Cationic Polyrotaxanes as Gene Carriers - American Chemical Society

May 7, 2009 - Observation of DNA Complexation, and Gene Transfection in Cancer ... Engineering, National UniVersity of Singapore, 9 Engineering DriVe ...
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J. Phys. Chem. B 2009, 113, 7903–7911

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Cationic Polyrotaxanes as Gene Carriers: Physicochemical Properties and Real-Time Observation of DNA Complexation, and Gene Transfection in Cancer Cells Chuan Yang,† Xin Wang,‡ Hongzhe Li,‡ Eunice Tan,† Chwee Teck Lim,†,§ and Jun Li*,†,‡ DiVision of Bioengineering, Faculty of Engineering, National UniVersity of Singapore, 7 Engineering DriVe 1, Singapore 117574; Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602; and Department of Mechanical Engineering, Faculty of Engineering, National UniVersity of Singapore, 9 Engineering DriVe 1, Singapore 117576, Singapore ReceiVed: February 12, 2009; ReVised Manuscript ReceiVed: April 21, 2009

Cationic polymers have been studied as promising nonviral gene delivery vectors. In contrast to the conventional polycations with long sequences of covalently bonded repeating units, we have developed a series of novel cationic polyrotaxanes consisting of multiple oligoethyleneimine-grafted β-cyclodextrin rings threaded on a poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock copolymer chain. In this study, these cationic polyrotaxanes with different oligoethyleneimine chain lengths were investigated for DNA binding ability, cytotoxicity, and gene transfection efficiency in cancer cells. Fluorescent titration assay results indicated that all the polyrotaxanes could completely condense plasmid DNA and form stable complexes at N/P ratio of 2, where the N/P ratio is the molar ration of amine groups in the cationic molecule to phosphate groups in the DNA. Particularly, tapping mode AFM imaging in aqueous environment was conducted to observe the morphology of the polyrotaxane/DNA complexes and their formation processes in real time. In both SKOV-3 and PC3 cancer cells, these polyrotaxanes showed low cytotoxicity and high transfection efficiency which is comparable to or significantly higher than that of high molecular weight branched polyethylenimine (25 kDa), one of the most effective gene-delivery polymers studied to date. In addition, the synthesized polyrotaxanes displayed sustained gene delivery capability in PC3 cells in the presence or absence of serum. Therefore, these cationic polyrotaxanes with strong DNA binding ability, low cytotoxicity, and high and sustained gene delivery capability have a high potential as novel nonviral gene carriers in clinical cancer gene therapy. Introduction Over the last few decades, cationic polymers have held great promise for their potential use as nonviral gene carriers. Compared with other gene delivery systems such as viral vectors and cationic lipids, cationic polymers for gene delivery are generally economical and stable, can be produced in a large scale, and show low host immunogenicity. By now a great number of polycations have been reported to be able to deliver genes, including homopolymers or derivatives of polyethylenimine (PEI),1-3 poly(L-lysine),4 polyamidoamine,5 poly(L-glutamic acid),6 polyphosphoester,7 and chitosan.8 Polyrotaxane is a mechanically interlocked supramolecular architecture consisting of multiple cyclic molecules (rings) threaded on a linear polymer chain (axis), and the dissociation of a ring from the axis is hindered by bulky groups (usually called stoppers).9 Since the first syntheses of polyrotaxanes with multiple R-cyclodextrin (R-CD) rings threaded over a polymer chain,10,11 increasing attention has been directed toward the study of these supramolecular structures10-20 and their properties for electronics21-23 and biomaterials applications.3,24-34 For the conventional polycations containing long sequences of covalently bonded repeating units, the mobility of their * To whom correspondence should be addressed at Division of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574. Phone: +65-6516-7273. Fax: +656872-3069. E-mail: [email protected]. † Division of Bioengineering. ‡ Institute of Materials Research and Engineering, A*STAR. § Department of Mechanical Engineering.

molecular chains will decrease with an increase in the number of repeating units and molecular weight in the solution state. In contrast, under the same conditions, the rings in polyrotaxanes will rotate and/or slide along the axis freely, which can improve the mobility of cationic ligands linked to the rings and enhance the interaction of the cationic ligands and receptor DNA. In 2006, Ooya et al. reported the use of dimethylaminoethylmodified R-CD rings threaded onto a poly(ethylene glycol) (PEO) chain and capped by cleavable end groups.33 The cleavage of the end groups caused the dethreading of R-CD rings and rapid release of DNA in cells, but the tertiary amines conjugated to the R-CD rings may not be efficient in DNA complexation and gene delivery. Our group also designed and synthesized cationic polyrotaxanes composed of multiple oligoethylenimine (OEI)-grafted β-CD rings threaded on a poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEO-PPO-PEO) triblock copolymer chain, these polyrotaxanes showed low cytotoxicity and high gene-transfection efficiency in HEK293 cells.26 In this study, we characterized the physicochemical properties of these cationic polyrotaxanes, conducted the observation of the morphology of the polyrotaxane/DNA complexes and their formation processes in real time using tapping mode AFM imaging in aqueous environment, and investigated their cytotoxicity and transfection efficiency in cancer cells. In both SKOV-3 and PC3 cancer cells, these polyrotaxanes showed low cytotoxicity and high transfection efficiency which is comparable to or significantly higher than that of high molecular weight branched PEI with molecular weight of 25 000 Da, or PEI

