Layer-by-Layer Assembly of Poly(ethyleneimine) and Plasmid DNA

The layer-by-layer assembly technique was used to adsorb alternately poly(ethyleneimine) and plasmid DNA onto the surface of a transparent electrode m...
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Layer-by-Layer Assembly of Poly(ethyleneimine) and Plasmid DNA onto Transparent Indium-Tin Oxide Electrodes for Temporally and Spatially Specific Gene Transfer Fumio Yamauchi, Koichi Kato, and Hiroo Iwata* Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Received February 25, 2005. In Final Form: June 3, 2005

The layer-by-layer assembly technique was used to adsorb alternately poly(ethyleneimine) and plasmid DNA onto the surface of a transparent electrode made of indium-tin oxide. The surface with adsorbed poly(ethyleneimine) and DNA was characterized by X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and contact angle measurements. These analyses revealed that the alternate adsorption process generated a multilayered assembly of cationic poly(ethyleneimine) and anionic DNA. For the spatially and temporally specific gene transfer, cells were cultured on the plasmid-loaded electrode and then a short electric pulse was applied to the cell-electrode system. It was shown that, upon electric pulsing, the plasmid was released from the electrode and transferred into the cells, resulting in efficient gene expression even in primary cultured cells. Transfection could be effected for hippocampal neurons after 3-day culture on the plasmid-loaded electrode, which indicated the feasibility of selecting the time of transfection. Our results also showed that electroporation could be performed in a spatially specific manner by using a plasmid-arrayed electrode, demonstrating the feasibility of the method for the fabrication of transfected cell microarrays.

Introduction Gene transfer is an important technique to analyze and manipulate gene functions. Gene transfer techniques have been used routinely in many fields of fundamental and therapeutic studies, such as functional genomics, drug development, and gene therapy. Various gene transfer methods that have been developed so far include lipofection, cationic polymer-based transfection, virus infection, and electroporation.1-4 Among them, electroporation is considered to be one of the most efficient nonviral methods. This method involves DNA transfer into the cytoplasm through transient pores created on the cell membrane upon electric pulsing,4,5 followed by the entry of DNA into the nucleus for gene expression. However, conventional electroporation is not practical when multiple genes are introduced in parallel, because a large number of cells and a large amount of gene expression constructs are usually needed. In addition, adherent cells are required to detach from the culture substrate before electroporation. A detachment procedure such as trypsinization often causes the damage of membrane-bound proteins, plasma membranes, and the cytoskeleton, resulting in low transfection efficiency and reduction of cell viability.6 * To whom correspondence should be addressed. Tel & Fax: +8175-751-4119. E-mail: [email protected]. (1) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413-7417. (2) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 72977301. (3) Sorge, J.; Wright, D.; Erdman, V. D.; Cutting, A. E. Mol. Cell. Biol. 1984, 4, 1730-1737. (4) Gehl, J. Acta Physiol. Scand. 2003, 177, 437-447. (5) Neumann, E.; Kakorin, S.; Toensing, K. Bioelectrochem. Bioenerg. 1999, 48, 3-16. (6) Zheng, Q.; Chang, D. C. Biochim. Biophys. Acta 1991, 1088, 104110.

Recently, substrate-mediated transfection methods were developed for efficient gene transfer into mammalian cells.7-11 In these methods, plasmids are adsorbed to the surface of a culture substrate, and then cells are cultured onto the plasmid-adsorbed surface to be transfected upon adhesion to the surface. In these systems, cationic lipids and polyelectrolytes are employed to enhance gene transfer into cells. These microarray-based transfection methods have several advantages: The methods require only an extremely small amount of DNA and cells and can be utilized for high-throughput and real-time monitoring of phenotypic changes caused by the forced expression of exogeneous DNA. However, these methods are often ineffective for delicate cells such as primary cultured cells due to the toxicity of the cationic enhancers. Another disadvantage associated with the current microarray methods includes their inability to control the time of transfection, which will be critical in cases where cells are required to adhere, differentiate, or proliferate prior to transfection. To overcome these problems, we previously developed an alternative microarray-based method that allowed efficient transfection of primary cultured cells in a temporally controlled manner.12 In this method, plasmids were spotted onto the surface of a cationically modified gold electrode in an array format. Cells were adhered to the microarray and then transfected by applying an electric pulse to the microarray-cell system. The application of (7) Segura, T.; Shea, L. D. Bioconjugate Chem. 2002, 13, 621-629. (8) Honma, K.; Ochiya, T.; Nagahara, S.; Sano, A.; Yamamoto, H.; Hirai, K.; Aso, Y.; Terada, M. Biochem. Biophys. Res. Commun. 2001, 289, 1075-1081. (9) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 893-895. (10) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107-110. (11) Yamauchi, F.; Kato, K.; Iwata, H. Biochim. Biophys. Acta 2004, 1672, 138-147. (12) Yamauchi, F.; Kato, K.; Iwata, H. Nucleic Acids Res. 2004, 32, e187.

