Using Magnetic Nanoparticles to Enhance Gene Transfection. on Magneto-electroporation Microchips

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1 Materials Science Forum Online: ISSN: , Vols , pp doi: / Trans Tech Publications, Switzerland Using Magnetic Nanoparticles to Enhance Gene Transfection on Magneto-electroporation Microchips Min Li 1, a, Yu-Cheng Lin 1, 2, b *, Kai-Chun Su 1, c 1 Department of Engineering Science, 2 Center for Micro/Nano Technology Research, National Cheng Kung University, 1 University Road, Tainan, Taiwan a felipa3@ksmail.seed.net.tw, b *yuclin@mail.ncku.edu.tw, c KC_Su@itri.org.tw Keywords: Microchip, Magnetic Nanoparticles, Transfection Abstract. This study demonstrated that DNA associated with magnetic nanoparticles can be attracted to specific areas of cell surfaces under magnetic fields, which highly increased the DNA concentration at specific areas and further enhanced the gene transfection in an electroporation (EP) method. The superparamagnetic nanoparticle s distribution could be operated by magnetic field, where the gravity effect could be neglected. Compared with the electroporation with and without electrostatic attracting force, the magneto-electroporation with magnetic attracting force showed higher delivery rate (63.05 %) in the electroporation processes. Simulating an asymmetric magnetic field helps to create experiment environment with different intensities of magnetic flux density. The resultant difference can be identified by the profile of fluorescence. This report focused on enhancement and targeting of gene transfection using 6 nm γ-fe 2 O 3 nanoparticles and electroporation microchips. Introduction Gene therapy is a potential therapeutic modality, which requires effective gene delivery into cells. Different types of gene delivery systems have been developed. Electroporation has been widely applied on gene delivery. The microchip used to gene transfection has been reported [1-3]. The electrostatic force has been used to attract DNA before electroporation process, because DNA carried negative charge [4]. For enhancing the delivery rate, nanoparticles have shown their potential on this domain. Using silica nanoparticles (diameter 225 nm) together with sedimentation methodology were employed to deliver gene. The calculated sedimentation rate is 8.3 mm/hr [5]. Magnetofection utilizing magnetic nanoparticles (400 nm-1 µm in diameter) have significant progress in delivering genes by means of magnetic forces [6]. In this study, 6 nm superparamagnetic iron oxide nanoparticles were manipulated under strong magnetic fields where the gravity effect can be neglected. It is helpful to verify that the magnetic nanoparticles migration would be influenced by the magnetic force in low viscosity solution, resulting in targeting and enhancement for electroporation. Experimental Microchip fabrication and superparamagnetic iron oxide nanoparticles preparation. The EP microchips were designed in two components, the well-defined cavity for cell accommodation as well as the interdigitated electrodes for providing the electroporation electric power, as shown in Fig. 1. The interdigitated Au/Ti thin film electrodes were fabricated on the glass slide by MEMS technology [4]. The microelectrodes with the dimensions of 100 µm wide and 200 µm spacing between electrodes were used for providing high electric field. The specific area made of PDMS helps to get desired cell density, save culture cell period and be convenient for detection. The iron oxides particles have the diameter of 5.8 ± 0.5 nm, as shown in Fig. 2. Experiments took about mm γ-fe 2 O 3 nanoparticles in sucrose solution as an electroporation buffer. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (# , Pennsylvania State University, University Park, USA-17/09/16,22:42:40)

2 662 Progress on Advanced Manufacture for Micro/Nano Technology 2005 Gene and cells preparation. The pegfp-n1 plasmids, 80 µg/ml, coding for green fluorescent protein (GFP) were transfected into mouse clone Osteoblast-like cells line (MC3T3E-1). In process of culture, the microchip was coated with 0.01 % poly-lysine in phosphate-buffered saline for cell adhesion, loaded into the cavity of the microchip for cell accommodation, and then the chip was washed with PBS. The concentration of 10 5 cell/ml of 50 µl was seeded onto the pretreated microchip. The cells were maintained at 37 C, under 5% CO 2, in Dulbecco s modified Eagle s medium for 24 hrs. The γ-fe 2 O 3 nanoparticles ( mm, 25 µl) combined with GFP plasmid (80 µg/ml, 25 µl) were put into the cavity before the experiment and washed out by medium after electroporation process, and then the medium was added for cell culture. The fluorescent detection was made after 36 hr culture. Magneto-electroporation and magnetic field simulation. The transfection process was under a magnetic field combined with an electroporation process using 1 3 V applied voltages. The asymmetrically magnetic fields were created to generate different magnetic particle s density profile. The schematic diagram of the DNA associated with magnetic nanoparticles attracted by an asymmetrically magnetic field is illustrated in Fig. 3. PDMS Au Electrodes Ti Glass Substrate (a) (b) Fig. 1 (a) 3D expanded view of the electroporation microchip (both the width and space of the interdigitated electrodes are 100 µm), (b) Photo image of 24 hour cultured MC3T3E-1 cell distribution before electroporation. Fig. 2 The γ-fe 2 O 3 nanoparticles picture captured by TEM.

