Mol Biotechnol (2010) 46:287–300 DOI 10.1007/s12033-010-9302-5
RESEARCH
Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents Seiichi Yamano • Jisen Dai • Amr M. Moursi
Published online: 29 June 2010 Ó Springer Science+Business Media, LLC 2010
Abstract This study compared six commercially available reagents (Arrest-In, ExpressFect, FuGENE HD, jetPEI, Lipofectamine 2000, and SuperFect) for gene transfection. We examined the efficiency and cytotoxicity using nine different cell lines (MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma), and primary cells (HEKn human epidermal keratinocytes) with two different plasmid DNAs encoding luciferase or b-galactosidase in the presence or absence of serum. Maximal transfection efficiency in MC3T3-E1, C3H10T1/2, HeLa, C2C12, Hep G2, and HCT116 was seen using FuGENE HD, in PT-30, 4T1, and HEKn was seen using Arrest-In, and in MCF-7 was seen using jetPEI. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetPEI and ExpressFect. These results suggest that FuGENE HD is the most preferred transfection reagent for many cell lines, followed by Arrest-In and jetPEI. These results may be useful for improving nonviral gene and cell therapy applications. Keywords Gene transfection Cationic lipid Polymer Polyethylenimine Activated-dendrimer S. Yamano (&) J. Dai Department of Prosthodontics, New York University College of Dentistry, 345 East 24th Street, 4W, New York, NY 10010, USA e-mail:
[email protected] A. M. Moursi Department of Pediatric Dentistry, New York University College of Dentistry, New York, NY, USA
Introduction Gene transfection is a widely used technique for molecular studies and therapeutics. Many of vectors have been developed with the purpose of delivering genes efficiently into target cells and protecting them from nuclease degradation. Viral vectors are generally most efficient for cell transduction, but they present several disadvantages related with immunogenicity, inflammation, DNA size restriction, and large-scale constraints [1]. Whereas, non-viral vectors are generally less efficient in delivering DNA and initiating gene expression when compared with viral vectors, particularly when used in vivo [2]. However, new non-viral vectors have been developed to overcome these limitations [3]. Non-viral vectors made of lipids [4], polymers [5], dendrimer [6], polyethylenimine [7], peptides [8], and nanoparticle-based compounds [9] have been receiving increasing attention, since they are safer and cheaper, can be produced easily in large quantities, and have higher genetic material carrying capacity [10]. They induce cellular uptake of DNA by complexing nucleic acids and generating particles with positively charged surfaces. Thus, these vectors bind to the negatively charged membrane through ionic interaction and enter the cell through spontaneous endocytosis, without significant cytotoxicity [11]. Although many new systems hold promises to achieve high-transfection efficiencies, a systematic evaluation in a large variety of cells has not, to our knowledge, been performed to date. In general, transfection efficiency of vectors is associated with cell types, kind of DNAs, and medium conditions. The aim of this study was to comparatively examine a panel of non-viral gene transfer systems commercially available in several established cell lines. We tested the hypothesis that, under optimized conditions, these gene transfer systems are capable
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of yielding high-transfection efficiencies. We chose six reagents of different formulations, Arrest-In (a lipo-polymeric formulation), ExpressFect (a cationic polymer), FuGENE HD (a lipid with other components), jetPEI (a linear polyethylenimine), Lipofectamine 2000 (a cationic lipid), and SuperFect (an activated-dendrimer). All of them were tested in nine cell lines, MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma, and in a primary culture of HEKn human epidermal keratinocytes. For these studies, two different plasmid DNAs encoding luciferase and b-galactosidase were used in the presence or absence of serum.
5,000 U/ml penicillin (Invitrogen) and 5,000 lg/ml streptomycin (Invitrogen) at 37°C in a humidified atmosphere with 5% CO2. These same mediums were used for transfections except in PT-30 cells in which we used aMEM instead of KGM.
Materials and Methods
Preparation of each transfection system
Materials
The plasmid DNA was mixed with different transfection reagents (TR) in various TR/DNA ratios (w/w). Complexes were prepared according to the manufacturer’s instructions. Solutions were combined, gently mixed, and incubated for the appropriate time to allow formation of complexes.