10.1021/jp901302f CCC: $40.75  2009 American Chemical Society Published on Web 05/07/2009

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(25K), one of the most effective gene-delivery polymers studied to date. In PC3 cells, the polyrotaxanes also displayed sustained gene delivery capability under both serum and serum-free conditions. Experimental Section Materials. Pluronic L64 PEO-PPO-PEO triblock copolymer was supplied by BASF, Germany. The copolymer has a chain composition of EO13PO30EO13 and a number-average molecular weight of 2900. The molecular characteristics were confirmed using GPC and 1H NMR spectroscopy, which were found to be within the specifications of the supplier. 2,4,6Trinitrobenzenesulfonic acid solution and pentaethylenehexamine (PEHA) were purchased from Fluka. Tri(2-aminoethyl)amine, ethylenediamine, and oligoethylenimine (polyethylenimine with low molecular weight, Mn ) 423) were obtained from Aldrich. β-CD was purchased from Tokyo Kasei Inc. D2O used as solvent in the NMR measurements was also obtained from Aldrich. Qiagen kit and Luciferase kit were purchased from Qiagen and Promega, respectively. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazodium bromide (MTT), ethidium bromide (EtBr), penicillin, and streptomycin were obtained from Sigma. Synthesis of Cationic Polyrotaxanes. The synthesis of cationic polyrotaxanes was performed as reported previously.26 Briefly, both hydroxyl ends of Pluronic L64 PEO-PPO-PEO triblock copolymer were activated with 1,1′-carbonyldiimmidazole (CDI), followed by reaction with large excess of tri(2aminoethyl)amine to give PEO-PPO-PEO tetra(amine). β-CD was selectively threaded over the PPO block of PEO-PPOPEO tetra(amine) to form inclusion complexes (ICs), and large excess of TNBS was conjugated to both ends of the block copolymer chain in the IC to prevent the threaded β-CD ring from dethreading. Finally, OEIs with different chain length were grafted to the β-CD rings to give the corresponding cationic polyrotaxanes. Synthesis of PEO-PPO-PEO Tetra(trinitrobenzene) (Polymer Control, PC). The above PEO-PPO-PEO tetra(amine) (0.1 g, 0.03 mmol) was dissolved in 5 mL of H2O, and TNBS (0.4 g) and NaHCO3 (0.06 g) were added and reacted overnight. Then, the reaction mixture was poured in 70 mL of THF to precipitate NaHCO3 and excess TNBS. The precipitate was centrifuged and washed for three times with THF. The supernatant was collected and combined, and concentrated to dryness. The resulting crude product was purified by size exclusion chromatography (SEC) on a Sephadex LH-20 column using MeOH as eluent. Finally, 0.115 g red solid was yielded (92.8%). 1H NMR (400 MHz, D2O, 5 °C): δ 8.47 (s, br, 8H, meta H of phenyl), 2.95-3.92 (m, 107H and 91H, -CH2CH2Oof PEO block and -CH2CHO- of PPO block), 0.83 (s, 91H, -CH3 of PPO block). 13C NMR (100 MHz, D2O, 5 °C): δ 75.47 (-CH(CH3)O- of PPO block), 72.32 (-OCH2- of PPO block), 69.88 (-CH2CH2O- of PEO block), 10.61 (-CH3 of PPO block). Measurements. The 1H NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at 400 MHz at room temperature (or at 5 °C for PC). The 1H NMR measurements were carried out with an acquisition time of 3.2 s, a pulse repetition time of 2.0 s, a 30° pulse width, 5208 Hz spectral width, and 32K data points. Chemical shifts were referenced to the solvent peaks (δ ) 4.70 ppm for D2O). The 13C NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at 100 MHz at room temperature (or at 5 °C for PC). The 13C NMR measurements were carried out using composite pulse decoupling with an acquisition time of 0.82 s,

