spie proceedings [spie lase - san francisco, california (saturday 23 january 2010)] frontiers in...

Fs- laser cell perforation using gold nanoparticles of different shapes

Markus Schomaker,1,* Holger Fehlauer,1 Willem Bintig,2 Anaclet Ngezahayo,2 Ingo Nolte,3 Hugo Murua Escobar,3 Holger Lubatschowski,1 and Alexander Heisterkamp1

1Laser Zentrum Hannover e.V. and Research Cluster of Excellence „REBIRTH“, Hollerithallee 8,

30419 Hannover, Germany 2Institute of Biophysics, Leibniz University, Herrenhaeuserstr. 2, 30419 Hannover, Germany

3Small Animal Clinic and Research Cluster of Excellence "REBIRTH", University of Veterinary Medicine Hanover, Bischofsholer Damm 15, D 30173 Hanover, Germany

*Corresponding author: [email protected]

ABSTRACT The resulting effects of the interaction between nanoparticles and laser irradiation are a current matter in research. Depending on the laser parameters as well as the particles properties several effects may occur e.g. bubble formation, melting, fragmentation or an optical breakdown at the surface of the nanoparticle. Besides the investigations of these effects, we employed them to perforate the membrane of different cell lines and investigated nanoparticle mediated laser cell perforation as an alternative optical transfection method. Therefore, the gold nanoparticles (GNP) of different shapes were applied. Furthermore, we varied the methods for attaching GNP to the membrane, i.e. co-incubation of pure gold nanoparticles and bioconjugation of the surface of GNP. The optimal incubation time and the location of the GNP at the cell membrane were evaluated by multiphoton microscopy. If these GNP loaded cells are irradiated with a fs laser beam, small areas of the membrane can be perforated. Following, extra cellular molecules such as membrane impermeable dyes or foreign DNA (GFP vectors) are able to diffuse through the perforated area into the treated cells. We studied the dependence of the laser fluence, GNP concentration, GNP size and shape for successful nanoparticle mediated laser cell perforation. Due to a weak focusing of the laser beam a gentle cell treatment with high cell viabilities and high perforation efficiencies can be achieved. A further advantage of this perforation technique is the high number of cells that can be treated simultaneously. Additionally, we show applications of this method to primary and stem cells. Keywords: perforation, transfection, nanoparticles, nanorods, ultrashort laser pulses, plasmonics, cell manipulation, biophotonics, membrane permeabilization, GFP

1. INTRODUCTION

The manipulation of cells is crucial for cell and gene therapy. To accelerate these arising therapies, new transfection methods are necessary to avoid critical issues of conventional methods [1-5]. These critical properties result in, depending on the method employed, low efficiency and/or low throughput, toxicity, complex handling and limitation to specific cell lines or high cell damages [2-5]. Lately, laser and especially NIR fs laser became an efficient and gentle tool for cell membrane permeabilization [6,7]. Herein, the laser beam disturbs the cell membrane due to a tight focusing and multiphoton absorption. Up to now a low troughput inhibits the broad use of this method.

Recently, the interaction of laser light and small particles became important for many biomedical applications and the resulting effects are suitable to perforate the cell membrane as reported by Yao et al. [8,9]. Especially the interaction between laser pulses and GNP plays an important role for cell manipulation due the properties of gold and the excited effects that occur. Different effects can appear at the GNP in liquid environment depending on the laser energy, wavelength as well as size and shape of the GNP [10,11].

In this study we used fs laser induced effects in GNP of different shapes (spheroids and rods) to perforate the cell membrane. Therefore, GNP loaded cells are irradiated with a weakly focused laser beam. If the cell membrane is perforated, impermeable molecules (e.g. RNA/DNA) can diffuse from the extra cellular medium into the cell. We present our investigation on nanoparticle mediated cell perforation for different cell lines and the interaction of fs laser pulses with GNP. The results show, that this alternative possibility for cell transfection promises high efficiency and cell viability with a high trough put even for cell line that are hard to transfect with common methods.

Frontiers in Ultrafast Optics: Biomedical, Scientific, and Industrial Applications X, edited by Alexander Heisterkamp,Joseph Neev, Stefan Nolte, Rick P. Trebino, Proc. of SPIE Vol. 7589, 75890C · © 2010 SPIE · CCC code:

0277-786X/10/$18 · doi: 10.1117/12.842446

Proc. of SPIE Vol. 7589 75890C-1

Downloaded From: http://proceedings.spiedigitallibrary.org/ on 09/04/2013 Terms of Use: http://spiedl.org/terms

2. MATERIALS AND METHODS 2.1 Cell preparation

The cells we applied in this study were cultivated in glass-bottom-dishes (MatTek). Besides a canine cell line (ZMTH3) which was cultivated in RPMI-1640 medium, we used primary cells from twelve weeks old albino mice (DRG neurons) and different human stem cells (embryonic and iPS cells).