Arrest-In (Thermo Scientific, Huntsville, AL), ExpressFect (Denville Scientific, Metuchen, NJ), FuGENE HD (Roche, Branchburg, NJ), jetPEI (Polyplus-transfection, New York, NY), Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and SuperFect (Qiagen, Valencia, CA) were used in this study. Cell culture Nine different cell lines, MC3T3-E1 (mouse preosteoblasts), C3H10T1/2 (mouse stem cells), PT-30 (human epithelial precancer), MCF-7 (human breast cancer), HeLa (human cervical cancer), C2C12 (mouse myoblasts), Hep G2 (human hepatocellular carcinoma), 4T1 (mouse mammary carcinoma), and HCT116 (human colorectal carcinoma) were used. Also, HEKn primary human epidermal keratinocytes (Lifeline Cell Technology, Walkersville, MD) was used. MC3T3-E1 (gift from Dr. Mani Alikhani, New York University College of Dentistry) was cultured in Gibco alpha minimal essential medium (aMEM; Invitrogen) with 10% Gibco fetal bovine serum (FBS; Invitrogen). C3H10T1/2 (gift from Dr. Mani Alikhani) was cultured in basal medium Eagle (BME; Invitrogen) with 10% FBS. PT-30 (gift from Dr. Peter Sacks, New York University College of Dentistry) was cultured in keratinocyte growth medium (KGM; Lonza, Walkerville, MD). MCF-7, HeLa, C2C12, Hep G2, 4T1, and HCT116 (gift from Dr. Xi Huang, New York University School of Medicine) were cultured in Gibco Dulbecco’s modified Eagle medium (DMEM; Mediatech, Manassas, MA) with 10% FBS. HEKn was cultured in DermaLife K Medium Complete Kit (Lifeline Cell Technology). All cells were cultured with
Plasmid DNAs Plasmid DNAs, encoding for luciferase (gWIZ luciferase) or b-galactosidase (gWIZ b-galactosidase) under the control of the cytomegalovirus promoter/enhancer obtained from Genlantics (San Diego, CA), were used. Vectors were propagated in competent Escherichia coli DH5a cells (Invitrogen). Ultrapure endotoxin-free plasmid DNA was prepared using the QIAfilter kit (Qiagen) according to the manufacturer’s instructions. Plasmid DNA was diluted in sterile water to a final concentration of 1 lg/ll DNA.
Arrest-In Arrest-In is a proprietary lipo-polymeric formulation. For optimization studies, 1 lg DNA was complexed with 5, 10, or 20 lg of the Arrest-In reagent [TR/DNA ratio (w/w) = 5:1, 10:1, 20:1] in the appropriate growth medium. ExpressFect ExpressFect is a new generation cationic polymer gene transfection reagent. For optimization studies, 1 lg DNA was complexed with 1, 3, or 6 lg of the ExpressFect [TR/ DNA ratio (w/w) = 1:1, 3:1, 6:1] in the appropriate growth medium. FuGENE HD FuGENE HD is a proprietary blend of lipids and other components. For optimization studies, 1 lg DNA was complexed with 2, 4, or 8 lg of the FuGENE HD [TR/ DNA ratio (w/w) = 2:1, 4:1, 8:1] in the appropriate growth medium. jetPEI jetPEI consists of a linear polyethylenimine and compacts the DNA into positively charged particles. For optimization
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studies, 1 lg DNA was complexed with 1, 2, or 4 lg of the jetPEI [TR/DNA ratio (w/w) = 1:1, 2:1, 4:1] in the appropriate growth medium.