Yang et al. a pulse repetition time of 5.0 s, a 30° pulse width, 20 080 Hz spectral width, and 32K data points. Wide-angle X-ray diffraction (XRD) measurements were carried out using a Siemens D5005 diffractometer using Nifiltered Cu KR (1.542 Å) radiation (40 kV, 40 mA). Powder samples were mounted on a sample holder and scanned from 5° to 35° in 2θ at a speed of 0.6° per minute. Plasmid. The plasmid used was pRL-CMV (Promega, USA), encoding Renilla luciferase, which was originally cloned from the marine organism Renilla reniformis. All plasmid DNAs were amplified in Escherichia coli and purified according to the supplier’s protocol (Qiagen, Hilden, Germany). The purity and concentration of the purified plasmid DNA were determined by absorption at 260 and 280 nm and by agarose gel electrophoresis. The purified plasmid DNA was resuspended in TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) and kept in aliquots at a concentration of 0.5 mg/mL. Cells and Media. All cell lines were purchased from ATCC (Rockville, MD). SK-OV-3 and PC3 cells were maintained in McCoy 5R or Ham’s F-12 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mg penicillin, 100 µg/mL streptomycin at 37 °C and 5% CO2. Opti-MEM reduced serum medium, and DMEM medium were purchased from Gibco BRL (Gaithersburg, MD). Fluorescence Titration Assay. Binding abilities of the cationic polyrotaxanes with pDNA were monitored by a fluorescent titration assay. Ten micrograms of pDNA was mixed with the polyrotaxanes in 200 µL of 1 × PBS (PBS, phosphatebuffered saline). N/P ratios used were 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0, where the N/P ratio means the molar relation of amine groups in the cationic molecule, which represent the positive charges, to phosphate groups in the DNA, which represent the negative charges. After incubation for 30 min at room temperature, 20 µL of 14 mM of EtBr (EtBr is an intercalating agent, and in this paper it is used as a fluorescent probe in agarose gel electrophoresis; when exposed to ultraviolet light, it fluoresced with an orange color, intensifying almost 20-fold after binding to DNA) solution was added to the polymer/DNA mixtures. The fluorescence of the intercalated dye was measured on a spectrofluorometer (RF-5301 fluorescence spectrophotometer, Shimadzu, Japan) by exciting at 530 nm while monitoring emission at 590 nm, with the slits set at 15 nm. The fluorescence intensity of the blank was subtracted from all values before data analysis. The relative fluorescence intensities of the pDNA/EtBr mixture solutions and EtBr solution without pDNA were taken as 100% and 0% reference, the relative fluorescence intensities were thereby measured for the three cationic polyrotaxanes and branched PEI (25K). Cell Viability Assay. Two cancer cell lines (PC3 and SKOV-3) were cultured in the McCoy 5R or Ham’s F-12 medium supplemented with 10% FBS at 37 °C, 5% CO2, and 95% relative humidity. For cell viability assay, the cells were seeded in a 96-well microtiter plate (Nunc, Wiesbaden, Germany) at a density of 15 000 cells/well for PC3 cells and 10 000 cells/well for SK-OV-3 cells. After 24 h, culture media were replaced with serum-supplemented culture media containing serial dilutions of the polymers, and the cells were incubated for 24 h. Then, 10 µL of sterile filtered MTT stock solution in PBS (5 mg/mL) was added to each well, reaching a final concentration of 0.5 mg/mL. After 5 h, unreacted dye was removed by aspiration. The formazan crystals were dissolved in DMSO (100 µL/well) and the absorbance was measured using a microplate reader (Spectra Plus, TECAN) at a wavelength of 570 nm. The relative cell viability (%) was related to control cells cultured

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SCHEME 1: Structure of Cationic Polyrotaxanes with Multiple OEI-Grafted β-CD Rings (CP1, CP2, and CP3), and Their Polymer Control (PC)

in media without polymer. All experiments were conducted using six samples and the data obtained were averaged. In Vitro Transfection and Luciferase Assay. Transfection studies were performed in SK-OV-3 and PC3 cancer cells using the plasmid pRL-CMV as reporter gene. In brief, 24 h before transfection, 24-well plates were seeded with cells at a density of 5 × 104/well. The polymer/DNA complexes at various N/P ratios were prepared by adding the polymer into DNA solutions dropwise, followed by vortexing and incubation for 30 min at room temperature before the transfection. At the time of transfection, the medium in each well was replaced with reduced-serum medium or normal medium. The complexes were added into the transfection medium and incubated with cells for 4 h under standard incubator conditions. After 4 h, the medium was replaced with 500 µL of fresh medium supplemented with 10% fetal bovine serum (FBS), and the cells were further incubated for an additional 20 h (longer incubation time of 44 and 68 h for sustained gene delivery study) under the same conditions, resulting in a total transfection time of 24 h (longer total transfection time of 48 and 72 h for sustained gene delivery study). Cells were washed twice with PBS and lysed in 100 µL of cell culture lysis reagent (Promega, Cergy Pontoise, France). Luciferase gene expression was quantified using a commercial kit (Promega, Cergy Pontoise, France) and a luminometer (Berthold Lumat LB 9507, Germany). Protein concentration in the samples was analyzed using a bicinchoninic acid assay (Biorad, CA). Absorption was measured on a microplate reader (Spectra Plus, TECAN) at 570 nm and compared to a standard curve calibrated with bovine serum albumin (BSA) samples of known concentrations. Results are expressed as relative light units per milligram of cell protein lysate (RLU/mg protein). Confocal Microscopy. For confocal microscopy, the plasmid pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA),

