Comparative studies on the genotoxicity and cytotoxicity of polymeric ...

Drug and Chemical Toxicology, 2010; 33(4): 357–366

RESEARCH ARTICLE

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Comparative studies on the genotoxicity and cytotoxicity of polymeric gene carriers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer in Jurkat T-cells Young Joo Choi1, Su Jin Kang2, Yang Jee Kim1, Yong-beom Lim3, and Hai Won Chung1 School of Public Health and Institute of Health and Environment, Seoul National University, Seoul, Republic of Korea, College of Pharmacy, Yeungnam University, Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea, and 3Department of Materials Science and Engineering, Yonsei University, Seoul, Republic of Korea

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Abstract A safe alternative to the viral system used in gene therapy is a nonviral gene delivery system. Although polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer are among the most promising genecarrier candidates for efficient nonviral gene delivery, safety concerns regarding their toxicity remain. The aim of this study was to scrutinize the underlying mechanism of the cytotoxicity and genotoxicity of PEI (25 kDa) and PAMAM (G4). To our knowledge, this is the first study to explore the genotoxic effect of polymeric gene carriers. To evaluate cell death by PEI and PAMAM, we performed propidium-iodide staining and lactate-dehydrogenase release assays. The genotoxicity of the polymers was measured by comet assay and cytokinesis-block micronucleus assay. PEI- and PAMAM-treated groups induced both necrotic and apoptotic cell death. In the comet assay and micronuclei formation, significant increases in DNA damage were observed in both treatments. We conclude that PEI and PAMAM dendrimer can induce not only a relatively weak apoptotic and a strong necrotic effect, but also a moderate genotoxic effect. Keywords: Comet assay; CBMN assay; apoptosis; necrosis; PEI; PAMAM

Introduction Numerous types of nanomaterials are being developed for use in cell-based bioapplications. The increased use and production of nanomaterials will possibly increase human exposure to them, which has created public concern about their potential health and environmental risks (Service, 2008). Nanoparticles composed of cationic polymers and DNA have been extensively explored as gene carriers for use in gene therapy (Pack et al., 2005; Mastrobattista et al., 2006). Therefore, it is imperative that experts thoroughly investigate their toxicity before using them in human gene-therapy trials. The aim of gene therapy is to

cure inherited or acquired diseases by delivering therapeutic genes to target cells, which results in the expression of therapeutic proteins (Azzam and Domb, 2006). There are two main categories of gene carriers: viral and nonviral vectors. Although viral vectors are generally superior to nonviral vectors in terms of transfection efficiency and tissue tropism, both in vitro and in vivo, there are significant problems associated with the use of viral vectors in human gene therapy (Luten et al., 2008). For example, retroviral vectors are limited because of the concern for carcinogenesis following nonspecific integration into host chromosomal DNA.

Address for Correspondence: Hai Won Chung, School of Public Health and Institute of Health and Environment, Seoul National University, 28 Yongon-dong, Jongno-gu, Seoul 110-460, Republic of Korea; Fax: 82-2-747-7082; E-mail: [email protected] (Received 06 October 2009; revised 12 November 2009; accepted 17 November 2009) ISSN 0148-0545 print/ISSN 1525-6014 online © 2010 Informa Healthcare USA, Inc. DOI: 10.3109/01480540903493507

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In this regard, a nonviral gene-delivery carrier system is a safer alternative to the use of viral systems in gene therapy (Okuda et al., 2004). Cationic polymers—such as linear- and branched-polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-lysine, cationic polyesters (Lim et al., 2001, 2000), and poly-β-amino esters—have been the subject of intensive interest as nonviral genecarrier molecules. Because the polyanionic nature of nucleic acids inhibits them from crossing the same charged cytoplasmic membrane barrier, cationic polymers are used to neutralize the negative charges of nucleic acids (Lim et al., 2008). PEI and PAMAM dendrimer are among the best known, most extensively studied cationic polymers and have been widely used in nonviral transfection protocols (Moghimi et al., 2005). The backbone of the PEI chain is composed of vinyl bonds (carbon–carbon linkages) that result in an almost nondegradable polymer (Figure 1). Consequently, a great deal of concern has been raised with respect to the accumulation of PEI within cellular compartments after completion of the gene-delivery process (Godbey et al., 1999; Suh et al., 2003). Another promising polymeric gene carrier, PAMAM dendrimer, is a synthetic spherical macromolecule with branches radiating from a central core (Figure 1). Contrary to PEI, which is nondegradable, PAMAM dendrimer is a biodegradable polymer with a backbone of polymer chains consisting of peptide bonds. For this reason, it exhibits comparatively less cytotoxicity than PEI. At physiological pH, positively charged terminal ammonium groups in PAMAM dendrimer can bind to negatively charged DNA phosphates to form a PAMAM/DNA complex (Hui et al., 2008). Studies