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among groups. P values less than 0.05 were considered significant. Cytotoxicity evaluation
Lipofectamine 2000 Lipofectamine 2000 is a novel proprietary cationic lipid formulation. For optimization studies, 1 lg DNA was complexed with 1, 2, or 4 lg of the Lipofectamine 2000 [TR/DNA ratio (w/w) = 1:1, 2:1, 4:1] in the appropriate growth medium. SuperFect This activated-dendrimer transfection reagent has a spherical architecture with branches radiating from a central core and terminating at positively charged amino groups. For optimization studies, 1 lg DNA was complexed with 2, 5, or 8 lg of the SuperFect [TR/DNA ratio (w/w) = 2:1, 5:1, 8:1] in the appropriate growth medium. In vitro DNA transfection Cells were plated in a 96-well cluster dish at a density of 2 9 105 cells/ml, cultivated in the appropriate growth medium with 10% FBS, and 100 ll was added to each well. After 24 h in culture, the cells were washed with phosphate-buffered saline (PBS), and 100 ll fresh growth medium with or without 10% FBS was added to the cells. TR/DNA complexes were then added to each well and incubated with the cells for 4 h at 37°C for the without FBS condition group. Then, 100 ll appropriate media with 20% FBS was added to the each well. The cells were cultured for 48 h at 37°C in 5% (v/v) CO2 after transfection. All transfection assays were carried out in quadruplicate simultaneously for all six transfection reagents and with no reagent for control purposes. Detection of transgene expression Transgene expression was detected at a standardized representative time point of 48 h after transfection. Luciferase expression level was measured with BrightGloTM Luciferase Assay System (Promega, Madison, WI) and b-galactosidase level was measured with BrightGloTM b-galactosidase Assay System (Promega) using a multi-detection microplate reader, SynergyTM HT (BioTek Instruments Inc., Winooski, VT). Relative light units (RLU) were recorded in duplicates with 10-s integration as their expression levels. Data are expressed as mean luciferase or b-galactosidase activity (RLU) per well ± standard deviation from quadruplicates. ANOVA was applied to determine the significance of differences
Cytotoxicity of the six transfection reagents in optimal condition for transfection was evaluated by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide, MTT assay. MC3T3-E1 cells (1 9 106/ml) in 100 ll of aMEM (Invitrogen) supplemented with 10% FBS were seeded in 96-well plates and incubated overnight. The 5 mg/ml MTT reagent in 1 9 PBS (10 ll/well) was added into the plates and incubated 4 h. After incubation, the medium was aspirated and dimethyl sulfoxide (100 ll/ well) was added to stop the reaction. The optical density was quantified in a multi-detection microplate reader, SynergyTM HT (BioTek Instruments Inc.) at 570 nm wavelength. The percentage of cell viability was calculated by comparing the appropriate luminescent signal to the signal obtained with non-transfected control cells. Each value represents the mean ± standard deviation from quadruplicates. As a control (100% viability), non-transfected cells were used.
Results Optimization of each transfection reagent/DNA ratio Before the transfection studies in the selected cell lines, optimal transfection conditions were evaluated by varying the amounts of plasmid DNA encoding luciferase gene at different TR/DNA ratios in order to form the complexes. This optimization study was performed with MC3T3-E1 cells. Optimized transfection efficiencies were obtained with the following ratios: Arrest-In in a ratio of 5:1, ExpressFect in a ratio of 1:1, FuGENE HD in a TR/DNA ratio of 4:1, jetPEI in a ratio of 2:1, Lipofectamine 2000 in a ratio of 1:1, and SuperFect in a ratio of 5:1. These ratios were used for subsequent transfection studies (Fig. 1). Luciferase in MC3T3-E1 cells Out of the six transfection systems tested, FuGENE HD promoted maximal luciferase expression with and without 10% FBS in MC3T3-E1 cells (34,977 ± 7,173 and 27,581 ± 11,266 RLU/well, respectively) compared to all other reagents tested (Fig. 2a). jetPEI resulted in the second highest luciferase expression with and without FBS (17,564 ± 7,173 and 12,438 ± 1,611 RLU/well, respectively) (Fig. 2a). The difference in luciferase expression between using FuGENE HD and jetPEI was significant both with and without 10% FBS conditions (p \ 0.05).