encoding a red-shifted variant of wild-type green fluorescence protein (GFP), was used to examine the GFP expression in PC3 cells. PC3 cells were seeded onto Lab-Tek 4-chambered coverglass (Nalge-Nane International, USA) at density of 5 × 104 cells/well in 500 µL of complete DMEM. After 24 h, transfection was undertaken with 2 µg of EGFP plasmid. Each chamber was transfected in 0.3 mL of reduced serum Opti-MEM media. Twenty microliters of the cationic polyrotaxane CP3/ DNA suspension was added per well. After 4 h, the transfection media were removed and the cells were washed. After 20 h of further incubation in serum-containing media, the wells were washed with phosphate-buffered saline (PBS) and imaged under a laser scanning confocal microscope (LSM 410, Carl Zeiss, Go¨ttingen, Germany). GFP fluorescence was excited at 488 nm and emission was collected using a 515 nm filter. Atomic Froce Microscopy (AFM). The nanoparticles were imaged using the AFM (MultiMode, Veeco, USA) in tapping mode on both dry sample and in aqueous environment. First, the nanoparticles were imaged on dry sample surface. In brief, silicon disks were soaked in 50% acetone for a minimum period of 2 h and rinsed with distilled water. After incubation for 30 min at room temperature, 20 µL of the cationic polyrotaxane CP3/DNA complex solutions containing 1 µg of pRL-CMV and at the N/P ratios of 0, 1, and 10 were each dropped on a dry and clean silicon disk surface. The above solutions were volatilized to dryness at room temperature prior to measurements. The AFM tips (PPP-NCH, Nanoscience Instruments, Inc., USA) used had a typical radius of 7 nm or less, and the images were recorded with a scan rate of 0.5 or 1 Hz over a selected area of 2 µm × 2 µm. Second, the nanoparticles were imaged in distilled water. Similar to the sample preparation for AFM imaging on dry sample surface, after incubation for 30 min at room temperature, 20 µL of the cationic polyrotaxane CP3/ DNA complexes solutions with the N/P ratios of 0, 1, and 10

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Figure 1.

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C NMR spectra of β-CD (a), PC (b), pentaethylenehexamine (c), and CP2 (d), in D2O.

were each dropped on a dry and clean silicon disk surface. Imaging was conducted in distilled water and recorded after 10 min incubation and at a time interval of 5 min. The AFM tips (DNP, Veeco, USA) used had a typical radius of 20-60 nm, and the cantilevers had a spring constant of 0.58 N/m. The images were recorded with a scan rate of 1 Hz over a selected area of 4 µm × 4 µm. All image analysis was performed using Nanoscope software after removal of the background slope by flattening images. Results and Discussion Synthesis and Characterization of Cationic Polyrotaxanes. Scheme 1shows the structure of the cationic polyrotaxanes (CP1, CP2, and CP3), as prepared according to our previous report.26 The results from NMR spectra revealed that 13 β-CD rings, on average, were covered and blocked on a Pluronic L64 PEO-PPO-PEO triblock copolymer chain in one cationic polyrotaxane molecule. For each threaded β-CD ring, OEIs with different chain length, ethylenediamine (k ) 1), pentaethylenehexamine (PEHA, k ) 5), and linear OEI with an average molecular weight of 423 g/mol (OEI-9, k ) 9), were grafted and gave the corresponding OEI-grafted cationic polyrotaxanes (CP1, CP2, and CP3). For comparison, in this study, PEO-PPO-PEO tetra(amine) was reacted with 2,4,6-trinitrobenzene sulfonate (TNBS) directly to give PEO-PPO-PEO tetra(trinitrobenzene) as polymer control (PC) compound without β-CD rings threaded as shown in Scheme 1. Figure 1 shows the 13C NMR spectra of the cationic polyrotaxane CP2 with reference to free β-CD, PC, and pentaethylenehexamine in D2O. In Figure 1d, all peaks attributed to L64 PEO-PPO-PEO triblock copolymer, β-CD ring, and grafting PEHA chain were observed significantly. The peak at

