Plasmonic perforation of living cells using ultrashort laser pulses and gold nanoparticles

Markus Schomaker*a, Judith Baumgarta, Anaclet Ngezahayob, Jörn Bullerdiekc, Ingo Noltec, Hugo

Murua Escobarc, Holger Lubatschowskia, Alexander Heisterkampa

aLaser Zentrum Hannover and Research Cluster of Excellence „REBIRTH“, Hollerithallee 8, 30419 Hannover, Germany

bInstitute of Biophysics, Leibniz University, Herrenhaeuserstr. 2, 30419 Hannover, Germany cSmall Animal Clinic and Research Cluster of Excellence "REBIRTH",

University of Veterinary Medicine Hanover, Bischofsholer Damm 15, D 30173 Hanover, Germany

ABSTRACT

Investigation on the interaction of small particles, e.g. gold nanoparticles with light is a current field of high interest. As light can be absorbed, enhanced or scattered by the nanoparticles a wide variety of possible applications become possible. If the electrons of such a nanoparticles oscillate with the incident light, plasmon resonances occur. Provided that these particles are brought very close to a cell, the cell membrane gets perforated due to the laser induced effect. We investigate nanoparticle mediated laser perforation as an alternative technique for cell transfection. By using weakly focussed femtosecond laser pulses, 150 nm gold particles were stimulated to perforate the cell membrane. Through the perforated area of the membrane macromolecules e.g. DNA are able to enter the cell. By this technique GFSHR-17 rat cells were successfully transfected with GFP vector and the dependence on laser parameters and concentration were studied. Even after 48 hours after manipulation the transfected cells show no indications of apoptosis or necrosis. This technique allows the transfection of cells by opto-perforation without the need of tight focusing conditions and single cell targeting- opening the way for a wide field of applications.

Keywords: ultrashort laser pulses, nanoparticles, cell manipulation, plasmon resonance

INTRODUCTION A key technology in molecular biology is the transfer of DNA/RNA molecules into target cells. Transfer of single or double-stranded genetic material into cells allows overexpression or silencing of target genes and studying the induced effects. The usage of DNA expression vectors coding for fluorescence protein or (GFP) fusion proteins in general [1], allows characterizing several internal cell processes such as protein in vivo behavior due to the GFP mediated visibility under a fluorescence microscope. Different techniques have been developed for cell membrane penetration in order to allow the uptake of genetic material. Those conventional methods are e.g. chemical transfection like calcium-phosphate-precipitation [2], transfection via viral vectors [3,4] or physical techniques like electroporation [5,6]. However, all listed methods show disadvantages e.g. low efficiency, low throughput, toxicity, complex handling, limitation to specific cell lines or high cell damages [2-6]. Especially primary tissue cultures and stem cells remain difficult to transfect with established techniques.

To avoid these problems optical transfection techniques have been developed during the last decades [7-11] showing very promising results, using pulsed as well as continuous wave lasers. By applying fs-lasers for cell permeabilization [12-14], the applied pulse energies can be reduced even further. Since this so called fs-optoporation is based on the multiphoton absorption, only in the focal plane the photon density is high enough to generate a low density plasma and thereby perforate the membrane [13,15]. This technique provides a very gentle cell treatment with a cell viability of 70 % to 80 % [13,14] accompanied with similar transfection efficiency [12]. However, this technique is based on tight focusing conditions, making cell and sample positioning difficult and leading to a limited number of transfected cells (single cell targeting). Tsampoula et al. published a less positioning-sensitive approach using an axicon fibre [16]. Nevertheless, none of the fs-optoporation methods allows high throughput cell permeabilisation. We propose an alternative approach for precise and gentle cell transfection relying on the use of gold nanoparticles in combination with loosely focused fs-laser pulses. Due to the use of a weakly focused beam this method allows targeting of many cells simultaneously ensuring high throughput and easy applicability.