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Luciferase expression (RLU/welll)
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Fig. 1 In vitro transfection efficiency of transfection reagent (TR)/ DNA complexes at different ratios (w/w) in MC3T3-E1 cells. Cells were transfected with TR/DNA complexes in the presence of serum
with various amounts of plasmid. AI Arrest-In, EF ExpressFect, FH FuGENE HD, JP jetPEI, Lipo Lipofectamine 2000, SF SuperFect
Luciferase in PT-30 cells
Luciferase in HeLa cells
Arrest-In and FuGENE HD mediated the highest luciferase expression with 10% FBS in PT-30 cells (131,172 ± 17,649 and 120,560 ± 27,984 RLU/well; p = 0.54) (Fig. 2b). Also, Arrest-In promoted the highest luciferase expression without FBS in PT-30 cells followed by FuGENE HD (203,690 ± 6,088 and 152,147 ± 19,628 RLU/well; p \ 0.05) (Fig. 2b).
FuGENE HD promoted the highest luciferase expression with 10% FBS in HeLa cells followed by jetPEI (709,135 ± 14,973 and 652,758 ± 13,095 RLU/well; p \ 0.05) (Fig. 2e). Also, Arrest-In produced the highest luciferase expression without FBS in HeLa cells followed by ExpressFect (517,334 ± 52,328 and 410,206 ± 41,810 RLU/well; p \ 0.05) (Fig. 2e).
Luciferase in C3H10T1/2 cells FuGENE HD achieved maximal luciferase expression with or without 10% FBS in C3H10T1/2 cells (43,408 ± 4,151 or 46,836 ± 14,020 RLU/well) compared to all other reagents tested (Fig. 2c). Lipofectamine 2000 resulted in the second highest luciferase expression with or without FBS (25,861 ± 7,416 or 23,894 ± 3,766 RLU/well) (Fig. 2c). The difference in luciferase expression between using FuGENE HD and Lipofectamine 2000 was significant both with and without FBS (p \ 0.05).
Luciferase in C2C12 cells FuGENE HD achieved maximal luciferase expression with or without 10% FBS in C2C12 cells (19,637 ± 3,309 or 6,142 ± 491 RLU/well) compared to all other reagents tested (Fig. 2f). jetPEI resulted in the second highest luciferase expression with or without 10% FBS (11,794 ± 1,472 or 4,628 ± 798 RLU/well) (Fig. 2f). The difference in luciferase expression between using FuGENE HD and jetPEI was significant both with and without 10% FBS conditions (p \ 0.05). Luciferase in Hep G2 cells
Luciferase in MCF-7 cells Arrest-In and jetPEI produced the highest luciferase expression with 10% FBS in MCF-7 cells (204,359 ± 26,873 and 199,136 ± 32,533 RLU/well; p = 0.81) (Fig. 2d). Also, jetPEI and Lipofectamine 2000 promoted the highest luciferase expression without FBS (63,677 ± 8,261 and 53,519 ± 2,646 RLU/well; p = 0.06) (Fig. 2d).
Arrest-In mediated the highest luciferase expression with 10% FBS in Hep G2 cells followed by jetPEI (194,791 ± 14,127 and 84,390 ± 8,856 RLU/well; p \ 0.05) (Fig. 2g). Also, Arrest-In promoted the highest luciferase expression without FBS in Hep G2 cells followed by FuGENE HD (167,307 ± 29,553 and 102,984 ± 26,338 RLU/well; p \ 0.05) (Fig. 2g).