using PEI and PAMAM dendrimer have indicated that these polymers have intrinsic nuclear targeting and localization activities (Lim et al., 1999; Wang et al., 2001; Xu et al., 2007; Patil et al., 2009). Cationic polymers may interact with negatively charged chromosomal DNA and adversely affect cell growth and nucleus function. Apoptosis and necrosis, the two major modes of cell death, have entirely different morphological and biochemical features (Arends and Wyllie, 1991). They also share several common pathways that can occur concurrently (Shimizu et al., 1996; Leist and Nicotera, 1997). p53 engages in many independent biological pathways that mediate apoptosis and induce G1 cell-cycle arrest (Moll and Zaika, 2001). Researchers of PEI treatment have reported the elevation of apoptosis markers, including the activation of caspase-3 and -9, and a significant increase in the level of cytochrome c production (Hunter, 2006). Cationic PAMAM dendrimer induced apoptosis in RAW 264.7 macrophage cells, but not in NIH/3T3 or BNL CL.2 cells (Kuo et al., 2005). Although PEI and PAMAM dendrimer can induce necrosis as well as apoptosis in human cells, very little is known about the detailed mechanisms of cell death (Xiong et al., 2007). Therefore, we investigated the cell-death pathway by analyzing differential mRNA levels of p53, p21, and Ataxia telangiectasia mutated (ATM) genes after PEI and PAMAM dendrimer treatment. To our knowledge, this study is the first to investigate the effects of PEI and PAMAM dendrimer on cellular genotoxicity and to discuss the correlation between genotoxic and cytotoxic effects. O

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Figure 1. The structures of PEI (polyethyenimine; 25 kDa) and PAMAM (polyamidoamine) G4 dendrimer, shown as partial structures.

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Materials and methods


Reagents Branched PEI [molecular weight (MW), 25,000 g/mol] and G4 PAMAM dendrimer were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). G4 stands for “generation 4” and refers to dendritic molecules that contain 64 peripheral primary amines. RPMI medium 1640, fetal bovine serum (FBS), 100 U/mL of penicillin, and streptomycin were purchased from Gibco-BRL (Carlsbad, California, USA). Cell culture and treatment Human acute T-cell leukemia Jurkat cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI medium 1640 supplemented with filtered 10% FBS,, along with penicillin and streptomycin (100 U/mL of each), in a humidified atmosphere at 37°C and 5% CO2. Cells were treated with various concentrations of PEI or PAMAM dendrimer. PEI stock solutions were stored at 4°C, whereas PAMAM stock solutions were stored at −76°C to prevent spontaneous degradation of the dendrimer (i.e., hydrolysis of peptide bonds). Evaluation of cell survival of polymers Cells (1 × 106 per mL) were seeded into 24-well Petri dishes and treated with PEI (1, 10, or 20 µg/mL) or PAMAM dendrimer (20, 100, or 200 µg/mL). Cells were cultivated for 24 hours at 37°C. The cell viability of PEI or PAMAM dendrimer was examined by using the trypan blue exclusion assay. The mixture of cell suspension and trypan blue solution was placed on a hemocytometer, and cells stained blue were considered to be dead. Single-cell gel-electrophoresis assay The subsequent steps of the single-cell gel-electrophoresis assay were performed as described by Singh et al. (1988). Jurkat T-leukemia cells were treated with PEI (1, 3, or 5 µg/mL) or PAMAM dendrimer (10, 30, or 50 µg/mL) for 4 hours. After being washed twice with phosphate-buffered saline (PBS) solution, the cells were maintained at 4°C to prevent DNA repair. Frosted slides had previously been dipped in 0.6% normal melting agarose for the first layer. The cells were then mixed with 85 µL of low-melting agarose and placed on the first layer. Another 85 µL of lowmelting agarose was added to form the top layer. The slides were placed in a lysis solution [2.5 µM NaCl,