2.2 Nanoparticle mediated laser cell perforation and transfection

For nanoparticle mediated cell perforation spherical gold particles from 80 nm – 250 nm in diameter (Kisker GmbH) or custom made PEG coated gold nanorods 12,5 nm with an aspect ratio of 4.0 added to the surrounding cell medium and incubated for several hours. During laser treatment Lucifer yellow (LY) was added to the medium to label successfully perforated cells. After laser exposure the cells were washed with PBS (phosphate buffer saline). The viability of the cells was evaluated by adding Propidium iodide (Pi) to the medium. To prove the successful transfection a non-recombinant pEGFP-C1 vector plasmid and a recombinant pEGFP-C1-HMGA2 vector plasmid was added to the medium at a concentration of 50µg/ml. The non-recombinant vector is labeling the whole cell and the recombinant vector is for specific nucleus labelling in case of successful transfection.

2.3 Multiphoton microscopy and nanoparticle localization

We imaged the GNP near the cell membrane with a custom build multiphoton microscope after different incubation times. For a better localization of the GNP we labeled the cells with different fluorescent dyes. The procedure was as follows: We incubated the cells with gold particles, thereafter the medium was removed from the cells. Then the dye was added to the cells and incubated for 15min. Finally, the cells were washed with PBS and imaged by the multiphoton microscope.

2.4 Experimental setup

The applied laser pulses (TEM00) were generated by a Ti:Sapphire laser (Thales, Bright system) with a pulse duration of 120 fs. The repetition rate of the laser system was tunable between 1 and 5 kHz at a fixed wavelength of 780 nm and kept at 5 kHz during all experiments. The laser beam was weakly focused by a lens with a focal length of 140 mm on the samples (see Fig. 1(a)). By moving the sample relative to the laser beam an area of 3 mm2 was scanned.

spot size

NIR fs laserpulses

foreign DNA (GFP)

c)

a) b)

Figure 1. (a) Experimental setup: The laser beam is scanning the sample by moving the stages relative to the laser beam. (b) Sketch of manipulation principle: Gold nanoparticles are located close to the cell membrane and irradiated by fs laser pulses. Due to plasmonic resonances and subsequent effects the cell membrane is disrupted. (c) Top view, scanning line is represented by the red arrow.



3. RESULTS 3.1 Effects based on GNP and laser pulse interaction

We studied the influence of NIR fs laser radiation on GNP. Therefore 150 nm GNP were located on a silicon wafer and irradiated with fs laser pulses. After the laser treatment the particles were removed from the wafer if the laser energy was high enough. Additionally, we observed that material was ablated and small holes appeared with a diameter smaller than the particle size.

Figure 2. SEM images of spherical 150 nm GNP located on a silicon wafer a) untreated GNP. b) effects of NIR fs laser pulse treatment, several particles are removed and small holes are generated in the silicon wafer due to material ablation.

3.2 GNP cell interaction and localizaztion

A multiphoton microscope was used to study the GNP- cell interaction. At different incubation times of the cells with the GNP, we washed the cells with PBS and labeled the membrane with a FM4-64 florescent dye. The results show a “bonding” of the GNP at the cell membrane after one hour. With an increasing incubation time more GNP are visible near the cell membrane. We determined an optimal incubation time for nanoparticle mediated cell perforation of 3 hours. After this time enough GNP are present at the membrane.

Figure 3. Multiphoton microscopy image of ZMTH 3 cells, the cell membrane is labeled with a FM4-64 fluorescent dye (green). The nanoparticles (bright spots) are located near the cell membrane, scale bar is 20 µm.

3.3 Nanoparticle mediated laser cell perforation

To perforate the cell membrane we incubated the cells with spherical GNP of different sizes for 3 hours. The cells were prepared as described above and treated with fs laser pulses while scanning an area of 3 mm2. A membrane impermeable fluorescent dye (Lucifer yellow) was used as an indicator for successfully perforated cells. The viability of the cells was checked with Propidium Iodide.

Perforation experiments were performed with different cell lines. The results show a successful perforation for human stem cells, human IPS cells and mouse DRG neurons using laser induced effects in 150 nm GNP. The highest perforation rate of 79.3 % was achieved using ZMTH3 cells, 200 nm GNP with a cell viability of 83.3 %. That means nearly every vital cell was perforated. Reason for the loss of ca. 20 % of the cells could be the temperature fluctuation during the preparation and laser treatment procedure. The parameters for the best rate of perforation were a GNP incubation concentration of 11.33 µg/ml, a laser fluence of 0.1 J/cm2 and a scanning velocity of 15 mm/s.



Figure 4. Perforation rate and viability of laser treated ZMTH3 cells using optimal set of process parameters for different sizes of spherical nanoparticles.

Next to the experiments with spherical GNP, essays with PEG coated, rod shaped GNP were performed. The preparation and laser treatment procedure was equal to the experiment with spherical GNP. As shown in Figure 5 successful perforation can be achieved. Additionally, we could observe that the fluorescent dye diffused only in the laser treated cells.

Figure 5. Perforated ZMTH3 cells using plasmon resonances induced in PEG coated gold nanorods a) bright field image: cells are growing on a coverslip b) fluorescence image, taken after laser exposure. Only in the laser treated area the cells are perforated. 1 mM lucifer yellow was used to label perforated cells. The cells were treated with a laser fluence of 0.1 J/cm2, a scanning velocity of 5 mm/s and the GNP concentration of 4.25 µg/ml.