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Fig. 2 Comparative analysis of luciferase expression for various cells with or without serum (a MC3T3-E1 mouse preosteoblasts, b PT-30 human epithelial precancer cells, c C3H10T1/2 mouse stem cells, d MCF-7 human breast cancer cells, e HeLa human cervical cancer, f C2C12 mouse myoblasts, g Hep G2 human hepatocellular carcinoma, h 4T1 mouse mammary carcinoma, i HCT116 human colorectal carcinoma, and j HEKn primary human epidermal keratinocytes)
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Luciferase in 4T1 cells Arrest-In and jetPEI mediated the highest luciferase expression with 10% FBS conditions in 4T1 cells (158,389 ± 22,712 and 129,119 ± 10,596 RLU/well; p = 0.06) (Fig. 2h). Also, FuGENE HD promoted the highest luciferase expression without FBS in 4T1 cells followed by Arrest-In (159,947 ± 27,835 and 94,175 ± 14,444 RLU/well; p \ 0.05) (Fig. 2h). Luciferase in HCT116 cells Arrest-In promoted the highest luciferase expression with 10% FBS in HCT116 cells followed by FuGENE HD (1,224,084 ± 36,613 and 962,654 ± 138,701 RLU/well; p \ 0.05) (Fig. 2i). Also, FuGENE HD produced the highest luciferase expression without FBS in HCT116 cells followed by Arrest-In (1,065,691 ± 44,374 and 730,415 ± 47,867 RLU/well; p \ 0.05) (Fig. 2i). Luciferase in primary epidermal keratinocytes FuGENE HD achieved the highest luciferase expression with 10% FBS in primary keratinocytes followed by Arrest-In (40,368 ± 4,304 and 31,838 ± 4,793 RLU/well;
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p \ 0.05) (Fig. 2j). Also, jetPEI promoted the highest luciferase expression without FBS in primary keratinocytes followed by ExpressFect (33,265 ± 1,311 and 28,999 ± 1,903 RLU/well; p \ 0.05) (Fig. 2j). b-galactosidase in MC3T3-E1 cells FuGENE HD led to maximal b-galactosidase expression with 10% FBS in MC3T3-E1 cells followed by Arrest-In (670,188 ± 39,815 and 608,099 ± 25,854 RLU/ well; p \ 0.05) (Fig. 3a). Also, Arrest-In and jetPEI produced the highest b-galactosidase expression without FBS in MC3T3-E1 cells (508,569 ± 23,141 and 462,838 ± 33,894 RLU/well; p = 0.07) (Fig. 3a). b-galactosidase in PT-30 cells Arrest-In mediated maximal b-galactosidase expression with 10% FBS in PT-30 cells followed by SuperFect (392,904 ± 8,547 and 288,212 ± 32,519 RLU/well; p \ 0.05) (Fig. 3b). Also, Arrest-In resulted in the highest b-galactosidase expression without FBS in PT-30 cells followed by jetPEI (402,909 ± 18,010 and 309,473 ± 52,315 RLU/well; p \ 0.05) (Fig. 3b).
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Fig. 3 Comparative analysis of b-galactosidase expression for various cells with or without serum (a MC3T3-E1 mouse preosteoblasts, b PT-30 human epithelial precancer cells, c C3H10T1/2 mouse stem cells, d MCF-7 human breast cancer cells, e HeLa human cervical cancer, f C2C12 mouse myoblasts, g Hep G2 human hepatocellular carcinoma, h 4T1 mouse mammary carcinoma, i HCT116 human colorectal carcinoma, and j HEKn primary human epidermal keratinocytes)
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b-galactosidase in C3H10T1/2 cells jetPEI achieved maximal b-galactosidase expression with 10% FBS in C3H10T1/2 cells followed by FuGENE HD (610,769 ± 22,737 and 504,992 ± 18,786 RLU/well; p \ 0.05) (Fig. 3c). Also, FuGENE HD led to the highest bgalactosidase expression without FBS in C3H10T1/2 cells followed by Lipofectamine 2000 (490,265 ± 24,690 and 381,831 ± 66,124 RLU/well; p \ 0.05) (Fig. 3c). b-galactosidase in MCF-7 cells jetPEI and Arrest-In produced maximal b-galactosidase expression with 10% FBS in MCF-7 cells (894,181 ± 20,530 and 883,933 ± 9,628 RLU/well; p = 0.40) (Fig. 3d). Also, Arrest-In and Lipofectamine 2000 resulted in the highest b-galactosidase expression without FBS in MCF-7 cells (668,641 ± 18,248 and 660,546 ± 10,862 RLU/well; p = 0.47) (Fig. 3d). b-galactosidase in HeLa cells FuGENE HD and Arrest-In achieved maximal b-galactosidase expression with 10% FBS in HeLa cells (611,625 ±
