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Nanosecond laser pulse induced stress waves enhanced magnetofection of human carcinoma cells in vitro

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Laser Physics Letters

Laser Phys. Lett. 9, No. 9, 678–681 (2012) / DOI 10.7452/lapl.201210064

Abstract: We have developed a novel platform for efficient gene delivery into cells using magnetic force for preconcentration of gene-magnetic nanoparticle complex on the surface of cells with subsequent nanosecond laser pulse for generation of stress waves in transfection chamber which is able to permeabilize cell membrane for the facilitated delivery of gene into the cell interior. Combination of these two physical factors increased the efficiency of three different human carcinoma cells transfection with plasmid coding green fluorescence protein from 43% to 67%, from 35% to 54%, and from 23% to 39%, for HeLa (cervical carcinoma), MCF-7 (breast carcinoma), and UCI-107 (ovarian carcinoma) cells, respectively, as compared with using only magnetofection. Proposed fast, simple, and efficient method may have far reaching applications for cancer gene therapy.

Nanosecond laser pulse

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Laser induced plasma Stress wave Transparent PET cover Magnetic nanoparticles with gene

Natural black rubber

Transfection chamber

Cell monolayer

Neodimium magnet

Experimental setup of our gene-transfer method using laserinduced stress waves (LISW) combined with magnetofection c 2012 by Astro, Ltd. ⃝

Nanosecond laser pulse induced stress waves enhanced magnetofection of human carcinoma cells in vitro ˇ Durd´ık, 1,2 M. Babincová, 3,∗ C. Bergemann, 4 and P. Babinec 3 S. 1

St. Elizabeth Cancer Institute, Heydukova 10, 812 50 Bratislava, Slovakia ˇ alska 24, 813 72 Bratislava, Slovakia Department of Oncological Surgery, Faculty of Medicine, Comenius University, Spit´ 3 Department of Nuclear Physics and Biophysics, Comenius University, Mlynská dolina F1, 842 48 Bratislava, Slovakia 4 Chemicell GmbH, Eressburg Straße 22-23, 12103 Berlin, Germany 2

Received: 4 May 2012, Accepted: 23 May 2012 Published online: 1 July 2012

Key words: gene therapy; pulsed laser; stress waves; magnetofection; human carcinoma cells

1. Introduction Gene therapy is the approach that can cure some of the human diseases using nucleic acids as therapeutic agents. Crucial step in gene therapy is cell transfection – the process of introducing nucleic acids into cells. Common methods for gene delivery are viruses [1], calcium phosphate [2], liposomes [3], gene gun [4], electroporation [5], and sonoporation [6]. Transfection of cells typically involves opening transient pores in the cell membrane, to allow the uptake of material. It is therefore not surprising that laser has been used for these purposes almost 30 years ago [7]. Typically, a laser is focussed to a diffraction limited spot (∼ 1 µm diameter) for short time (from femtoseconds to seconds), generating a transient pore on ∗

the membrane, to allow a gene present in the surrounding medium to be transferred into cell [8–16]. The generation of high-amplitude stress waves with short pulses of laser radiation was first investigated a few years after the first laser became operational [17, 18]. These early studies predicted that high amplitude stress waves could be generated in materials by impinging the laser beam on an unconfined surface of the body and vaporizing a small amount of surface material. As has been demonstrated DNA can be successfully delivered to mammalian cells by applying nanosecond-pulsed laser-induced stress wave (LISW) [19–22]. Another system used successfully in gene therapy are multifunctional magnetic nanoparticles which have a di-

Corresponding author: e-mail: [email protected] c 2012 by Astro, Ltd. ⃝


Laser Phys. Lett. 9, No. 9 (2012)

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(UCI-107) cells were cultured as monolayer in 96 well plate (Corning Inc., Corning, NY, USA ) in a humidified atmosphere of 95% air and 5% CO2 at 37◦ C in complete Roswell Park Memorial Institute culture medium RPMI1640. The cells were seeded at a density of 104 cells/well, 24 hours before the experiments.

