1.

Introduction

Light-absorbing micro- and nanoparticles, which are bound to cell membranes and irradiated by lasers, have been proven to create highly localized cell damage. By this method, membranes can be permeabilized¹ and our previous results demonstrated the ability to introduce exogenous molecules into mammalian cells using this technique.^{2, 3}

The introduction of exogenous materials into cells of multicellular organisms using different techniques has been proven to be a powerful approach for the study of gene function and their regulation in bacterial, fungi, animal, and plant cells. Any efficient method for gene transfer could be of considerable value in the continuing progress of genetic engineering. The most common methods for gene transfer involve either the viral vector method or nonviral physical methods such as electroporation. The former is widely used because of its efficiency; but problems associated with safety and limited targeting characteristics exist.⁴ Recently, laser-mediated gene transfection has drawn much attention because it is a noncontact and noncontaminating method.^{1, 5, 6, 7, 8}

Laser irradiation parameters, such as irradiance wavelength and pulse duration, and the nature of the absorbing structure are major factors for targeting cells and tissue.⁹ Different kinds of lasers that operate with unique pulse durations targeting specific structures were used to introduce exogenous molecules into cells. Examples include the following: $20\text{-}\mathrm{ns}$ $532\text{-}\mathrm{nm}$ Q-switched Nd:YAG laser pulse absorbed by gold nanoparticles,¹ latex microparticles targeted by a $532.5\text{-}\mathrm{nm}$ Q-switched Nd:YAG laser,⁵ $532\text{-}\mathrm{nm}$ Q-switched Nd:YAG pulse laser irradiating a strongly absorbing layer,^{6, 7, 8, 10} femtosecond $800\text{-}\mathrm{nm}$ titanium-sapphire pulse laser,¹¹ and $cw$ $488\text{-}\mathrm{nm}$ argon laser,¹² which both targeted the cell membrane directly. These different lasers carry out three transfection methods: laser microinjection, laser-induced stress wave, and selective cell targeting with light-absorbing particles.

In previous studies we had shown that a permeability of the cell membrane can be caused by laser irradiation of selectively bound gold nanoparticles. Following irradiation, a relatively small $10\text{-}\mathrm{kDalton}$ fluorescent labeled dextran was able to penetrate into cells. By comparing the results from different cell lines, the universality of this approach was confirmed.² In the present study, we used different laser sources operating at different pulse durations to evaluate their influence on membrane permeability of the human lymphoma cell lines L-428 and Karpas-299. For the first time also, a delivery of a $150\text{-}\mathrm{kDa}$ monoclonal IgG antibody into targeted cells was demonstrated.

2.

Materials and Methods

3.

Results

Three kinds of lasers with different parameters and different irradiation manners were used to irradiate the lymphoma cell lines Karpas-299 and L-428. Four different types of conjugates were prepared from two differently sized gold nanoparticles (15 and $30\mathrm{nm}$ ) and the two antibodies BerH2 and ACT-1, against CD30 and CD25. Using different irradiation modes and different pulse durations, the influence of these parameters could be separated.

First, cells were irradiated with nanosecond single pulses (Surelite I laser) and nanosecond multipulses in scanning mode (Spectron laser). The influence of pulse energy on the permeabilization efficiency was tested on both cell lines with BerH2 and ACT-1 gold conjugates. The results for the Karpas-299 cell line with $30\text{-}\mathrm{nm}$ ACT-1 gold conjugates irradiated by single pulses are shown in Fig. 1 and after irradiation with scanning multipulses in Fig. 2 . A schematic of the scanning pattern is given in Fig. 2f. In the dot plots, three subpopulations of cells can be separated: intact viable cells that exhibited no FITC-D and no PI fluorescence (lower left square), transiently permeabilized cells that contained only FITC-D but no PI (lower right square), and permanently damaged or dead cells that were positive for FITC-D and/or PI, which was added at least $40\min$ after irradiation (upper right square). From the dot plots, a fluorescence threshold for FITC-D was chosen in a way that in the control experiment, less than 5% of cells were positive for FITC-D. The threshold for the PI fluorescence is halfway between the average fluorescence of viable and damaged cells. With these thresholds, the fractions of permeabilized and dead cells were calculated as shown in Figs. 1f and 2e, corresponding to the different pulse energies. Both the fraction of transiently permeabilized cells (lower right square) and the damaged or dead cells (upper right square) increased with increasing pulse energy for both laser sources. Results with the L-428 cell line and $15\text{-}\mathrm{nm}$ conjugates are comparable (data not shown), except that the $15\text{-}\mathrm{nm}$ particles needed slightly more energy than the $30\text{-}\mathrm{nm}$ particles for permeabilization or cell killing.

Fig. 1

$10\text{-}\mathrm{kDa}$ FITC-Dextran uptake by Karpas-299 cells after irradiation with nanosecond single pulses. The cells were incubated with $30\text{-}\mathrm{nm}$ ACT-1 immunogold particles and irradiated with different energies: (a) control, no laser; (b) $90\mathrm{mJ}∕{\mathrm{cm}}^2$ ; (c) $125\mathrm{mJ}∕{\mathrm{cm}}^2$ ; (d) $170\mathrm{mJ}∕{\mathrm{cm}}^2$ ; and (e) $250\mathrm{mJ}∕{\mathrm{cm}}^2$ . The percentages of dead (PI positive) and permeabilized cells are given in (f).