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5,724 and 582,224 ± 30,534 RLU/well; p = 0.11) (Fig. 3e). Arrest-In led to highest b-galactosidase expression without FBS in HeLa cells followed by ExpressFect (567,382 ± 6,810 and 521,361 ± 15,791 RLU/well; p \ 0.05) (Fig. 3e). b-galactosidase in C2C12 cells Arrest-In promoted maximal b-galactosidase expression with 10% FBS in C2C12 cells followed by jetPEI (535,040 ± 37,738 and 406,252 ± 42,337 RLU/well; p \ 0.05) (Fig. 3f). Also, Arrest-In and jetPEI produced the highest b-galactosidase expression without FBS in C2C12 cells (397,526 ± 3,122 and 389,320 ± 8,864 RLU/ well; p = 0.13) (Fig. 3f). b-galactosidase in Hep G2 cells Arrest-In and FuGENE HD achieved maximal b-galactosidase expression with 10% FBS in Hep G2 cells (475,453 ± 106,902 and 470,912 ± 90,197 RLU/well; p = 0.95) (Fig. 3g). Also, FuGENE HD resulted in highest b-galactosidase expression without FBS in Hep G2 cells followed by Arrest-In (602,069 ± 28,287 and 507,250 ± 43,152 RLU/well; p \ 0.05) (Fig. 3g).
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Cell v viability (%)
Fig. 4 Cell viability with six transfection reagents in optimal condition for transfection evaluated by MTT assay using MC3T3-E1 cells
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b-galactosidase in 4T1 cells Arrest-In produced maximal b-galactosidase expression with 10% FBS in 4T1 cells followed by jetPEI (598,541 ± 28,733 and 546,577 ± 9,920 RLU/well; p \ 0.05) (Fig. 3h). Also, Arrest-In and FuGENE HD achieved the highest b-galactosidase expression without FBS in 4T1 cells (591,871 ± 10,640 and 585,649 ± 6,745 RLU/well; p = 0.36) (Fig. 3h). b-galactosidase in HCT116 cells FuGENE HD promoted maximal b-galactosidase expression with or without 10% FBS in HCT116 cells (699,424 ± 9,591 or 687,052 ± 10,980 RLU/well) compared to all other reagents tested (Fig. 3i). Arrest-In resulted in the second highest b-galactosidase expression with or without 10% FBS (675,208 ± 3,093 or 657,464 ± 15,205 RLU/well) (Fig. 3i). The difference in b-galactosidase expression between using FuGENE HD and ArrestIn was significant both with and without 10% FBS conditions (p \ 0.05). b-galactosidase in primary epidermal keratinocytes Arrest-In promoted maximal b-galactosidase expression with 10% FBS in primary keratinocytes followed by ExpressFect (127,174 ± 9,424 and 101,366 ± 7,841 RLU/ well; p \ 0.05) (Fig. 3j). Also, Arrest-In and Lipofectamine 2000 produced the highest b-galactosidase expression without FBS in primary keratinocytes (169,540 ± 25,703 and 147,809 ± 41,978 RLU/well; p = 0.41) (Fig. 3j). Effects of transfection on cell viability For the determination of cytotoxicity, composition and preparation of complexes as well as transfection conditions were the same as those used in the efficiency experiments.
ExpressFect
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Lipofectamine 2000
SuperFect
The largest amount of viable cells was found after transfection with jetPEI and ExpressFect in MC3T3-E1 (109.5 ± 18.9 and 109.1 ± 16.8%, respectively; p = 0.91) (Fig. 4). In contrast, Lipofectamine 2000, Arrest-In, and SuperFect showed the lowest amount of cell viability (69.1 ± 17.8, 70.5 ± 19.7, and 71.4 ± 25.4%; p = 0.80) (Fig. 4). The cell viability with jetPEI and Lipofectamine 2000 was significantly lower than other reagents (p \ 0.05). Toxic effects of transfection with SuperFect included a visible change in cell morphology. Cells disintegrated and eventually detached from the plate surface. None of the other transfection reagents had a similarly distinctive effect on cell morphology.