Plasmonics in Biology and Medicine VI, edited by Tuan Vo-Dinh, Joseph R. LakowiczProc. of SPIE Vol. 7192, 71920U · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.809316

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Gold is a biocompatible material and suitable to stimulate collective electron oscillation in combination with laser radiation. For example, the plasmon resonance wavelength for a small single gold sphere (<50nm) is λabs≈ 520 nm but this can shift to higher wavelength if the size or the number of absorbing particles changes [17-20]. Using two photon absorption, the relatively high absorbance of gold nanoparticles between 350 nm - 450 nm can be employed. Combined with this plasmonic effect the field enhancement at the surface of the particle can be used to ablate material in the nanometer regime, as shown recently by Eversole et al. [21]. This group found, that especially 150-160 nm size gold particles exhibit a very strong scattering of light into the near field. Similarly, Imura et al. found high near field emission at near infrared wavelength from 100 nm gold particles [22]. For this, the wavelength, particle size and the number of particles are the fundamental factors. Therefore, fs laser systems with infrared wavelengths around 800 nm might be suitable instruments to stimulate plasmon resonances and near field emission in clusters and nanoparticles of larger size.

In the following we present nanoparticle mediated cell perforation by ultrashort laser pulses as an alternative transfection tool. Previous approaches to perforate the cell membrane employing nanoparticles and laser radiation were demonstrated by Yao et al. [23] using laser at wavelengths near the plasmon resonance and longer laser pulses. It was shown that when conjugated 15 nm and 30 nm gold particles were irradiated with 532nm laser light, a change in membrane permeability can be achieved and can be used to label cells with Propidium Iodide (PI) and Isothicoyanate (FITC). The reached perforation efficiency in this study was up to 68 % with 27 % of dead cells. Another work from Pitsillides et al. used similar parameters for 20 nm gold particles to successfully permeabilize the membrane [24]. Based on this and our preliminary work [13] we aimed at combining the effects of low energy fs-laser pulses and nanoparticles mediated permeabilization to transfer large molecules (DNA expression plasmids) as well as dye molecules into the cell. For this, the influence of laser fluence and concentration of the gold nanoparticles on the transfection efficiency were studied.

MATERIAL AND METHODS 2.1 Laser system

A Ti:Sapphire laser (Spectra-Physics, Spitfire Pro II) with a tunable repetition rate of 1 to 5 kHz was used for our experiments. The generated laser pulses had a pulse duration of 120 fs and a maximum output power of 2.5 W which was controlled by a tunable filter wheel. Another Ti:Sapphire laser system (Coherent, Chameleon ultra II) was used for multiphoton imaging (see section 2.5).

2.2 Nanoparticles

For our experiments we used 80 nm and 150 nm gold particles (Kisker GmbH). We used a laser wavelength of 700-900 nm to induce plasmon resonance at the surface of a nanoparticles as the result of two photon absorption. The absorbance is about 0.38 between 350 and 450 nm for 150 nm particles and roughly 0.5 in the same range for 80 nm particles. It is also expected that strong near-field scattering by the particles would lead to field enhancement at their surface in the near infrared wavelength range [21].

2.3 Experimental setup and manipulation principle

To target the laser radiation onto the sample a positioning system for x, y, and z direction (Aerotech, Inc.) was used. This axis system was implemented into a computerized numerical control machine (CNC) (3D Micromac®, Kugler) allowing to control the process velocity and scanning distance via a computer. The laser beam was guided from the laser system through a shutter (Thorlabs, SC10), into the CNC machine. An IR coated lens with a focal length of 140 mm was weakly focusing the laser beam onto the samples (see Fig. 1(a)). By moving the sample at different velocities relative to the laser beam in x and y direction we scanned an area of 3 mm2 with a spot size of 30 µm. The distance in y direction between the lines was 30 µm such as the spot size of the laser focus.

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y

x

f= 140 mm

foreign DNA (GFP)

weakly focused

NIR fs laser pulses

Fig. 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 near field enhancement the cell membrane is disrupted.

2.4 Cell preparation

GFSHR-17 granulosa rat cells were cultivated in glass-bottom-dishes (MatTek) in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 5% fetal calf serum (FCS), penicillin, streptomycin and partricin. For nanoparticle mediated laser perforation, 80 nm and 150 nm gold particles were added to the surrounding cell cultivation media and incubated for 6 hours. The cells were washed with PBS (phosphate buffer saline) and relabelled by 1µM PI.