10.1021/la0505059 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/28/2005

Layer-by-Layer Assembly of PEI and Plasmid DNA

an electric pulse facilitated detachment of plasmids from a microarray and also translocation of the detached plasmids into the cells. We demonstrated that the method was particularly effective for transfecting primary cultured embryonic fibroblasts and hippocampal neurons under optimized conditions. In the present study, we intended to further improve our method by employing a transparent electrode made of electrically conductive indium-tin oxide (ITO) instead of gold as we previously used. It is expected that microscopic observation of transfected cells is much easier on a thin layer of ITO than gold using a standard microscope with a transmission light path.13 The strong quenching of fluorescent molecules in the vicinity of the metallic surfaces,14,15 such as silver or gold, can be avoided using ITO, which is important for detecting small differences in expression of fluorescently active proteins in reporter assays. For loading plasmids, poly(ethylenimine) (PEI) and plasmid DNA were adsorbed sequentially to the ITO surface by the layer-by-layer electrostatic adsorption technique.16 The ITO surface with adsorbed PEI and plasmid DNA was characterized by X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle measurements. The efficiency of cell elecroporation was evaluated using a plasmid designed to express a fluorescent protein. To prepare transfectional microarrays, we employed the photoassisted micropatterning of a selfassembled monolayer (SAM) of organosilanes formed on the ITO surface. We will show that the method studied here is useful for temporally and spatially specific gene transfer into adherent cells and the fabrication of transfectional microarrays with adequate transparency of the substrate. Materials and Methods ITO Electrodes. ITO electrodes, donated by Canon Inc. (Tokyo, Japan) had a thin layer of ITO sputtered to a thickness of 200 nm onto one side of a glass plate (thickness 0.7 mm) or a poly(ethylene terephthalate) (PET) film (thickness 188 µm). The sheet resistance of the ITO layer was 7-10 and 15-20 Ω per square on a glass plate and a PET film, respectively. The electrodes were cut into rectangular specimens with a size of 22 mm × 26 mm, rinsed with acetone and water, and dried under a stream of nitrogen gas. Then the electrodes were irradiated with oxygen plasma at a pressure of 5 Pa at 30 W for 5 min using a plasma generator (PA300AT, Okuma Engineering, Co., Ltd., Fukuoka, Japan). The plasma treatment for surface cleaning was critical for efficient adsorption of cationic PEI. The cleaned electrodes were washed with ethanol and water, sterilized with 70% ethanol, and air-dried in a sterile laminar flow hood. Plasmid DNA. Plasmids used in this study were pEGFP-C1 and pDsRed-C1 (4.7 kbp; Clontech Laboratories, Palo Alto, CA), which encoded an enhanced green fluorescent protein (EGFP) and Discosoma sp. red fluorescent protein (DsRed), respectively, under the control of a cytomegalovirus promoter. The plasmids were amplified in Escherichia coli DH5R and purified using an EndoFree Plasmid Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored in Tris-EDTA buffer (pH 8.0). Layer-by-Layer Assembly of PEI and DNA. A multilayer consisting of PEI and plasmid DNA was formed onto an ITO electrode by layer-by-layer adsorption under sterile conditions. As shown in Figure 1, a silicone frame (Figure 1B) with a thickness (13) Raptis, L.; Balboa, V.; Hsu, T.; Vultur, A.; Turkson, J.; Jove, R.; Firth, K. L. Anal. Biochem. 2003, 317, 124-128. (14) Kuhn, H. J. Chem. Phys. 1970, 53, 101-108. (15) Chance, R. R.; Prock, A.; Silbey, R. Adv. Chem. Phys. 1978, 37, 1-65. (16) Decher, G. Science 1997, 277, 1232-1237.

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Figure 1. Schematic diagram of electroporation on the plasmidloaded ITO electrode. (A) counter electrode (anode), (B) silicone frame, (C) adherent cells under PBS, (D) multilayer of plasmid and PEI, (E) thin ITO layer (cathode), (F) glass or PET substrate, (G) electric pulse generator, and (H) steel clip. The positive (+) and negative (-) signs in the bottom diagram indicate the electrical connecting points through which the electric pulse is delivered to the electrodes from the pulse generator. The electrodes (A and E) were connected via the steel chip (H) to the pulse generator (G). of 1 mm was fixed to the electrode in order to confine the cell culture area (13 mm × 13 mm). Branched PEI (Aldrich Chemical Co., Milwaukee, WI) with an average molecular weight of 800 was dissolved in 0.01 M phosphate buffered saline (PBS; pH ) 7.4, [NaCl] ) 138 mM, [KCl] ) 2.7 mM) to a concentration of 10 mg/mL. The solution pH was adjusted to 7.4 with HCl. The solution was then sterilized by filtration though a Millipore membrane (Millex GP, 0.22 µm). The ITO surface within the fixed frame was exposed to the PEI solution and kept for 30 min at room temperature to adsorb electrostatically the PEI onto the surface.17 The PEI-adsorbed surface was then washed with PBS to remove unbound PEI. The PEI-adsorbed surface was then exposed to a plasmid solution (50 µg/mL) in PBS (pH 7.4) for 30 min at room temperature to adsorb electrostatically the plasmid onto the surface, followed by washing with PBS. The sequential adsorption of PEI and the plasmid was repeated to obtain a multilayer. Finally, the electrode was washed with PBS and immediately used for further experiments. Our preliminary experiments showed that a further increase in the polyelectrolyte concentration and adsorption time did not significantly alter the water contact angle of each surface. The pH value of the polyelectrolyte solutions used for layer-by-layer assembly was adjusted to 7.4 so that PEI was protonated, while the plasmid retains its native structure.2 Since the transfection efficiency was slightly decreased when the DNA-coated surface was dried (data not shown), drying of the surface was avoided till the cells are plated. In this paper, a layer number is defined as the number of procedures for the adsorption of either PEI or plasmid DNA. For example, five layers correspond to the adsorption of PEIplasmid-PEI-plasmid-PEI. The multilayer was assembled of up to seven layers. The amount of plasmid loaded on the glass-based ITO electrode surface was determined in the following way. A rhodaminelabeled plasmid (5.7 kbp; pGeneGrip Rhodamine & GFP, Gene Therapy Systems, Inc., San Diego, CA) was loaded by the same procedure as described above but in the dark and visualized with a fluorescence microscope (MZ FLIII, Leica, Germany) equipped with a cooled-CCD camera (ORCA-ER; Hamamatsu Photonics K.K., Hamamatsu, Japan). The average fluorescence intensity was measured on the plate surface with an area of 13 mm × 13 mm using an AQUA-Lite digital imaging software (17) Wang, L. Z.; Omomo, Y.; Sakai, N.; Fukuda, K.; Nakai, I.; Ebina, Y.; Takada, K.; Watanabe, M.; Sasaki, T. Chem. Mater. 2003, 15, 28732878.