Transparent PET cover Magnetic nanoparticles with gene

Natural black rubber

Transfection chamber

Cell monolayer

Neodimium magnet

Figure 1 (online color at www.lasphys.com) Experimental setup of our gene-transfer method using laser-induced stress waves (LISW) combined with magnetofection

verse potential applications in many biological and medical applications such as cell separation [23] and drug targeting [24–28]. Magnetic nanoparticle-based gene transfection (magnetofectionTM ) has also been shown to be effective in combination with both viral vectors and with non-viral agents [29–31]. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target cells via high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. Our aim in this study is to develop a new in vitro gene delivery method combining magnetofection for preconcentration of gene-magnetic nanoparticle complex on the surface of cells with subsequent nanosecond laser pulse for generation of stress waves in transfection chamber which is able to permeabilize cell membrane for the facilitated delivery of gene into the cell interior.

2. Material and methods 2.1. Chemicals If not otherwise stated all chemicals were purchased from Sigma (St. Louis, MO, USA). Enhanced green fluorescent protein (pEGFP-C1) coding reporter plasmid used for the study of gene delivery, was provided by Amersham Bioscience (London, UK). This plasmid expresses GFP when delivered to cell nucleus.

2.2. Cell culture Human cervical carcinoma (HeLa) cells, human breast carcinoma (MCF-107) cells, and human ovarian carcinoma

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2.3. Setup for generation of laser induced stress waves The experimental setup is shown in Fig. 1. LISWs were generated by irradiating a target, a 6.4 mm diameter, 0.6 mm thick black natural rubber disk, covering the well of a plate (total volume of the plate well was 0.36 ml) with a 532 nm Q-switched Nd:YAG 6 ns laser (Surelite I-10, c Continuum⃝ , Santa Clara, CA, USA). Laser pulse was focused with a plane-convex lens (f = 60 mm) to a 2.5 mm diameter spot on the target. A 0.5 mm thick transparent polyethylene terephthalate sheet with diameter 6.4 mm was adjusted on the top of the target to confine laserinduced plasma for increasing LISW impulse.

2.4. Transfection and viability determination Before transfections the cells were washed with phosphate-buffered saline (PBS) and 150 µl of incomplete RPMI 1640 (without FBS, L-glutamine and penicillin/streptomycin) per well was added. 0.5 µl of polyethylenimine functionalized magnetic nanoparticles Polymag (Chemicell GmbH, Berlin, Germany) with 0.5 µg of plasmid DNA were added to each well. For magnetofection the 96-well plate was then placed on top of a magnetoFACTOR plate (Chemicell GmbH, Berlin, Germany) and for 30 min incubated at room temperature. In combined treatment, after 15 min of incubation LISW was applied and after pulse application the plate was next 15 min left on magnetoFACTOR plate. After transfection medium was replaced with 200 µl of complete RPMI 1640 and incubation was continued for 24 hours in a humidified atmosphere of 95% air and 5% CO2 at 37◦ C, and cells were then analyzed for transfection efficiency and viability. Transfection efficiency is defined as the fraction of transfected cells that are alive to the total number of cells including dead cells. Cells successfully transfected with pEGFPC1 were counted according to their fluorescence using Meiji Techno 5040 fluorescent biological microscope. For the assessment of cell viability crystal violet staining assay was used 48 h post-transfection. Cells were washed with PBS and 50 µl of 0.5% crystal violet solution in methanol was added to each well. The cells were incubated for 10 min at room temperature. The staining solution was discarded and the 96-well plate washed gently with tap water. The plate was placed upside down on

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ˇ Durd´ık, M. Babincová, et al.: Enhanced magnetofection of human carcinoma cells S.