3.4 Nanoparticle mediated laser cell transfection

Since impermeable dye molecules, with a molecular weight of 457 Dalton, diffuse into the cells, transfection experiments with pEGFP-C1 vectors were performed. These plasmid vectors have a size of 4.7 kbp and are therefore much bigger than the LY molecules. Even though, these GFP plasmids diffused into the cells after laser treatment but compared to the Ly molecule with a lower efficiency. The transfection was proved with a fluorescent microscope 48 h after laser treatment. In case of successful GFP expression the whole cell got fluorescent. To demonstrate successful transfection for primary cell, mouse DRG neurons were transfected. Therefore a pEGFP-C1-HMGA2 vector, a laser fluence of 0.37 J/cm2 and a scan velocity of 50 mm/min were used.

laser treated area

0102030405060708090

neg. 80 nm 150 nm 200 nm 250 nm

perforation rate [%] viability [%]



Figure 6. With pEGFP-C1-HMGA2 transfected neuron. The used parameters were a laser fluence of 0.37 J/cm2 and a scan velocity of 50 mm/min

4. CONCLUSION AND DISCUSSION The GNP mediated laser cell perforation is suitable to perforate the cell membrane with a high efficiency and high cell viability. This method requires an interaction of nanoparticles with the cell membrane, as the laser induced effects for perforation are located at or in close vicinity of the particle surface. This laser pulse- particle interaction leads to a permeabilization of the cell membrane with thicknesses of some nanometer. Furthermore the laser induced effects are strong enough to create holes in solid materials but underlying mechanisms are not fully investigated yet.

Once the membrane is perforated, impermeable molecule diffuse into the cell using fs laser pulses and either spherical or rod shaped GNP. Lucifer Yellow molecules with a molecular weight of 457 Da enter the cells with a higher efficiency than 4.7 kbp plasmid vectors. However, the possibility to transfer genes into living cells using GNP mediated laser cell perforation is very promising for high troughput cell transfection. Furthermore, this method is suitable for primary and stem cells permeabilization.

REFERENCES

[1] F.L. Graham, A.J. van der Eb, “A new technique for the assay of infectivity of human adenovirus 5 DNA,” Virology 52, 456–467 (1973). [2] D.A. Rubinson, C.P. Dillon, A.V.Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, D.L. Rooney, M. Zhang, M.M. Ihrig, M.T. McManus, F.B. Gertler, M.L. Scott, L. Van Parijs, “A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference,” Nat. Genet. 33, 401-406 (2003). [3] H. Tahara, H. J. Z. Zeh, W. J. Storkus, I. Pappo, S. C. Watkins, U. Gubler, S. F. Wolf, P. D. Robbins, M. T. Lotze, "Fibroblasts genetically engineered to secrete interleukin-12 can suppress tumor growth and induce anti-tumor immunity to a murine melanoma in vivo," Cancer Res. 54, 182–189 (1994). [4] T.K. Wong, E. Neumann, “Electric- field mediated gene-transfer,” Biochem. and Biophys. Res Commun. 107, 584-587 (1982). [5] W. Chen, J. McCluskey, "Electroporation of antigen-presenting cells for T-cell recognition and cytotoxic T-lymphocyte priming," Methods Mol. Biol. 48, 73–81 (1995). [6] J. Baumgart, W. Bintig, A. Ngezahayo, S. Willenbrock, H. Murua Escobar, W. Ertmer, H. Lubatschowski, A. Heisterkamp, “Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53a cells,” Opt. Express 16, 3021-3031 (2008). [7] D. Stevenson, B. Agate, X. Tsampoula, P. Fischer, CTA. Brown, W. Sibbett, A. Riches, F. Gunn-Moore, K. Dholakia, “Femtosecond optical transfection of cells: viability and efficiency,” Opt. Express 14, 7125–7133, (2006). [8] C. Yao, R. Rahmanzadeh, E. Endl, Z. Zhang, J. Gerdes, G. Hüttmann, „Elevation of plasma membrane permeability by laser irradiation of selectively bound nanoparticles,” J. Biomed. Opt. 10, 064012-1 (2005). [9] C. Yao, X. QU, Z. Zhang, “Laser-based transfection with conjugated gold nanoparticles,” Chinese Opt. Lett. 7, No. 10 (2009). [10] C. M. Pitsillides, E. K. Joe, X. Wei, R. R. Anderson, C. P. Lin, “Selective cell targeting with light-absorbing microparticles and nanoparticles,” Biophys. J. 84, 4023–4032 (2003). [11] V.K. Pustovalov, A.S. Smetannikov, V.P. Zharov, “Photothermal and accompanied phenomena of selective nanophotothermolysis with gold nanoparticles and laser pulses,” Laser Phys. Lett. 5, 775-792 (2008)

Acknowledgement This work was funded by the German Research Foundation (DFG) within the Transregio 37 and the excellence cluster REBIRTH.



spie proceedings [spie lase - san francisco, california (saturday 23 january 2010)] frontiers in...

Documents