δ 158.2 ppm corresponds to the carbon of carbonyl groups, which conjugated OEI chains to β-CD rings. Moreover, comparing to free β-CD, the peak of C-6 on the β-CD rings of CP2 was moved toward downfield and shifted from 60.9 to 64.1 ppm. This is evidence that the grafting of PEHA chains mainly occurred at the 6-position hydroxyl groups. In fact, of the three types of hydroxyl groups of β-CD, those at the 6-position (primary hydroxyl groups) are the most nucleophilic and are thought to be modified under the weak basic conditions.35 In addition, the 13C NMR spectroscopy of PC was carried out at 5 °C since the strong hydrophobic TNBS end groups of PC make it insoluble in water at room temperature. For CP2, in contrast, the large amount of hydrophilic OEIgrafted β-CD rings counteract the effect of the hydrophobic TNBS ends and enable the cationic polyrotaxanes to dissolve well in water at room temperature. Figure 2 shows the 1H NMR spectra of the cationic polyrotaxane CP2 with reference to free β-CD, PC, and pentaethylenehexamine in D2O. In Figure 2d, the peaks for β-CD ring, EO, and PO segments of the triblock copolymer, and grafting PEHA chain were all observed. The peaks were broader as compared to their respective free counterparts in Figure 2, a and c. This is due to the restricted molecular movement of the components in the cationic polyrotaxane. Similar to the 13C NMR, the proton NMR spectroscopy of PC was also carried out at 5 °C attributing to the influence of strongly hydrophobic TNBS end groups. In Figure 2b, the peaks attributed to TNBS ends and methyl groups of PO segments of the triblock copolymer were much broadened since the amphiphilic PC could form micelles at this temperature and the movements of these components are limited and restricted in a confined environment.

Cationic Polyrotaxanes as Gene Carriers

Figure 2.

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H NMR spectra of β-CD (a), PC (b), pentaethylenehexamine (c), and CP2 (d), in D2O.

Formation of Cationic Polyrotaxane/DNA Complexes. The ability of the cationic polyrotaxanes to condense plasmid DNA (pDNA) into particulate structures was examined by fluorescence titration assay and AFM imaging. It is known that the DNA condensation capability of cationic polymers is one of the prerequisites for gene transfer in most types of cells because the complexation of DNA with cationic polymer can protect DNA from extracellular nuclease degradation.39,40

Figure 3. X-ray powder diffraction diagrams of β-CD (a), β-CD-PEOPPO-PEO polyrotaxane (b), and cationic polyrotaxanes CP1 (c), CP2 (d), and CP3 (e).

Figure 3 shows the wide-angle X-ray powder diffraction patterns of the β-CD-PEO-PPO-PEO-TNB polyrotaxane and the three cationic polyrotaxanes with reference to free β-CD. Figure 3a gives the cage structure of free β-CD.36In contrast, all the patterns of β-CD-PEO-PPO-PEO-TNB polyrotaxane are significantly different from those of free β-CD, but they are quite similar to those of inclusion complexes between β-CD and some polymers, which have been reported to adopt a channel-type structure.34,37,38 Nevertheless, in Figure 3c-e, broad and diminished reflections were observed, probably due to the conjugation of amorphous grafting OEI chains to β-CD rings after the conjugation reaction.

To confirm the formation of the cationic polyrotaxane/DNA complexes, fluorescence titration assay was performed and the fluorescence signal of fluorescent probe ethidium bromide (EtBr) was recorded. The fluorescent probe EtBr can intercalate into DNA, leading to an increase in the fluorescence quantum yield of the dye. Upon complexation of DNA with cationic polymers, EtBr is expelled and the fluorescence signal decreases dramatically.41-43 Hence, the compaction of DNA by cationic polymers can result in a distinct change in EtBr fluorescence intensity, and thus the DNA condensation capability of cationic polymer can be examined. Figure 4 shows the fluorescence titration assay results of the cationic polyrotaxane/DNA complexes with increasing N/P ratios in comparison with branched PEI (25K). As shown in Figure 4, at N/P 2 the relative fluorescence intensities of all three cationic polyrotaxanes and branched PEI was significantly decreased to a value below 5%, indicating that almost all DNA molecules were compacted and condensed into small nanoparticles by these cationic polymers. This result is in good agreement with the gel electrophoresis results we reported previously.26 Furthermore, at N/P ratio ranging within 0-2, the slopes of CP1 and CP2 fluorescence intensity curves (see Figure 4) are much higher than that of CP3 and PEI (25K), indicating their strong DNA binding ability. In comparison with gel electrophoresis assay, fluorescence titration assay could