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Transfection efficiency Cell viability

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Transfection efficiency and cell viability, %


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Magnetofection

HeLa cells

Figure 2 (online color at www.lasphys.com) Transfection efficiency and cell viability obtained for human cervical HeLa cells using magnetofection, laser induced stress wave (LISW), and their combination

LISW


MCF-7

Figure 3 (online color at www.lasphys.com) Transfection efficiency and cell viability obtained for human breast carcinoma MCF-7 cells using magnetofection, laser induced stress wave (LISW), and their combination

2.5. Statistical analysis


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paper towels to drain any remaining water. 100 µl of 1% SDS solution was added to each well to solubilize the stain. The plate was agitated until a uniform color was obtained. Absorbance at 570 nm was measured with a microplate spectrophotometer (Bio-Rad Benchmark Plus, Biorad, Hercules, CA, USA). Cell viability (%) was computed as (A/B) × 100, where A is the optical density derived from wells containing Polymag-DNA complexes and B is the mean optical density value derived from control wells.


90 80 70 60 50 40 30 20 10 0 Magnetofection

All measurements are given as a means of 3 – 10 separate experiments with an average of five samples per experiment. Differences between all samples were assessed by one-way factorial ANOVA. A value of p < 0.05 was considered to be statistically significant.

LISW


UCI-107

Figure 4 (online color at www.lasphys.com) Transfection efficiency and cell viability obtained for human ovary carcinoma UCI-107 cells using magnetofection, laser induced stress wave (LISW), and their combination

3. Results and discussion The goal of these experiments was to investigate the feasibility and efficacy of combination of two methods of cell transfection - magnetofection and LISW, using three different human carcinoma cell lines. We were guided by the observation, known especially from electrotransfection, that 99% DNA uptake into cell corresponds to the membrane bound DNA [32]. That the diffusion of plasmid in the cytoplasm is very slow, especially for DNA molecules larger than 2000 base pairs was also shown using HeLa cells, where only very few DNA molecules diffused away


from the site of injection after few hours [33]. As has been shown by [34], magnetic nanoparticle bound DNA is not able to penetrate cell membrane, therefore magnetofection just preconcentrate plasmid-nanoparticle complex on the cell surface. Shear force induced by LISW might cause disruption in cell membranes, through which DNAmagnetic nanoparticle complex diffuse into the cytoplasm under the influence of magnetoforetic force and concentration gradient. Disruption of the plasma membrane explain also the cytotoxicity of the method. Results are shown in Fig. 2 – Fig. 4.

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Laser Phys. Lett. 9, No. 9 (2012)

Specifically, we have obtained enhancement of transfection efficiency from 43% to 67%, from 35% to 54%, and from 23% to 39%, for HeLa cells, MCF-7 cells, and UCI-107 cells, comparing magnetofection and combination LISW + magnetofection, respectively. For all three human cell lines we have found substantial enhancement of transfection efficiency, when we have applied LISW during the magnetofection process, which supports our working hypothesis. LISW alone is also able to transfect cells with efficiency only about 5%.

4. Conclusion In this paper we have presented a novel method for the transfection of cells based on the combination of magnetofection and LISW. On the example of human cervical carcinoma (HeLa) cells, human breast carcinoma (MCF107) cells, and human ovarian carcinoma (UCI-107) cells, widely used for the evaluation of efficiency of cell transfection methods, we have demonstrated significant improvement when compared to usage of magnetofection or LISW alone. Viability of magnetofection, which is now already routinely used in gene therapy, was lowered only by few percents, therefore our method, may be of particular importance, especially for hard-to-transfect cells, e.g. neurons. A human ovarian carcinoma UCI-107 can be genetically engineered using proposed method to secrete the cytokine interleukin-2 by retroviral-mediated gene transduction and these cells can be used as vaccines for the treatment of advanced epithelial ovarian cancer. Moreover, there are also potential applications of these two methods for in vivo cell transfection, where especially LISW is very powerful, therefore their combination can be an interesting alternative for e.g. in vivo electroporation. Acknowledgements This work was supported by VEGA grant 1/0642/11.

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