To prove the success of transfection a non-recombinant pEGFP-C1 vector plasmid and a recombinant pEGFP-C1-HMGB1vector plasmid were added respectively to the medium at a concentration of 50µg/ml. The non-recombinant vector labels the complete cell and the recombinant vector labels specifically the nucleus in case of successful transfection. Before starting of the manipulation the cells were incubated with 80 nm and 150 nm gold nanoparticles respectively for 6 hours and then washed with PBS.

2.5 Multiphoton microscopy and nanoparticle localization

Multiphoton microscopy images were taken to observe the uptake, location and the possibility of a passive bonding of the particles. Therefore, the granulosa rat cells were incubated at 37°C with 150 nm gold particles for either 6 h or 24 h, allowing the nanoparticles to attain close to the cell membrane. Thereafter, the cells were washed with PBS. An incubation in 20 µM FM4-64 or 8 µM HOECHST for 15 minutes followed to label the membrane or the nucleus respectively. After a washing with PBS, the cells were observed using a multiphoton microscope. This microscope was operating with a fs laser system (Chameleon ultra 2, Coherent GmbH) tunable from λ= 690 nm to 1040 nm. The imaging was performed by using a 100x oil objective (Carl Zeiss AG, “Plan-Neofluar”, NA=1.3).

2.6 Nanoparticle mediated laser cell perforation and transfection

For nanoparticle mediated laser perforation a weakly focused laser beam was targeted onto the sample with the cells and the gold particles. Directly after manipulation the cells were washed with PBS. After this, we observed the uptake for the normally membrane impermeable dye, propidium iodide (PI), into the cells by detection of the fluorescence using a fluorescence microscope (Carl Zeiss AG, Axiovert 200). Following this, cells were washed, incubated for a time of 1.5 h at 37°C. Afterwards, the viability of the cells was tested by PI again [13].

For the transfection experiments we weakly focused the laser beam onto the sample and an area of 3 mm2 was treated, as described above. A non recombinant GFP expression vector or a recombinant expression vector coding for a GFP fusion protein, with the HMGB1 protein for specific nucleus labelling, were used, respectively. Particles of 80 nm and 150 nm size were irradiated to treat the cells. After washing the cells with PBS again we incubated them for 24 h and monitored the expression of the DNA by the fluorescence microscope.

a) b)

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RESULTS 3.1 Visualization of the location of nanoparticles near the cell membrane

Figure 2 shows the HOECHST 33342 stained cell nuclei of GFSHR-17 granulosa cells. The two-photon excitation wavelength for this dye is in the range between λexc= 600 nm and 750 nm. With a fs laser we stimulated the fluorescence at an excitation wavelength of λexc= 700 nm and took images of the cell nuclei (Fig. 2(a)). Images of cells which were incubated for 24 h with 150 nm gold nanoparticles were taken additionally and presented in Figs. 2(c) and 2(e). The incubation time was near the time period (24 h) which was used from Tsai [20] to allow the particles getting close to the cells. The nanoparticles are visible around the cell nucleus due to the plasmon resonance stimulated at λexc= 700 nm by two-photon absorption. To verify the presence of the nanoparticles we switched the excitation wavelength to λexc=900 nm which is out of the excitation range of HOECHST 33342. As shown in Figs. 2(d) and 2(f) the nuclei are not visible anymore but the nanoparticles are still present (marked by the arrows). Multiphoton microscopy allows a separation between the signal of the fluorescent dye and the nanoparticles.

GFSHR-17 cells

GFSHR-17 cells

with gold nanoparticles

GFSHR-17 cells

with gold nanoparticles

λexc.=700 nm / λexc.= 900 nm

Fig. 2. Multiphoton microscopy images of granulosa cells, the cell nuclei are labeled with a Hoechst 33342 fluorescent dye

(λexc= 650-700 nm). (a, d) cells without nanoparticles at different excitation wavelength (b, c) cells incubated with gold nanoparticles for 24 h and imaged with an excitation wavelength of 700 nm (e, f) incubated cells (24 h) with nanoparticles. The excitation wavelength was changed to λexc.= 900 nm, so that only the nanoparticles are visible (highlighted by the arrows).

a) b)

c) d)

e) f)

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To verify whether the nanoparticles are located at the surface of cells, we labeled the cell membrane using a FM4-64 fluorescent dye to observe their exact location and to get a better insight in the “bonding” between the gold nanoparticles and the cells. Figure 3(a) shows multiphoton microscopy images of the cell membrane of granulosa cells without nanoparticles. In fig. 3(b) nanoparticles were added to the cell media and incubated for 6 hours. The images show bright spots near the cell membrane which represent clusters of nanoparticles. Furthermore the images prove that the nanoparticles are attached near the cell membrane in less than 6 hours.