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(Hamamatsu Photonics) and converted to the density of a loaded plasmid using a calibration curve that was obtained by depositing the plasmid of known amounts on the PEI-adsorbed ITO electrodes. Surface Characterization. The elemental composition of the electrode surfaces was analyzed by XPS using ESCA-850 (Shimadzu Corp., Kyoto, Japan). A magnesium-anode source was used to generate Mg KR X-ray at 8 kV and 30 mA. The pressure in the sample chamber was kept below 5 × 10-5 Pa throughout the analysis. The takeoff angle of photoelectrons from the surface was fixed to 90°. The position of the carbon 1s peak, assigned to hydrocarbon C-C and C-H, was set to a binding energy of 285.0 eV to compensate for any charge-induced shifts. ATR-FTIR absorption spectra were acquired using a Spectrum One (Perkin-Elmer Inc., Wellesley, MA) instrument equipped with a universal ATR sampling accessory and a MIR-TGS detector. All spectra were recorded at a resolution of 4 cm-1 and an accumulation of 32 scans. A bare ITO electrode, cleaned by the procedure described above, was used as a reference surface. Static contact angles of water were measured on the surface of the PEI/plasmid multilayers by the sessile drop method using a contact angle meter (CA-X, Kyowa Interface Science Co., Ltd., Saitama, Japan). The measurements were performed at five randomly chosen positions on the surface, and these data were averaged. Analyses by XPS, ATR-FTIR spectroscopy, and contact angle measurements were all carried out after glass-based samples were rinsed with water and kept in a desiccator at room temperature for 12 h. Electroporation. A human embryonic kidney cell line, HEK293 (Health Science Research Resources Bank, Tokyo, Japan), was grown in minimal essential medium (MEM; Invitrogen Corp., Carlsbad, CA) supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37 °C and 5% CO2 under a humidified atmosphere. Primary hippocampal neurons were obtained from fetal Wistar rats (E18) and cultured in Neurobasal (Invitrogen) medium supplemented with B-27 (Invitrogen), 25 µM glutamic acid, and 0.5 mM L-glutamine.18,19 The electroporation setup is schematically depicted in Figure 1. The plasmid-loaded electrode was placed in a 35 mm polystyrene cell culture dish. In the electroporation experiment, the outmost layer of the multilayer surface was always PEI (number of layers ) 1, 3, 5, 7, and 9) to enhance cell adhesion and to protect DNA from nuclease-mediated degradation. A suspension of HEK293 cells was transferred onto the plasmidloaded surface within the silicone frame at a density of 3.0 × 104 cells/cm2 (70-80% confluency). Cells were cultured in MEM supplemented with 10% FBS and antibiotics at 37 °C under a 5% CO2 atmosphere for 24 h so that they could attach to the plasmid-loaded electrode surface. Prior to pulse application, the attached cells were washed with PBS, and the inner space of the silicone frame was filled with cold PBS for sufficient permeabilization for DNA transfer.4 A counter electrode (Figure 1A; a glass plate having a thin gold layer with a thickness of 19 nm) was placed above the frame with the gold surface down, and then lower (Figure 1E; ITO, cathode) and upper (gold, anode) electrodes were connected to a pulse generator (Figure 1G; ElectroSquarePorator T820, BTX, San Diego, CA). The silicone frame insulates the anode from direct contact with the cathode. A steel clip (Figure 1H) was used for the connection of an electrode to the pulse generator via an electrical cord. Cells were treated with a single electric pulse at a field strength of 50-400 V/cm for a duration of 10 ms. After a 10-min incubation at room temperature, PBS was replaced with a growth medium containing 10% FBS and antibiotics prewarmed at 37 °C, and the cells were further cultured. Electroporation of primary hippocampal neurons was carried out by a similar fashion as described above, but cells were seeded at a density of 1.3 × 105 cells/cm2 and cultured for 72 h before electric pulsing. A single electric pulse was applied to the cells at 40% confluence at a field strength of 200 V/cm for 10 ms duration. (18) Evans, M. S.; Collings, M. A.; Brewer, G. J. J. Neurosci. Methods 1998, 79, 37-46. (19) Brewer, G. J.; Price, P. J. NeuroReport 1996, 7, 1509-1512.