0.1 M Na2-EDTA, 0.01 M Tris-HCl, 1% Triton X-100, and 10% dimethyl sulfoxide (DMSO) adjusted to pH 10] for 1 hour at 4°C, rinsed with distilled water, placed in the electrophoresis buffer (300 mM of NaOH and 1 mM of Na2-EDTA; pH 13) for 20 minutes to allow for DNA unwinding, and electrophoresed for 25 minutes at 0.78V/cm and 300 mA. The slides were neutralized with 0.4 M of Tris-HCl buffer (pH 7.5) to remove the alkali and detergent. Slides were then dried and fixed by immersion in absolute ethanol. Before examination, ethidium bromide, diluted to 5 µg/mL in distilled water, was placed on top of the slide and covered with a coverslip. Finally, DNA damage was evaluated by visually scoring images of 60 randomly selected cells from each sample. The tail length of each cell was measured under a fluorescent microscope (Nikon, Tokyo, Japan), equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm, and analyzed by using the Komet 5.5 image-analysis system (Kinetic Imaging Limited, Nottingham, UK). Cytokinesis-block micronucleus assay Jurkat T-leukemia cells were treated with PEI (1, 10, or 20 µg/mL) or PAMAM dendrimer (10, 100, or 200 µg/mL) polymers for 4 hours. Cytochalasin B (4 µg/mL; Sigma-Aldrich) was added 20 hours after the start of the culture, and incubation was continued for an additional 28 hours. After culturing for 48 hours, the cells were harvested and treated twice with 0.075 M of KCl hypotonic solution for 1 minute and fixation solution (a mixture of methanol and acetic acid; 3:1). Air-dried cell preparations were stained with Giemsa solution (5%). A total of 1,000 binucleated cells with well-preserved cytoplasm slides were scored according to standard criteria (Fenech, 2000), and the nuclear division index (NDI) was evaluated in 500 cells from each sample to determine the percentage of cells with mono-, bi-, and multinuclei. The NDI was calculated by using the standard formula (Eastmond and Tucker, 1989). Quantitative real time-PCR of ATM, p53, and p21 Total RNA was extracted from Jurkat cells after treatment with PEI or PAMAM dendrimer for 24 hours, using the AxyPrep Multisource Total RNA Miniprep Kit (Axygen Biosciences, Union City, California, USA), according to the manufacturer’s instructions. For the first cDNA synthesis, total RNA (1 µg) was subjected to reverse transcription, using a random primer


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and the Reverse Transcription System (Promega, Madison, Wisconsin, USA). Real-time polymerase chain reaction (PCR) of each sample was obtained by using Express Start SYBR Green qPCR Supermix (Invitrogen, Burlington, Ontario, Canada) and was performed by using the thermal cycling condition: incubation at 94°C for 5 minutes, cycled 40 times at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Each assay was performed in triplicate, using the Applied Biosystems 7300 Real-Time PCR system (Foster City, CA, USA). GAPDH was used as an endogenous control to normalize expression. The following primers were used for each gene: GAPDH, 5′-GGAAGGTGAAGGTCGGAGTCA-3′ and 5′-GTCATTGATGGCAACAATATCCACT-3′; ATM, 5′-CGAGGTGAGCGGATCACAA-3′ and 5′-TTGGCCCACAGCAACCTT-3′; p53, 5′-GTGAGCGCTTCGAGATGTTC-3′ and 5′-ATGGCGGGAGGTAGACTGAC-3′; and p21, 5′-TGGACCTGTCACTGTCTTGT-3′ and 5′-TCCTGTGGGCGGATTAG-3′ (Bose et al., 2009; Kanellou et al., 2008). The expression fold changes (i.e., ratio) of each gene were measured relative to the control and each treatment, using the comparative threshold cycle method (◿◿Ct).