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Figure 4. Fluorescence titration assay of plasmid DNA in the complexes between cationic polyrotaxanes and plasmid DNA with reference to that of PEI/DNA complex at various N/P ratios.

provide more detailed information on the formation of cationic polymer/DNA complexes. The morphology of naked plasmid DNA has been extensively characterized using AFM and it adopts a distinctly different conformation after complexation with cationic polymer.44-46 Thus, AFM topographic images could be carried out to characterize the morphology of the cationic polyrotaxane/DNA complexes and this confirms the condensation of DNA due to the formation of cationic polyrotaxane/DNA complexes. Figure 5 shows representative tapping mode AFM images of naked DNA and cationic polyrotaxane CP3/DNA complexes at N/P ratios of 1, 2, and 10 on dry sample surface. Results from the AFM study on a dry sample in air revealed that the complexation of DNA by CP3 led to the formation of compact nanoparticles even at N/P ratio of 2. In Figure 5a, loose, supercoiled structure of pDNA could be found when the pDNA was not condensed by polymer. At N/P ratio of 1, supercoiled plasmid DNA could still be detected under AFM while some of the pDNA was condensed to nanoparticles by CP3 (Figure 5b). In Figure 5, c and d, all the pDNA packed tightly and formed CP3/pDNA complexes completely at N/P ratios of 2 and 10, respectively. The particle size of the cationic polyrotaxane/DNA complex ranged from 70 to 150 nm. For further understanding of the condensation process of pDNA by the cationic polyrotaxane and the kinetics of the complex formation, real-time AFM imaging of the cationic polyrotaxane/DNA complexes formed in aqueous environment was carried out. Figure 6 shows tapping mode AFM images of cationic polyrotaxane CP3/DNA complexes at N/P ratios of 1, 2, and 10. Imaging was conducted in distilled water and recorded after 10 min incubation and at a time interval of 5 min. To avoid the conformation change induced by immobilization chemicals, in this study, immobilization had been achieved simply on a clean and bare silicon disk without any substrate

Figure 6. Real-time AFM images of the complexes between CP3 and DNA at N/P ratios of 1, 2, and 10, in distilled water.

modifications or addition of multivalent cations. As shown in Figure 6, there were no nanoparticles observed at N/P ratio of 1, indicating that the complexes between CP3 and pDNA did not form. In fact, uncondensed pDNA was never visualized on the silicon surface since the silicon surface is negatively charged in neutral aqueous solution, which leads to the electrostatic repulsion for highly negatively charged pDNA.47,48 At N/P ratio of 2, four consecutive AFM images recorded from 15 to 30 min showed an explicit and representative condensation process of pDNA by CP3. After 15 min condensation, an obscure spot, which represents a CP3/pDNA nanoparticle, was observed. With an increase in time, this nanoparticle became clearer and its particle size was determined as 148 nm. When N/P ratio reached 10, many CP3/DNA nanoparticles were observed. In the

Figure 5. Atomic force microscopy (AFM) images of the supercoiled plasmid DNA (a), and the complexes between CP3 and DNA at N/P ratios of 1 (b), 2 (c), and 10 (d), on dry sample surface.

Cationic Polyrotaxanes as Gene Carriers

Figure 7. Cell viability assay in PC3 (a) and SK-OV-3 (b) cancer cell lines. The cells were treated with various concentrations of CP1, CP2, and CP3, and PEI (25K) for 24 h in a serum-containing medium. Data represent mean ( standard deviation (n ) 6).

consecutive images, these nanoparticles shifted slowly and their conformations changed slightly due to the influence of AFM tips. Cytotoxicity of Cationic Polyrotaxanes. Cytotoxicity of polymeric gene vector may be an important factor that affects