Yamauchi et al. The number of cells expressing fluorescent proteins (EGFP or DsRed) was counted 48 h after electroporation at five randomly chosen areas under an epifluorescence microscope (BX-51; Olympus, Tokyo, Japan). Our preliminary results showed that prolonged cultivation for more than 48 h did not improve the efficiency. HEK293 cells reached confluence just 48 h after electroporation, while the confluency of primary hippocampal neurons remained unchanged. To evaluate both transfection efficiency and viability at the same time points, viable cells were determined from the number of trypan blue-excluding cells 48 h after electric pulsing. To assess viability, cells were cultured on the plasmid-loaded electrode for 72 h without electric pulsing, and the number of viable cells was determined as a control in the same manner as just described. The transfection efficiency and cell viability were determined according to the following equations:

transfection efficiency (%) ) (the number of fluorescently positive cells)/ (the number of viable cells 48 h after pulsing) × 100 cell viability (%) ) (the number of viable cells 48 h after pulsing)/ (the number of viable cells cultured without pulsing) × 100 All experiments were performed more than five times, and transfection efficiency and cell viability were expressed as mean ( standard deviation (SD). Plasmid Release. Using a similar set up as described above, an electric pulse was applied to the PET-film-based ITO surfaces loaded with plasmids on which no cells were cultured. Then the amount of released plasmid in the medium (PBS) was determined with an intercalating fluorescent dye, PicoGreen (Molecular Probes, Inc. Eugene, OR). The fluorescence intensity was measured with a fluorescence spectrophotometer (F-2500, Hitachi, Ltd., Tokyo, Japan) at excitation and emission wavelengths of 480 and 525 nm, respectively. All experiments were performed three times, and data were expressed as mean ( standard deviation (SD). Parallel Electroporation on Microarrays. The silanization of the glass-based ITO surface was carried out according to the method by Mooney et al.20 with minor modifications. Briefly, a cleaned ITO electrode was immersed in 1% dry toluene solution of octadecyltriethoxysilane (OTS; Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) containing 0.5% n-butylamine as the catalyst for 2 h at room temperature to form a self-assembled monolayer of OTS onto the ITO surface. The surface was then rinsed with toluene, dried under a stream of nitrogen gas, and kept at 80 °C in an oven for 12 h. Then the ITO surface was rinsed with ethanol and water. By the reaction with OTS, the ITO surface became highly hydrophobic, exhibiting a contact angle of water of approximately 100°. Photoassisted patterning of the OTS layer was performed by ultraviolet irradiation through a chromium photomask.20 A mask having a pattern of 10 × 10 circular holes with a diameter of 1 mm was overlaid on the surface of the monolayer, and then a UV light from an ultrahigh-pressure mercury lamp (180 mW/cm2, Optical Modulex SX-UI500HQ, Ushio Inc., Osaka, Japan) radiated the OTS surface through the photomask under atmospheric conditions. After 1-h irradiation, the electrode was washed with ethanol to remove photocleaved OTS, followed by drying with a nitrogen stream. These processes generated a hundred circular dots, presenting a pristine ITO surface surrounded by the hydrophobic OTS monolayer. Immediately after the above procedures, PEI and plasmid DNA were adsorbed by spotting sequentially each solution onto the dots using a micropipet. The electroporation of cells on the plasmid-arrayed electrode was performed in the same manner as described above to examine the possibility of parallel electroporation. (20) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1228712291.

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Figure 2. Relative atomic concentration of a PEI/plasmid multilayer assembled on the glass-based ITO electrode determined by XPS analysis. (O) C 1s, (b) N 1s, (0) O 1s, (9) P 2p, (4) In 3d, and (2) Sn 3d.

Figure 3. ATR-FTIR analysis of PEI/plasmid multilayers assembled on the glass-based ITO surface. (A) ATR-FTIR spectra of the multilayer with a layer number of (a) 1, (b) 2, (c) 4, and (d) 6. (B) Peak areas of absorption bands at (b) 1230, (O) 1080, and (9) 960 cm-1 as a function of layer numbers.

