capability of thecationic polyrotaxane 4b was also obtained by fluo-rescence microscopy. Plasmid pEGFP-N1 encodingGFP was used to examine the GFP expression inMES-SA cells. As shown in Figure 10, strong fluores-cence signal could be observed when transfectionwas mediated by 4b at an N/P ratio of 10. In con-trast, GFP expression could not be detected whenthe transfection was mediated by ND, which wasused as a negative control.

CONCLUSIONS

In the present study, a series of cationic polyrotax-anes consisting of multiple OEI grafted a-CD ringsthreaded and blocked on a PEO chain have beensynthesized and characterized, as well as investi-

Figure 9. In vitro gene transfection efficiency of the complexes of cationic polyrotaxanes/DNA in comparison with thatof PEI (25 kDa) or naked DNA (ND), in BHK-21 (a,b) and MES-SA (c,d) cells in the absence and presence of serum. Datarepresent mean 6 standard deviation (n 5 3).

Figure 8. Cell viability assay in BHK-21(a) and MES-SA(b) cell lines. The cells were treated with various concen-trations of 4a, 4b, and 4c, and PEI (25 kDa) for 24 h in aserum-containing medium. [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]



gated for gene delivery. In vitro cytotoxicity studiesshowed that these cationic polyrotaxanes exhibitedmuch less cytotoxicity than PEI (25 kDa) because oftheir low density of amino groups. In both BHK-21and MES-SA cells, generally speaking, the transfec-tion efficiencies mediated by the cationic polyrotax-anes synthesized increased with an increase of anN/P ratio. Particularly, under complete serum me-dium condition, cationic polyrotaxane 4b displayedcomparable or much higher transfection efficiencythan PEI (25 K) at an N/P ratio of 10. Hence, highertransfection efficiencies along with lower cytotoxicitymake these new cationic polyrotaxanes promisingcarriers for gene delivery.

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Figure 10. Confocal microscopy images of transfected MES-SA cells. The transfection was mediated by cationic polyro-taxane 4b at an N/P ratio of 10 in the absence of serum, using green fluorescence protein (GFP) gene as a reporter gene.The same field of cells was observed with Nomarski optics (a) and with fluorescence microscope (b) to visualize GFPexpression. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Cationic supramolecules consisting of oligoethylenimine-grafted α-cyclodextrins threaded on poly(ethylene oxide) for gene delivery