J. Phys. Chem. B, Vol. 113, No. 22, 2009 7909 the transfection efficiency. Figure 7 showed the results of in vitro cytotoxicity of the cationic polyrotaxanes analyzed by MTT method in two cancer cell lines (PC3 and SK-OV-3). As shown in Figure 7a, all three cationic polyrotaxanes and PEI (25K) showed a strong dose-dependent effect on cytotoxicity and the cytotoxicity of the polymers was much lower than that of PEI (25K) in PC3 cell lines. For example, at the concentration of 0.025 mg/mL, PC3 cells only showed approximately 5% cell viability when incubated with PEI (25K). Whereas, in the case of CP1, CP2, and CP3, their relative growth rate in PC3 cells showed more than 65% viability under the same conditions. In SK-OV-3 cell lines (Figure 7b), the relative cell viability for all three cationic polyrotaxanes decreased slightly with an increase of concentration. Even at the high concentration of 0.1 mg/mL, all three polyrotaxanes showed above 40% cell viability. In contrast, at the concentration of 0.05 mg/mL, merely 4% of PC3 cells incubated by PEI (25K) survived. These results may be attributed to the introduction of cyclodextrin and block copolymer, which leads to reduction of amino density of the cationic polyrotaxanes. High amino density is considered as an important factor contributing to the high cytotoxicity of PEI. Transfection Efficiency of Cationic Polyrotaxanes. In vitro transfection efficiency of complexes formed between pDNA and cationic polyrotaxanes was assessed utilizing a transient expression of luciferase reporter in both SK-OV-3 and PC3 cancer cells. Figure 8 shows the gene transfection efficiency of cationic polyrotaxanes for DNA delivery compared with those of branched PEI (25K) and naked pDNA (ND) in the presence and absence of serum, in both SK-OV-3 and PC3 cells. The structure of polymers plays an important role in the transfection efficiency. In SK-OV-3 cells, the transfection efficiency mediated by the cationic polyrotaxanes was dependent upon the chain length of the OEI conjugated to β-CD. Generally, increased OEI length produced greater transfection efficiency following the order CP3 > CP2 > CP1, but an opposite order was obtained in the presence of serum and at lower N/P ratios. Moreover, the transfection efficiency mediated by each cationic polyro-

Figure 8. In vitro gene transfection efficiency of the complexes of cationic polyrotaxanes/DNA with reference to that of PEI (25K) or naked DNA (ND), in SK-OV-3 (a and b) and PC3 (c and d) cells in the absence and presence of serum. Data represent mean ( standard deviation (n ) 3).

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Figure 9. In vitro gene transfection efficiency of the complexes of CP3/DNA with reference to that of PEI (25K) at various N/P ratios in the absence and presence of serum at different expression duration (24, 48, and 72 h) in PC3 cells. Data represent mean ( standard deviation (n ) 3).

taxane increased with an increase of N/P ratio. At higher N/P ratios the transfection efficiency mediated by CP2 and CP3 was comparable to or slightly higher than that of branched PEI (25K) in the presence or absence of serum. The relationship between transfection efficiency and the chain length of OEI was not strong in PC3 cells. It is noteworthy that, in the presence or absence of serum and over the entire range of N/P ratios examined, the synthesized cationic polyrotaxanes displayed excellent in vitro transfection efficiency that was comparable to or significantly higher than that of branched PEI (25K). Kinetics of expression is also important for gene delivery. Figure 9 shows the time-dependent changes of gene expression of the cationic polyrotaxane CP3 in comparison with that of PEI (25K) at various N/P ratios in PC3 cells, and the transfection efficiency was monitored for 3 days. Generally, in the absence or presence of serum, the transfection efficiency mediated by PEI (25K) decreased slowly with the increasing expression duration, while CP3 showed a sustained gene delivery capability. In both serum and serum-free conditions, increases in transfection efficiency could be found in PC3 cells transfected with CP3/pRL-CMV when the expression duration increased from 24 to 48 h, and then to 72 h. Only at higher N/P ratios, the transfection efficiency mediated by CP3 decreased after 48 h expression in the presence of serum. These results may be mainly attributed to the supramolecular structure of the polyrotaxane: in these cationic polyrotaxanes, the OEI-grafted β-CD rings can rotate and/or move along the block copolymer chain freely, and this flexibility may enhance the interaction of the cationic polyrotaxane with DNA and/or cellular membrane. Confirmation of the gene delivery capability of cationic polyrotaxane CP3 was also obtained by laser confocal scanning fluorescence microscopy in comparison with that of PEI (25K) (Figure 10). Plasmid pEGFP-N1 encoding green fluorescence protein (GFP) was used to examine the GFP expression in PC3 cells. Similar strong fluorescence signal could be observed when transfections were mediated by either CP3 or PEI at N/P ratio

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Figure 10. Confocal microscopy images of transfected PC3 cells. The transfections were mediated by the CP3 (a) and PEI (25K) (b) at N/P ratio of 10 in the absence of serum using green fluorescence protein gene as a reporter gene. The same field of cells was observed by Nomarski optics (right panel) or by fluorescence microscope (left panel) to visualize GFP expression.