Estimated release of lactate dehydrogenase (LDH) assay The release of LDH from Jurkat cells exposed to various concentrations of PEI and PAMAM dendrimer was determined by using a Cytotoxicity Detection KitPLUS (Roche, Indianapolis, IN, USA), according to the manufacturer’s protocols. This assay measures the LDH released from necrotic cells into the medium upon the rupture of the plasma membrane. Absorbance at 490 nm (reference wavelength, 600 nm) was read by using a microplate reader (Tecan, Männedorf, Switzerland). LDH release is shown as a percentage of the LDH released from the total LDH in each sample. Statistical analysis The damaging effect of PEI and PAMAM dendrimer on DNA, in terms of micronuclei (MN) formation and change in comet length between the control and treatment groups, was tested using Mann-Whitney’s U nonparametric test. Intergroup differences were tested by analysis of variance. Statistical analysis was performed with SPSS statistics v17 (SPSS Inc., Chicago, Illinois, USA).

Flow cytometry To measure apoptosis, we collected 1 × 106 cells, washed them with PBS, fixed them with 1 mL of icecold 70% ethanol in PBS at −20°C, and left them overnight. The cell pellets were harvested after washing with PBS, followed by the addition of 500 μL of propidium iodide solution containing propidium iodide and RNase A solution. The mixture was incubated at room temperature in the dark for 30 minutes. The nuclei were analyzed by flow cytometry (FACSCalibur; Becton-Dickinson, San Jose, California, USA), and data were analyzed by using CellQuest Pro software (Becton-Dickinson). All experiments were performed at least three times. Mitochondrial membrane potential (MMP; Δψm) was also determined by flow cytometry. Flow cytometric analysis of Jurkat T-leukemia cells stained with 3,3’-dihexyloxacarbocyanine (DiOC6; Molecular Probes, Eugene, Oregon, USA) was performed to determine changes in Δψm (Conrad et al., 2008). Jurkat T-cells were exposed to PEI or PAMAM polymers for 6 hours and then incubated with DiOC6 (40 nM) for 30 minutes at 37°C. Cells were washed twice with PBS and analyzed by using flow cytometry (FACSCalibur; Becton-Dickinson) (Kang et al., 2008). All experiments were performed at least three times.

Results Cell-death assay We examined the effect of various concentrations of PEI and PAMAM dendrimer on the cell viability of Jurkat T-cells by using the trypan blue exclusion assay (Figure 2A). The results revealed a dose-dependent decrease in cell viability. Of note is that a more significant decrease in cell viability was observed in the PEI-treated group (28%) than in the PAMAM-treated group (93%) at an equivalent concentration (20 µg/ mL). Overall, PEI showed an approximately 5- to 10-fold higher cytotoxic effect than PAMAM dendrimer (55% for PEI at 10 µg/mL vs. 47% for PAMAM at 100 µg/mL; 82% for PEI at 1 µg/mL vs. 93% for PAMAM at 20 µg/mL). The release of LDH, a sensitive marker of cytotoxicity, was also evaluated. The amount of LDH released increased in a dose-dependent manner for both PEI- and PAMAM-treated groups (Figure 2B), which was consistent with the results of the trypan blue exclusion assay. Overall, PEItreated groups released more LDH than PAMAMtreated groups.

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Cytogenetic toxicity of PEI and PAMAM dendrimer 361

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Figure 2. Viability assay for PEI- or PAMAM-induced cytotoxicity in Jurkat T-cells. Jurkat cells were incubated on a 24-well culture plated for 24 hours, treated with various concentrations of PEI or PAMAM, and viability was determined by trypan blue exclusion assay (A) and released LDH activity assay (B). Data are shown as percentages of control and represented the mean ± standard error of the mean. Each experiment was performed three times. Results were statistically analyzed with the Student’s t-test. *P