3 Materials Science Forum Vols Fig. 3 Schematic drawings of the model setup. To further investigate the magnetic fields in the cell-culture cavity, 3-D magnetic field simulations were performed. The magnetic field distributions of different positions under the EP microchip were simulated and shown in Fig. 4. The finite element method was used to establish an experimental model for magnetic force analysis by ANSYS 5.5 software. The positions of the magnet edge between the cell-culture cavities were full, half, quarter and at boundary, respectively. To further evaluate the magnetic field influence on the enhancement of gene transfection, the position of the magnet should be at the half (X = 0.3 mm) of the cell-culture cavity to have a significantly asymmetric magnetic field. Cell wall Magnet (a) (b) Fig. 4 Magnetic field distribution of an EP chip, (a) example of magnetic field in a section of the EP chip, (b) magnetic density is along the center-line from the edge of a cell-culture cavity toward the center. The magnet located at the half position of cell-culture cavity is found to generate the highest asymmetric magnetic fields. Results and discussion Magneto-nanoparticles enhance gene transfection. The γ-fe 2 O 3 nanoparticles can be suspended in low viscosity solution and the suspension situation can be kept for a long time. For the nano-size of magnetic particles, the influence of magnetic force can dominate that of the gravity. With the same electroportion parameters, 3V, 2 pulses, the case with γ-fe 2 O 3 nanoparticles inside under magnetic fields shows better transfection results than that without particles inside. And there are high survival rate found in the cells, shown as Fig. 5. The γ-fe 2 O 3 nanoparticles delivered into the cell could be detected by a transmission electron microscope, as shown in Fig. 5c.

4 664 Progress on Advanced Manufacture for Micro/Nano Technology 2005 (a) (b) (c) Fig. 5 Transfection gene distribution of an EP chip, (a) The disperse of fluorescence cells without γ-fe 2 O 3, (b) The disperse of fluorescence cells and γ-fe 2 O 3, and (c) The TEM image about the γ-fe 2 O 3 on the cell. Quantify the transfection rate of magnetic nanoparticles. The amount of γ-fe 2 O 3 nanoparticles delivered into a cell could be measured by Inductively Coupled Plasma Atomic Emission Spectrometer, ICP-AES, (SHIMADZU/ICPS-1000IV, Japan). The muriatic acid was added into the samples, electroporated cells and residual buffer, respectively. And then the resultant acidified samples were vaporized as an aerosol. The aerosol of the sample was transported to the plasma torch where excitation occurred. Characteristic atomic-line emission spectra were produced by a radio-frequency inductively coupled plasma (ICP). Based on the ICP analysis, the delivery rate of γ-fe 2 O 3 nanoparticles was shown in Table 1. The initial electroporation buffer (50 µl) consisted of mm γ-fe 2 O 3 and cell medium (mixture ratio 1:1). Due to the detection limit of ICP, the measured samples were the sum of several experiments. The average weight of Fe element in the electroporation buffer was about 3.00 ppm (15 µg) by calculation and viewed as the original weight. In the experiments, there were two physical forces, magnetic force or electrostatic force, were used for attracting γ-fe 2 O 3. In this experiment, the delivery rate was defined as the ratio of the γ-fe 2 O 3 weight in the electroporated cell to the original weight. To ensure the experimental errors were within acceptable range, the difference between the original weight of the γ-fe 2 O 3 and the total weight of nanoparticles in the cells and the residual buffer was within 1% of the original weight. The measurement results were shown in Fig. 6, and the delivery rate of the Sample 1 electroporated without magnetic and electrostatic force was %. The electroporation process of the Sample 4 was applied with 0.6 V attracting voltage and kept 30 sec followed by a 3 V, 2 pulse electroporation voltage. The delivery rate of the Sample 4 was increased 8.33 % compared to the that of the Sample 1 due to the negative charged γ-fe 2 O 3 nanoparticles-dna complexes were attracted to the cell surfaces by the electrostatic force. However, the magnetic field (3 kg, 30 sec) was used to attract γ-fe 2 O 3, and the delivery rate was increased an addition to % compared to the electroporation without any attracting force. When the attracting time increased by 10 fold, the delivery rate of the Sample 2 (63.05 %) was two-fold higher than that of the Sample 1 with only electroporation. Table 1 The parameters and ICP measurement of the Magneto-Electroporation experiment. Attracting force Average (Fe) weight delivery rate Sample 1 No 4.72±0.02 µg % Sample 2 Magnetic field 3KGuass, 300 sec 9.46±0.02 µg % Sample 3 Magnetic field 3KGuass, 30 sec 7.09±0.02 µg % Sample 4 Electrostatic field 0.6V, 30 sec 5.97±0.02 µg %