Results and Discussion Preparation of Plasmid-Loaded Electrodes. It was reported that bare ITO has a negatively charged surface due to the presence of dangling In-O- and Sn-Obonds.21,22 This surface attracts polycations, such as poly(diallyldimethylammonium) chloride,23-25 poly(allylamine),26 and, as in our case, PEI,17 producing a cationic surface by charge overcompensation.27 The resulting surface is expected to attract in turn oppositely charged DNA, most likely driven by electrostatic interactions,28 producing again a negatively charged surface. The sequential adsorption of PEI and DNA consequently gives rise to a multilayered thin film attached firmly to the ITO surface. To monitor such processes, elemental composition was determined by XPS for the samples obtained after each step of electrode preparation. Figure 2 shows changes in the composition of six characteristic elements, including carbon, nitrogen, oxygen, phosphorus, indium, and tin, determined from the counts of photoelectrons from C1s, N1s, O1s, P2p, In3d, and Sn3d. On the bare ITO surface (corresponding to layer number zero), indium, tin, oxygen, and carbon were detected, while no signal was observed (21) Li, L. S.; Li, A. D. Q. J. Phys. Chem. B 2001, 105, 10022-10028. (22) Zotti, G.; Zecchin, S.; Schiavon, G.; Vercelli, B.; Berlin, A.; Porzio, W. Chem. Mater. 2004, 16, 2091-2100. (23) Li, L. S.; Wang, R.; Fitzsimmons, M.; Li, D. Q. J. Phys. Chem. B 2000, 104, 11195-11201. (24) Li, L. S.; Li, A. D. Q.; Jia, Q. X. Appl. Surf. Sci. 2003, 219, 199202. (25) Shi, L.; Lu, Y.; Sun, J.; Zhang, J.; Sun, C.; Liu, J.; Shen, J. Biomacromolecules 2003, 4, 1161-1167 (26) Chu, J.; Li, X.; Zhang, J.; Tang, J. Biochem. Biophys. Res. Commun. 2003, 305, 116-121. (27) Decher, G. In Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Decher, G., Schlenoff, J. B., Eds.; WileyVCH: Weinheim, 2002; Chapter 1, p 1-46. (28) Sukhorukov, G. B.; Montrel, M. M.; Petrov, A. I.; Shabarchina, L. I.; Sukhorukov, B. I. Biosens. Bioelectron. 1996, 11, 913-922.

for phosphorus and nitrogen. A minor signal of C1s detected on the bare ITO is likely due to surface contamination with organic substances. The composition of nitrogen and carbon increased upon adsorption of PEI (first layer) and plasmid (second layer), while phosphorus composition remained almost zero after PEI adsorption but increased after plasmid adsorption. These changes were associated with a decrease in the composition of oxygen, indium, and tin, indicating that signals from the ITO surface were obscured upon adsorption of PEI and plasmid. Although oxygen is also contained in a plasmid, its contribution to the total composition is small, because oxygen content in a DNA molecule (ca. 32%) is much lower than that in ITO (ca. 70%). These results as a whole suggest the presence of PEI and plasmid sequentially adsorbed to the ITO surface. The composition of carbon, nitrogen, and phosphorus increased with the number of cycles, while that of oxygen, indium, and tin decreased. These changes reached rather a plateau level after adsorption of four or five layers, suggesting the formation of a PEI/plasmid multilayer with a thickness of at least ca. 5 nm, which corresponds to an effective mean free path for C1s photoelectrons in an organic matrix.29 The multilayer assembly was further assessed by ATRFTIR spectroscopy. Figure 3A shows the ATR-FTIR spectra of the PEI/plasmid multilayer. Strong absorption is observed at 1230, 1080, and 960 cm-1 on the surface with a layer number of two, four, and six (spectra b, c, and d), while a spectrum obtained from a surface only with PEI does not show absorption in the same wavelengths (spectrum a). The absorption bands at 1230 and 1080 cm-1 are assigned to, respectively, antisymmetric and symmetric stretching vibration of phosphate groups of DNA (29) Clark, D. T.; Dilks, A. J. Polym. Sci. Polym. Chem. Ed. 1977, 15, 2321-2345.

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Figure 4. Contact angles of water measured on the PEI/ plasmid multilayer surface of the glass-based ITO electrode as a function of layer numbers.

backbone. The absorption band at 960 cm-1 is due to the C-C stretching vibration of deoxyribose.30 In addition, absorption peaks seen in the 1600-1750 cm-1 region are attributed to the vibration mode of amines contained in PEI and nucleotide bases. These assignments are consistent with literature values for DNA adsorbed to the cationically modified solid surfaces.31,32 In Figure 3B, peak areas were plotted for absorption bands at 1230, 1080, and 960 cm-1 as a function of layer numbers. An increase in the layer number gives rise to an increase in the peak areas. This result indicates the accumulation of plasmid DNA onto the ITO electrode by the layer-by-layer assembly process. The contact angle measurement was performed to further confirm the alternate adsorption of PEI and plasmid by the layer-by-layer adsorption process (Figure 4). The contact angle of the ITO surface increased by 10° when PEI was adsorbed (first layer). The formation of the second layer (plasmid) resulted in a small reduction in the contact angle, compared to the surface with the first layer. In the following adsorption cycles, the contact angle fluctuated alternately between 45° and 32°, depending on the molecules adsorbed. The alternate changes of the contact angle confirmed that layers of PEI and plasmid were built up alternately onto the ITO surface. To support the accumulation of DNA onto the ITO surface, the amount of loaded DNA was determined using a rhodamine-labeled plasmid. Figure 5A shows the fluorescence microscopic images of the ITO surface onto which the rhodamine-plasmid conjugate and PEI were adsorbed alternately. The surface density of plasmid was determined from the fluorescent intensity measured for a 13 mm × 13 mm area (Figure 5B). The plasmid density increased with the number of layers and reached 0.5-0.6 µg/cm2 when measured after assembly of seven layers. However, the increment of DNA loading slightly deviated from linearity, especially in the first two layers. This is probably due to truncation of the excess charge profile by the substrate.33 In fact, as demonstrated by the contact angles (Figure 4), deposition of the primary PEI layer was insufficient for totally covering the ITO surface. After the third layer, the steady state DNA accumulation was observed at a rate of approximately 0.3 µg/cm2/DNA layer. We used a circular plasmid having a supercoiled structure. (30) Banyay, M.; Sarkar, M.; Gra¨slund, A. Biophys. Chem. 2003, 104, 477-488. (31) Luo, L.; Liu, J.; Wang, Z.; Yang, X.; Dong, S.; Wang, E. Biophys. Chem. 2001, 94, 11-22. (32) Zhou, Y.; Li, Y. Biophys. Chem. 2004, 107, 273-281. (33) Schlenoff, J. B. In Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Decher, G., Schlenoff, J. B., Eds.; WileyVCH: Weinheim, 2002; Chapter 4, p 99-132.