Discussion The present study comparatively analyzed the efficacy of transfection of six nonviral transfection systems in several cells of different origins. The data indicate that relatively high levels of transfection were achieved using FuGENE HD, Arrest-In, and jetPEI. The results obtained using two genes (luciferase and b-galactosidase) indicated that the nature of the cell itself is a critical factor that determines the level of gene expression following transfection. Generally, transfection using b-galactosidase achieved higher efficiency than using luciferase. Also, transfection without serum achieved higher efficiency than with serum. These results may be associated with zeta potential, electric potential in the interfacial double layer at the location of the slipping plane versus a point in the bulk fluid away from the interface. We found that values of zeta potential of b-galactosidase plasmid DNA was significantly less negative than those of luciferase (-21.3 and -35.2 mV, respectively). The zeta potential values without serum were also significantly less negative than those with serum (data not shown). These indicated that zeta potential of the DNA complex condition was one of the important factors which
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control gene transfection. Therefore, it is important to carefully characterize the most suitable transfection system for each cell type prior to functional studies in order to achieve maximal transgene expression. Transfection efficiencies for MC3T3-E1 preosteoblasts have been reported using cationic liposome [12], cationic polymer [13], cationic lipid, gelatin, and PEI [14], nanostructured calcium phosphates [15], and gold nanoparticles [16]. In the present study, the highest transfection efficiency in MC3T3-E1 cells using luciferase gene in the presence and absence of serum was seen with FuGENE HD (lipids and other components). Using the b-galactosidase gene, FuGENE HD also promoted the highest efficiency in the presence of serum. Conversely, in the absence of serum Arrest-In (lipo-polymeric formulation) and jetPEI achieved highest efficiency using b-galactosidase gene. In the presence of serum, these data suggested that lipid based reagents (FuGENE HD) are potentially highly selective as a predictable nonviral vector for MC3T3-E1 cells in a variety of gene transfection studies. Transfection efficiencies have been reported in epithelial cells using PEI [17], PEI grafted N-maleated chitosan copolymers [18], nanostructured lipid carrier with cetylated PEI and Lipofectamine 2000 [19], cationic gemini lipids [20], and poly(disulfide amine)s [21]. A comparison study using lung tumor and normal epithelial cells showed that Lipofectamine 2000 (cationic lipid) produced higher transfection efficiency than Effectene (cationic lipids) or SuperFect (activated-dendrimer) [22]. In the present study, the highest transfection efficiency in PT-30 epithelial precancer cells using luciferase gene in both the presence and absence of serum was seen with Arrest-In (lipo-polymeric formulation). Also, the highest transfection efficiency with FuGENE HD in the presence of serum or with jetPEI in the absence of serum in primary epidermal keratinocytes using luciferase gene was seen. Using the b-galactosidase gene, Arrest-In promoted the highest efficiency in both PT-30 cells and primary epidermal keratinocytes in both the presence and absence of serum. These data suggested that lipo-polymeric formulation based reagents (Arrest-In) are potentially highly efficient in epithelial cells for gene transfection studies. C3H10T1/2 multipotent mesenchymal cells have been transfected with various nonviral vectors, including cationic liposome [23], PEI [24], polycationic dendrimers [25], Lipofectamine and DOSPER [26], Effectene and SuperFect [27], LipofectAMINE PLUS [28] and FuGENE 6 [29]. Several comparable studies with fibroblasts have reported that cationic liposomes mediated greater transfection efficiency than PEI [30] and Metafectene produced higher transfection activity any other reagents, including Nanofectin 1 and 2, Superfect, JetPEI, GeneJammer, Effectene, TransPass D2, FuGENE 6, Lipofectamine 2000,