5 Materials Science Forum Vols % 70% Fe2O3 in cell Residual Fe2O3 in buffer 60% Fe2O3 weight (%) 50% 40% 30% 20% 10% 0% Sample number Fig. 6 The percentage of γ-fe 2 O 3 in cell samples and residual buffers. Magnetic operation of the γ-fe 2 O 3 nanoparticles induces targeting effect. The magnetic field would attract the magnetic nanoparticles-dna complex to the cell surfaces. The magnet with a strong magnetic field, 3 kg, was placed at 0.3 mm from the left side of cavity for 5 minutes. Under an asymmetric magnetic field with maximum magnetic flux density 2.5-fold greater than the minimum, the successfully transfected cells expressed green fluorescence which was detected on the right side (higher magnetic field) of cavity and decayed on the left side (higher magnetic field). Fig. 7 shows that the delivery rate under a stronger field is much higher. For further verifying that the magnetic nanoparticles operation could influence the gene targeting, an electromagnet was used instead of a magnet. Using a electromagnet generating the high magnetic field in an annular region, a large amount of transfected cells, expressing green fluorescence, were observed in the annular area with a high magnetic field, as illustrated in Fig. 8. The performance of the magnetic particles carrying the genes into the target cells could be attributed to the occurrence of the interaction, such as hydrogen bonding, between the nucleic acids and the OH groups on the iron oxide surfaces. Fig. 7 The dispersion of the cells with fluorescence and γ-fe 2 O 3 under an asymmetrically magnetic field.

6 666 Progress on Advanced Manufacture for Micro/Nano Technology 2005 Weaker Stronger Fig. 8 The dispersion of the cells with fluorescence and γ-fe 2 O 3 under a non-uniform magnetic field generated by an electromagnet. Conclusions We demonstrated that as an attracting force the magnetic force could enhance γ-fe 2 O 3 nanoparticles delivery. The delivery rate (63.05 %) of electroporation with magnetic force was 2-fold higher than that (31.45 %) of electroporation without any attracting force. The pegfp-n1 DNA delivery rate is highly dependent on the intensity of the magnetic field. The performance of the magnetic particles carrying the genes into the targeted cells could be attributed to the occurrence of the interaction, such as hydrogen bonding, between the nucleic acids and the OH groups on the iron oxide surfaces. This study successfully demonstrates the magnetic nanoparticles associated with DNA can help gene delivery and a specific region targeting utilizing a guiding magnetic field. Acknowledgement The authors would like to thank the Center for Micro/Nano Technology Research, National Cheng Kung University, Tainan, Taiwan, R.O.C. for access to their equipment and for their technical support. Funding from the Ministry of Education and the National Science Council of Taiwan, R.O.C. under contract no. (NSC B ) is gratefully acknowledged. References [1] Y.C. Lin, C.M. Jen, M.Y. Huang, X.Z. Lin, Sen. and Act. B: Chem. Vol.79 (2001), p [2] Y.C. Lin, M. Li, and C.C. Wu, Lab on a Chip Vol. 4 (2004), p [3] Y.C. Lin, M. Li, C.S. Fan, L.W. Wu, Sen. and Act. A: Phys. Vol.108 (2003), p. 12. [4] C.P. Jen, W.M. Wu, M. Li, and Y.C. Lin, J. MEMS Vol.13 (2004), p. 1. [5] Luo, et al., U.S Patent, US B1, (2001). [6] F. Scherer, et al., Gene Therapy Vol.9 (2002), p. 102.