Figure 5. Quantification of plasmid loaded on the glass-based ITO electrode. (A) Fluorescent microphotographs of the rhodamine-conjugated plasmid loaded on the ITO surface. (B) Surface density of the plasmid as a function of layer numbers.

It is known that such structure is very compact compared to the random coil structure of typical linear vinyl polymers frequently used in the studies of layer-by-layer assembly. It is interesting to speculate that the supercoiling of DNA may restrain molecular rearrangements in the polyelectrolyte complex, promoting charge reversal and thus accumulation of PEI in the successive layer. However, further studies are required to elucidate such a structural effect. Electrotransfection on Plasmid-Loaded ITO Electrodes. One of the most fundamental requirements for the transfectional microarrays may be the surface design by which plasmid DNA is stably adsorbed to the substrate to a sufficient amount. In addition, the adsorbed plasmid is necessary to be released at a preferred moment when any exogenous stimuli are applied to the system. These were achieved here by making use of the layer-by-layer assembly technique.16 The sequential adsorption of polycations and polyanions has emerged as an efficient and simple technique to create various substrates incorporating bioactive molecules, such as drugs, proteins, and DNAs.34 Recently, Zhang et al.35 used this technique to prepare the substrates for the sustained release of polyanionic DNA during biodegradation of polycationic components. In contrast, our study employed a highvoltage electric pulse to flush the loaded DNA at a preferred moment after cellular organizations are formed. Figure 6 A,B shows the fluorescence and phase contrast images of HEK293 cells electroporated on the glass-based ITO surfaces with a layer number of seven under the optimal pulsing conditions determined as described below. These photographs demonstrate that the cells were effectively electroporated to express EGFP. The transfection efficiency was determined to be 80% with a viability of 79%. In this case, approximately 65% of the total cells expressed EGFP. There are differences in the degree of fluorescence between individual 293 cells. This is probably due to different levels of EGFP expression and/or different numbers of plasmid copies electroporated per cell. Moreover, cell density and cell shape had an effect on the transfection efficiency; cells grown at a low density or (34) Decher, G.; Lehr, B.; Lowack, K.; Lvov, Y.; Schmitt, J. Biosens. Bioelectron. 1994, 9, 677-684. (35) Zhang, J.; Chua, L. S.; Lynn, D. M. Langmuir 2004, 20, 80158021.

Layer-by-Layer Assembly of PEI and Plasmid DNA

Figure 6. Electroporation of HEK293 cells on the plasmidloaded electrode. (A) Fluorescence image of EGFP-expressing HEK293 cells treated with a single pulse for 10 ms duration at the constant field strength of 200 V/cm on the glass-based ITO surface (layer number ) 7). (B) Phase-contrast image of the same site as in A. Scale bar ) 200 µm. (C) Effect of electric field strength on (b) transfection efficiency and (O) cell viability. HEK293 cells were cultured on the PET-film-based ITO surface having five layers of PEI and DNA and then treated with a single pulse for 10 ms duration at varying field strengths. (D) Effect of layer number on (b) transfection efficiency and (O) cell viability. The effect of layer numbers was assessed at the constant field strength of 250 V/cm. Transfection efficiency and cell viability were determined 48 h after electric pulsing. The data are expressed as mean ( standard deviation for three independent experiments.

spread cells required a lower field strength for optimal electroporation than those at a high density or clumpy cells (data not shown). The use of ITO electrodes allows direct observation of transfected cells with the standard light and fluorescent microscopes, since ITO has excellent optical transparency and low incidence of fluorescent quenching.13,36,37 The nontoxicity of ITO13,37 further enhances the usefulness of the electrode as a platform for cell transfection. In the conventional electroporation, transfection efficiency depends on several parameters, including field strength, pulse length, and the number of pulses.38 Therefore, we examined the effect of the electric field strength on the electroporation of HEK293 cells cultured on the PET-film-based ITO surfaces with a layer number of five. As shown in Figure 6C, transfection efficiency and cell viability were considerably influenced by the field strength. The efficiency increased with the field strength up to 250 V/cm without a significant loss in cell viability. The cell viability decreased gradually with the field strength, indicating that higher field strength caused severe cell damage. From these results, we chose 250 V/cm as the optimum field strength under which transfection efficiency and cell viability were optimally balanced. Increasing the pulse length and the pulse number resulted in no improvement in transfection efficiency, but the cell viability decreased (data not shown). Therefore, we (36) Yamada, H.; Imahori, H.; Nishimura, Y.; Yamazaki, I.; Ahn, T. K.; Kim, S. K.; Kim, D.; Fukuzumi, S. J. Am. Chem. Soc. 2003, 125, 9129-9139. (37) Raptis, L.; Firth, K. L. DNA Cell Biol. 1990, 9, 615-621. (38) Chang, D. C. In Guide to Electroporation and Electrofusion; Chang, D. C., Chassy, B. M., Saunders, J. A., Sowers, A. E., Eds.; Academic Press: New York, 1992; Chapter 26, p 429-455.