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Dreamfect, Escort III, and calcium phosphate [31]. In the present study, the highest transfection efficiency in C3H10T1/2 cells using the luciferase gene in the presence and absence of serum was seen with FuGENE HD (lipids and other components). Using the b-galactosidase gene, FuGENE HD also promoted the highest efficiency in the absence of serum. Conversely, in the presence of serum jetPEI achieved the highest efficiency using b-galactosidase gene. In the serum condition, these data suggested that lipid based reagents (FuGENE HD) are effective for C3H10T1/2 cells in transfection studies. Generally, transfection of tumor cells has been easy to achieve. Transfection efficiencies for MCF-7 and 4T1 breast cancer cells have been reported using cationic lipids [32], chitosan cationic polymer [33, 34], PEI [35, 36], and gas plasma [37]. In the present study, the highest transfection efficiency in MCF-7 cells using both luciferase and bgalactosidase genes in the presence and absence of serum was seen with Arrest-In and jetPEI. In 4T1 cells, the highest transfection efficiency with Arrest-In in the presence of serum or with FuGENE HD in the absence of serum using luciferase gene was seen. These data suggested that ArrestIn, jetPEI, and FuGENE HD are effective in mammalian tumor cells for gene transfection studies. Other tumor cell lines, HeLa cervical cancer, Hep G2 hepatocellular carcinoma, and HCT116 colorectal carcinoma, have been used for transfection studies with PEI [38, 39], cationic lipids [40], and cationic liposome [41, 42]. In the present study, the highest transfection efficiency in HeLa cells using both luciferase and b-galactosidase genes in the presence of serum was seen with FuGENE HD and in the absence of serum was seen with Arrest-In. In Hep G2 cells, the highest transfection efficiency with Arrest-In using luciferase gene and with FuGENE HD using bgalactosidase gene in both the presence and absence of serum was seen. FuGENE HD also achieved maximal transfection efficiency in HCT116 cells using both luciferase and b-galactosidase genes. These data suggested that Arrest-In and FuGENE HD are most effective in HeLa, Hep G2, and HCT116 tumor cells for gene transfection studies. C2C12 myoblasts have been transfected with various nonviral vectors, including cationic polymer [43], PEI [44], and cationic lipid [45]. In the present study, the highest transfection efficiency with FuGENE HD using luciferase gene and with Arrest-In using b-galactosidase gene in both the presence and absence of serum was seen in C2C12 cells. These data suggested that FuGENE HD and Arrest-In are potentially highly efficient in C2C12 cells for gene transfection studies. Similar to transfection efficiency, toxic effects due to the applied transfection reagents also depend on the TR used as well as on the conditions of the transfected cells [46].
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Significant differences in cell viability were seen after transfection of mouse osteoblastic cells. The largest amount of viable cells was found after transfection with jetPEI and ExpressFect. In contrast, the lowest amount of cell viability with Lipofectamine 2000, Arrest-In, and SuperFect was seen after transfection. There was a statistically significant difference between jetPEI and Lipofectamine 2000. The results of cell viability tests were relatively correlated with the results of transfection efficiency. Other factors thought to influence cytotoxicity as well as transfection efficiency are the presence or absence of serum and the charge ratios of TR/DNA complexes, incubation time and reagent doses [47, 48]. However, we could not determine significant changes in toxicity caused by increasing the incubation time or altering charge ratios or reagent doses. In this study, a suitable gene transfer system has been evaluated and optimized for in vitro conditions. These studies are a first step in the development of an in vivo gene transfer system. Here, transfection efficiency and cytotoxicity have been analyzed according to cells of interest, ratio of transfection reagent to DNA, kind of DNA, and transfection medium. Taken together, our results indicate that these high efficiency reagents can be sufficiently optimized to offer a feasible approach for gene delivery into a wide range of cells. Also, the techniques might be of potential value for functional gene analyses and for gene therapy approaches in tissue engineering. Acknowledgments We thank Dr. Mani Alikhani for providing the MC3T3-E1 and C3H10T1/2 cells, Dr. Peter Sacks for the PT-30 cells, and Dr. Xi Huang for providing the MCF-7, HeLa, C2C12, Hep G2, 4T1, and HCT116 cells.
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