Fig. 3. Multiphoton microscopy images of granulosa cells. The cell membrane is labelled with a FM4-64 fluorescent dye

(λexc= 800 nm). (a) incubated cells without nanoparticles. (b) Cells were incubated for 6 h with nanoparticles. The nanoparticles are visible near the cell membrane.

3.2 Nanoparticle mediated laser cell perforation

In analogy to the imaging experiments we prepared the cells for membrane perforation. As a proof of principle experiment we used PI as a maker for perforated cells, to monitor the cell membrane permeability due to the laser-nanoparticle interaction. The chosen fluences were far below the laser induced optical breakdown threshold in water of 1.25 J/cm2 [15] to ensure, that no large scale breakdown at the 30 µm focus occurred and that the perforation of the cells is not due to laser induced shockwaves emanating from the breakdown region, as for example used in the work of Soughayer or Yamaguchi [25,26]. After the manipulation PI molecules have entered the granulosa cells (see Fig. 4).

Fig. 4. Perforated granulosa cells using plasmon resonances induced in 150 nm gold particles due to a laser fluence of 0.3

J/cm2. Cells are growing on a coverslip in a 1.5 µM PI solution. a) bright field image. Perforated cells are highlighted with dashed lines. b) fluorescence image, taken 1,5 h after laser exposure.

3.3 Nanoparticle mediated laser cell transfection

Transfection was performed either with a non-recombinant pEGFP-C1 vector or a recombinant pEGFP-C1-HMGB1 vector. The pEGFP-C1 vector labels the whole cell when successfully transfected into the cells. The HMGB1 protein is an architectural transcription factor naturally located in the nucleus, therefore successful transfection with pEGFP-HMGB1 vector specifically results in labeling the nucleus. The nucleic pEGFP-HMGB1 staining proves that the cell is still able to synthesize proteins after the laser exposure and process them correctly. All parameter combinations lead to successful transfection. The transfection using the different vectors shows, that the functionality of the DNA was not disturbed (see Fig. 5).

20 µm 20 µm

a) b)

a) b)

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Fig. 5. Cells subjected to nanoparticle mediated laser transfection and observed 28 h to 48 h after transfection. The scanning

velocity in this experiment was about 50 mm/min. (a) GFP positive cells had been transfected with a laser fluence of 0.8 J/cm2 using 150 nm gold nanoparticles. (b) GFP cell transfection using 80 nm gold particles and a laser fluence of 0.3 J/cm2. (c) GFP- HMGB1 transfection using a laser fluence of 0.4 J/cm2 and a particle size of 150 nm.

3.4 Control experiments

To prove that the cell transfection takes place due to the laser-nanoparticle interaction we performed two different control experiments. In the first one we illuminated the cells of six dishes with the laser without the use of any nanoparticles. The results show, an overall of three cells were transfected in the six control measurements which leads to 0.5 ± 0.84 transfected cell per dish. For the second control experiment, we incubated the cells in two dishes with 150 nm gold particles and the pEGFP-C1 vector for approximately 15 minutes without laser activation. Samples were then washed with PBS and observed 24 h later. No cells were transfected by this treatment.

3.4 Transfection rate in dependence of laser fluence

A critical parameter with respect to the viability of the targeted cells and thus of the achieved transfection efficiency is the laser fluence. We performed the transfection experiments in presence of 80 nm particles and and 150 nm particles. In case of 80 nm particles it seems like the number of transfected cells goes to saturation at a laser fluence of 0.2 µJ. For 150 nm particles the number of transfected cells rises with increasing laser fluence.