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selected a single pulse of 10 ms duration as the standard conditions for the electroporation on the ITO electrodes. We further assessed the effect of layer number on transfection efficiency and cell viability (Figure 6D). The result showed that the highest transfection efficiency was achieved by increasing the number of layers at a fixed field strength of 250 V/cm. The data also indicate that the cell viability was independent of the number of layers; a further increase in the layer number (more than eight layers) had no effect on the enhancement of transfection efficiency. A control experiment in which cells were pulsed on the substrate with no DNA loading (one layer) resulted in negligible reduction in cell viability, indicating the nontoxicity of PEI after electroporation (data not shown). According to the above results, we determined the optimal conditions for HEK293 cells: a single pulse at a field strength of 150-250 V/cm for pulse length of 10 ms on the PET-film-based ITO onto which five layers of PEI and DNA were assembled. It was further found that the optimal electroporation on the glass-based ITO could be conducted at a slightly lower field strength (optimum at 100-200 V/cm) than that for the PET-based electrode, probably due to the difference in the sheet resistance of thin ITO films. Unlike the ITO on the PET film, that on the glass substrate was finished with the thermal oxidation process, which gave rise to an improvement in conductivity.39 The rigid and planar surface of a glass substrate as well as less autofluorescence enhances its usefulness. On the other hand, the mechanical flexibility and high ease of processibility of the PET substrate will be advantageous in fabricating DNA-loaded electrodes with a variety of shapes. However, burning of the ITO coating was observed under the higher field strength (>200 V/cm). To address this problem, further improvement of the chamber design and electrodes is required.13 To gain insights into the mechanism of gene transfer on the DNA-loaded electrode, we examined the release of loaded plasmids from the surface upon application of an electric pulse. As shown in Figure 7A, an increase in the number of layers and field strength resulted in an increase in the amount of released plasmids. The released DNA reached 15-30% of initial loading. No plasmid was released without pulse application, regardless of layer numbers (closed bars in Figure 7A). Although the detailed mechanism of DNA release remains unclear at this moment, results show that the released plasmid retains its activity for gene expression even after pulsing with an intensive electric field. The results shown in Figures 6 and 7A led us to suppose that transfection efficiency was primarily governed by the amount of plasmids released upon electric pulsing. To examine this hypothesis, the transfection efficiency was plotted against the amount of released plasmids. As clearly demonstrated in Figure 7B, transfection efficiency was directly proportional to the amount of released plasmids. From these findings, we concluded that the efficiency of electroporation on the plasmid-loaded ITO electrode is governed primarily by the amount of the released plasmid. We also found that at least five layers were needed to detach the plasmid with sufficient amounts for the efficient electroporation under the conditions employed in this work. Since biological studies often require cotransfection of different plasmids, for instance, a gene of interest and a fluorescent reporter, into the same cell, we attempted to introduce two plasmids coding for EGFP and DsRed into primary hippocampal neurons. Neuronal cells were cul(39) Morikawa, H.; Fujita, M. Thin Solid Films 1999, 339, 309-313.

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Figure 7. Effect of the amount of released plasmid on the transfection efficiency. (A) Amount of plasmid released from the PET-film-based electrode by applying an electric pulse at 0 (closed bar), 200 (open bar), and 250 V/cm (hatched bar). (B) Transfection efficiency plotted as a function of the amount of the released plasmid. All data are expressed as mean ( standard deviation for three independent experiments.

Figure 8. Electric-pulse-triggered gene transfer into cells on the plasmid-loaded ITO electrodes. (A-C) Gene transfer into hippocampal neurons. The pEGFP-C1 and pDsRed-C1 were coassembled with PEI on the glass-based ITO electrode. An electric pulse (200 V/cm, 10 ms) was applied 72 h after cell seeding. Fluorescence images of neurons were acquired 48 h after electroporation using an interference filter for (A) EGFP and (B) DsRed. (C) Phase-contrast image of the same site as in A and B. (D) Parallel transfection of HEK293 cells on the plasmid-arrayed ITO electrode. Scale bar: (A, B, C) 100 µm, (D) 1 mm.