of 10. GFP expression could not be detected when the transfection was mediated by naked DNA, which was used as a negative control (data not shown). Conclusions In this study, cationic polyrotaxanes consisting of multiple OEI-grafted β-CD rings threaded on a PEO-PPO-PEO triblock copolymer chain were investigated for their physicochemical properties, cytotoxicity and gene transfection efficiency in cancer cells with reference to branched PEI (25K). Fluorescence titration assay results showed that, resembling branched PEI (25K), these catonic polyrotaxanes could completely condense pDNA and form stable complexes at N/P ratio of 2. The representative tapping mode AMF images in air characterized the morphology of the complexes between the cationic polyrotaxane and pDNA and comfirmed the complex formation. Moreover, real-time tapping mode AFM imaging in aqueous environment was successfully achieved, elucidating the condensation process of pDNA by the cationic polyrotaxane and formation of the complexes. In addition, cytotoxicity studies showed that these cationic polyrotaxanes displayed significantly low cytotoxicity in SK-OV-3 and PC3 cancer cells in comparison with branched PEI (25K), owing to the low positive charge density resulted from the supramolecular structure of the polyrotaxanes. In SK-OV-3 cells, generally, in the presence or absence of serum, the increased OEI length or N/P ratio produced greater transfection efficiency. In PC3 cells, in the presence or absence of serum and over the entire range of N/P ratios examined, the synthesized cationic polyrotaxanes displayed excellent in vitro transfection efficiency that was comparable to or significantly higher than that of branched PEI (25K). In addition, in the absence or presence of serum, the transfection efficiency mediated by PEI (25K) decreased slowly with the increasing expression duration, while cationic polyrotaxane showed a sustained gene delivery capability in PC3 cells. Hence, these cationic polyrotaxanes show strong DNA binding ability, low cytotoxicity, and high and sustained gene

Cationic Polyrotaxanes as Gene Carriers delivery capability and they have a high potential for efficient gene transfer in cancer cells. Abbreviations CD, cyclodextrin; DMEM, Dulbecco’s Modified Eagle’s Medium; EtBr, ethidium bromide; GFP, green fluorescence protein; HEK293, cell line derived from human embryo kidney; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide; N/P ratio, molar ratio of amine groups in cationic molecule to phosphate groups in DNA; OEI, oligoethyleneimine; PBS, phosphate-buffered saline; PC3, human prostate adenocarcinoma; pDNA, plasmid DNA; PEHA, pentaethylenehexamine; PEI, polyethylenimine; PEI (25K), polyethylenimine with molecular weight of 25 000 Da; PEO, poly(ethylene glycol); PPO, poly(propylene glycol); SK-OV-3, cell line derived from human ovary adenocarcinoma; TNBS, trinitrobenzenesulfonic acid. Acknowledgment. We acknowledge the financial support from Ministry of Education Academic Research Fund Tier 2 Grant (R-397-000-031-112) and Institute of Materials Research and Engineering, Singapore. References and Notes (1) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297. (2) Yang, C.; Li, H. Z.; Goh, S. H.; Li, J. Biomaterials 2007, 28, 3245. (3) Li, J.; Loh, X. J. AdV. Drug DeliVery ReV. 2008, 60, 1000. (4) Zauner, W.; Ogris, M.; Wagner, E. AdV. Drug DeliVery ReV. 1998, 30, 97. (5) KukowskaLatallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897. (6) Dekie, L.; Toncheva, V.; Dubruel, P.; Schacht, E. H.; Barrett, L.; Seymour, L. W. J. Controlled Release 2000, 65, 187. (7) Wang, J.; Mao, H. Q.; Leong, K. W. J. Am. Chem. Soc. 2001, 123, 9480. (8) Leong, K. W.; Mao, H. Q.; Truong-Le, V. L.; Roy, K.; Walsh, S. M.; August, J. T. J. Controlled Release 1998, 53, 183. (9) Wenz, G.; Han, B. H.; Muller, A. Chem. ReV. 2006, 106, 782. (10) Harada, A.; Li, J.; Kamachi, M. Nature 1992, 356, 325. (11) Wenz, G.; Keller, B. Angew. Chem.sInt. Ed. Engl. 1992, 31, 197. (12) Wenz, G. Angew. Chem.sInt. Ed. Engl. 1994, 33, 803. (13) Nepogodiev, S. A.; Stoddart, J. F. Chem. ReV. 1998, 98, 1959. (14) Raymo, F. M.; Stoddart, J. F. Chem. ReV. 1999, 99, 1643. (15) Li, J.; Li, X.; Zhou, Z. H.; Ni, X. P.; Leong, K. W. Macromolecules 2001, 34, 7236. (16) Li, J.; Ni, X. P.; Leong, K. Angew. Chem.sInt. Ed. 2003, 42, 69. (17) Li, J.; Chen, B.; Wang, X.; Goh, S. H. Polymer 2004, 45, 1777. (18) Liu, K. L.; Goh, S. H.; Li, J. Macromolecules 2008, 41, 6027.

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