0

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30

40

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60

0 0.1 0.2 0.3 0.4

laser fluence [J/cm^2]

num

ber o

f tra

nsfe

cted

cel

ls

80 nm150 nm

Fig. 6. Total number of transfected cells after 6 measurements [18mm2] for different laser fluences.

3.5 Transfection rate in dependence of nanoparticle concentration

As a further important factor for nanoparticle mediated laser cell transfection we analyzed the dependence of the nanoparticle concentration and the laser fluence with respect to the number of transfected cells. Therefore we incubated the cells with different concentrations (up to 2.8 µg/ml) of gold nanoparticles of about 80 nm size and applied laser fluences between 0.2 and 0.6 J/cm2. The treated cells were observed 28 hours after laser exposure. We found that the number of transfected cells is increasing with an increasing concentration of nanoparticles. As an example, the highest number of transfected cells was found to be 40 cells in a dish at a concentration of 2.8 µg/ml. This leads to a roughly estimated transfection rate of 1%.

20 µm 20 µm 20 µm

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CONCLUSION AND DISCUSSION In our study, we investigated the potential of plasmon-resonance based cell perforation in combination with fs laser pulses as an alternative optical transfection method. The presented multiphoton images show a specific localization of nanoparticles near the cell membrane. We assume that the nanoparticles stick to the membrane and the membrane forms a cavity to enclose the particles as described by Tsai et al. [20]. The results show that a bonding between gold nanoparticles and cells is possible even without the use of any linker such as antibody or other particle coating for specific binding to the cell membrane. This can be an advantage considering the complex particle preparation or the binding efficiency between linker and particle. Moreover, the use of linker such as antibodies can be limiting and expensive. However, special conjugated particles may help to increase the transfection efficiency as shown by Yao et al. [23] and can be useful in situations where cell specific transfection is needed e.g. specific transfection of tumour cells within a mixed culture or even in tissue.

With the uptake of PI into the cell we can observe the membrane perforation directly because for living cells, the intact membrane is impermeable for PI molecules. We checked the vitality by restaining the cells with PI. In case of successful membrane perforation, cells are fluorescent under the microscope due to the uptake of the dye. Further we showed that even larger molecules such as foreign DNA are able to diffuse into the cell as a result of nanoparticle mediated laser perforation (Fig 5). Since we observed the protein products of DNA introduced into the cells, we can conclude that the laser treatment is not damaging the DNA, that the distortion of the membrane by the nanoparticle induced breakdown is reversible and that the cells are able to synthesize and process recombinant proteins after treatment and are still vital 24 h after laser exposure.

To establish the nanoparticle mediated laser transfection as an alternative transfection method the number of transfected cell has to be increased. In comparison with other opto-perforation methods the transfection efficiency is quite low. Until now only an estimated transfection rate of roughly 1% could be achieved. Further studies which evaluate the cell viability and transfection efficiency have to be done. An advantage of the nanoparticle mediated cell perforation is the possible high throughput. In comparison to other fs-optoperforation methods, this method has the potential to treat more cells simultaneously. In our particle concentration experiments we could show that the number of transfected cells increases with an increasing concentration of gold nanoparticles and might therefore be improved by higher particle concentrations. Contrarily we have to consider that a higher concentration of gold particles may have negative side effects on some cell types.

The mechanism of nanoparticle mediated cell perforation is not fully investigated yet. There are several effects that occur during the interaction of laser light and gold nanoparticles (GN) in context with living cells. The main effects are: selective thermal denaturation of proteins around GN, thermal expansion of GN, water vaporation and bubble formation, melting, evaporation of GN, optical breakdown and explosion with fragmentation of GN [27].

Our studies about the cell perforation using nanoparticles showed that this method is very promising for cell transfection. The used parameters, as nanoparticle concentration, particle size as well as the laser fluence and the wavelength still have to be optimized in further studies, however, the nanoparticle mediated cell perforation has the potential to compete with the conventional transfection methods offering a better alternative in terms of a gentle cell treatment, high throughput and high efficiency. Nevertheless, more detailed studies about underlying mechanisms, transfection efficiencies and cell viability have to be done.

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ACKNOWLEDGMENTS

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

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