tured for 3 days on a glass-based ITO having a multilayer of PEI with the two plasmids, and then an electric pulse was applied at a field strength of 200 V/cm. As shown in Figure 8A-C, green (EGFP) and red (DsRed) fluorescent proteins were coexpressed in the cells. These cells maintained their morphology with a viability of 90%. The transfection efficiency was determined to be approximately 30%, corresponding to approximately 27% of the total cells, for both genes 48 h after electroporation. In a separate experiment, we confirmed that no fluorescent proteins were expressed in neurons, during a 7-day culture on the plasmid-loaded electrode without pulse application (data not shown). Importantly, the multilayer assembly serves to protect DNA from degradation in the serum-containing medium, as evidenced by the fact that EGFP expression occurred even after 72 h of cell culture (Figure 8A-C), with marked contrast to our observation that a free plasmid lost most of its activity within 12 h in the serumcontaining medium (data not shown). Both the layer-bylayer assembled structure and the existence of the PEI layer at the outmost surface might restrict the approach

of nucleases to plasmid DNA, resulting in an increased nuclease resistance. These features of our method prove the feasibility of selecting the time of transfection, if it is up to 3 days after cell seeding. The possibility of controlling transfection will be of great advantage to the studies of genes whose functions are required to be synchronized closely with the specific state of cells or their organization. It is likely that electric-pulse-triggered gene transfer on the plasmid-loaded electrode involves four crucial steps: detachment of plasmids from the electrode surface, electropermeabilization of cell membranes,4,5 translocation of the plasmids through the permeabilized membranes into the cytoplasm by electrophoresis,40 and entry of the plasmids into the cell nucleus for gene expression. The higher electric field is expected to promote these steps except the nuclear entry of the DNA, whereas intensive electropermeabilization would cause severe damage to the cells, reducing cell viability. Cell viability is of practical importance and was not affected by the layer number but by the electric field strength (Figure 6C,D). More detailed studies on the effect of pulsing media and serum on the transfection efficiency, cell viability, the voltage tolerances, and DNA degradation will serve to improve the method for the efficient introduction of exogenous nucleotides into various types of cells. Site-Specific Parallel Electroporation. The viability of spatially controlled gene transfer was also demonstrated in this study. The plasmid-arrayed ITO electrode was prepared by the photoassisted patterning technique and used in an attempt to transfect cells precisely at the defined regions. Under the optimal field strength (200 V/cm), HEK293 cells were electroporated on the glass-based electrode that was spotted with three types of plasmids (pEGFP-C1, pDsRed-C1, or both). As clearly demonstrated in Figure 8D, cells on the plasmid-loaded spots expressed the corresponding fluorescent proteins in an array format, whereas no transfection was observed in the surrounding regions between spots. These results demonstrate that electroporation could be spatially controlled in the length scale of a few hundred micrometers. Plasmid DNA does not go through gap junctions,41 and HEK293 cells have no gap junctions,42 a property which offers advantages for the restricted gene expression at defined regions in the (40) Sukharev, S. I.; Klenchin, V. A.; Serov, S. M.; Chernomordik, L. V.; Chizmadzhev, Y. Biophys. J. 1992, 63, 1320-1327. (41) Flagg-Newton, J.; Simpson, I.; Loewenstein, W. R. Science 1979, 205, 404-407. (42) Toyofuku, T.; Yabuki, M.; Otsu, K.; Kuzuya, T.; Hori, M.; Tada, M. J. Biol. Chem. 1998, 273, 12725-12731.

Layer-by-Layer Assembly of PEI and Plasmid DNA

2-D culture of cells. For cells having gap junctions, the distance between the spots may be important for parallel transfection, or the area between the spots should be modified to be made resistant to cell adhesion. The size, shape, and integrity of DNA-loaded islands can be readily designed using an appropriate photomask. A single island created in this study had an area of 0.8 mm2, to which approximately 2000 cells adhered in the typical case with HEK293. In an integrated transfectional microarray, cross-contamination between different plasmids spotted to the juxtaposed islands should be avoided. The results shown in Figure 8D indicate that transfection is sufficiently restricted within the islands on the ITObased microarray. The uniformity of the electric field strength would also be very important for parallel electroporation in a microarray fashion. In this study, no significant difference in transfection efficiency was observed over an area of 13 × 13 mm, as demonstrated in Figure 8D. However, modifications of the electroporation assembly, such as incline of the top electrode as proposed by Raptis et al.,13,37 will be introduced to obtain a uniform electrical field for future quantitative studies. These results illustrate that this method will be advantageous in expressing multiple genes in parallel for the highthroughput functional analysis at a cellular level. Conclusions In the present study, we demonstrated that efficient transfection of plasmid DNA was achieved by applying a

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short electric pulse to the cells cultured on the plasmidloaded ITO electrode. The sequential assembly process was efficient for the stable loading of plasmids onto the ITO surface under physiological conditions. The loaded plasmid was shown to be released from the electrode upon application of an electric pulse and transferred into cells cultured directly on the electrode. Our data showed that transfection efficiency was dependent on both the electric field strength and the number of layers. These effects can be well-explained by considering the availability of a detached plasmid to the cells. Because the plasmids incorporated in the multilayer are released only when an electric pulse is applied, electroporation on the plasmidloaded electrode provides the possibility of temporally controlled gene transfer into adherent cells. Furthermore, the electroporation method reported here enables siteaddressable, parallel gene transfer into cells, taking advantage of photoassisted micropatterning of the ITO surface. The technique should be a powerful way to create a transfected cell microarray for functional characterization of genes on a genome scale. Acknowledgment. This study was partly supported by Kobe Cluster, the Knowledge-Based Cluster Creation Project, Ministry of Education, Culture, Sports, Science and Technology (MEXT). LA0505059