Fig. 2

$10\text{-}\mathrm{kDa}$ FITC-Dextran uptake by Karpas-299 cells after irradiation with nanosecond pulses in scanning mode. 180 pulses per sample were applied with different energies: (a) control, no laser; (b) $64\mathrm{mJ}∕{\mathrm{cm}}^2$ ; (c) $102\mathrm{mJ}∕{\mathrm{cm}}^2$ ; and (d) $153\mathrm{mJ}∕{\mathrm{cm}}^2$ . The percentages of dead cells (PI positive) and permeabilized cells are given in (e). A schematic of the scanning pattern is given in (f). One well (circle) was irradiated with 180 overlapping pulses (dashed lines).

Both irradiation modes showed comparable results and permeabilization efficiencies between 40 to 50%. The number of permeabilized cells and dead cells both increased with pulse energy and number of applied pulses. More permeabilized cells than dead cells were observed under the appropriate conditions, but when energy and the number of pulses exceeded a certain value, the fraction of permeabilized cells decreased and the fraction of dead cells greatly increased.

To investigate the influences of pulse number for single pulse and scanning mode, the cells were irradiated with the same energy but with differing pulse numbers. For single pulse mode, the samples were irradiated with $10\mathrm{mJ}$ and 1-to 50 pulses. With increasing number of pulses, the percentage of dead cells increased, whereas the fraction of successfully permeabilized and resealed cells was optimal after 5 pulses and decreased again after irradiation with more pulses [Fig. 3a ]. For scanning mode, irradiation energy of $100\mathrm{mJ}∕{\mathrm{cm}}^2$ was used, and pulse numbers were multiples of 180 when a whole well was irradiated fully. Samples were irradiated with 180, 360, 540, and 720 pulses. However, differing from single pulse irradiation, the percentage of the dead cells and the successfully permeabilized cells did not increase with the number of pulses [Fig. 3b].

Fig. 3

The percentage of permeabilized and dead cells as a function of pulse number (L-428 cells with BerH2-gold conjugate). (a) Irradiation with nanosecond single pulses of $170\mathrm{mJ}∕{\mathrm{cm}}^2$ . (b) Irradiation with nanoseconds in scanning mode with a maximum pulse energy of $100\mathrm{mJ}∕{\mathrm{cm}}^2$ .

To compare the influence of the different irradiation manners on membrane permeabilization, the results of scanning mode irradiation under optimal conditions are given in Table 1 . The permeabilization efficiencies for the different cell lines are in a range from 35 to 65%. For both cell lines, $30\text{-}\mathrm{nm}$ conjugates show better transfection results than $15\text{-}\mathrm{nm}$ conjugates. These results are very much comparable with the results from single pulse irradiation,² with permeabilization efficiencies ranging between 30 and 70%.

Table 1

The best efficiencies for scanning mode irradiation for the transfer of 10-kDa FITC-Dextran into Karpas-299 and L-428 cells.

To investigate the influence of pulse duration on the transfection efficiency, we compared nanosecond irradiation with the irradiation of a picosecond laser. Both lasers were used for scanning irradiation, and the results are given in Fig. 4 . The $30\text{-}\mathrm{nm}$ conjugates show better permeabilization results than the $15\text{-}\mathrm{nm}$ conjugates when picosecond irradiation is used. The results in L-428 cells with BerH2 gold particles using similar experimental conditions yielded comparable permeabilization rates (data not shown). Based on these results, we conclude that no dramatic dependence on pulse duration exists when the experimental conditions are correctly adjusted for picosecond or nanosecond irradiation.

Fig. 4

Influence of pulse duration on membrane permeabilization and cell death for Karpas-299 cells with $30\text{-}\mathrm{nm}$ BerH2 gold conjugates. Upper diagrams show irradiation with picosecond pulses with radiant exposure of 15, 25, and $40\mathrm{mJ}∕{\mathrm{cm}}^2$ . Lower diagrams show the results of nanosecond irradiation with $64\mathrm{mJ}∕{\mathrm{cm}}^2$ , $102\mathrm{mJ}∕{\mathrm{cm}}^2$ , and $153\mathrm{mJ}∕{\mathrm{cm}}^2$ . All irradiations are performed in scanning mode.

While the experiments with the dextran served for an optimization of the procedure, the cellular uptake of monoclonal IgG antibodies into cells was investigated as one possible application of a targeted transfer of macromolecules. Karpas-299 cells were incubated with $30\text{-}\mathrm{nm}$ Au-BerH2-conjugates, and before irradiation, the Alexa 488 labeled antibody MIB-1 was added. MIB-1 is directed against the nuclear protein Ki-67, which is strongly overexpressed in tumor cells.^{17, 18} To receive a more distinct separation between surviving and irreversible damaged cells, the PI addition and flow cytometer analysis were performed $12\mathrm{h}$ after irradiation. With increasing radiant exposure, also the fraction of permeabilized cells increased. The best rate of permeabilization was reached with an irradiance of $90\mathrm{mJ}∕{\mathrm{cm}}^2$ using a broad nanosecond beam. 50.5% of Karpas-299 cells were positive for MIB-1-Alexa 488 without being permeable for PI. The fraction of permeabilized and PI-positive cells is here 35.5% (Fig. 5 )

Fig. 5

Cellular uptake of Alexa 488 labeled MIB-1 antibody after single pulse irradiation. Karpas-299 cells were incubated with 30-Au-BerH2-conjugates and irradiated after addition of MIB-1 Alexa 488 with nanosecond single pulses with different energies: (a) control, no laser; (b) $35.4\mathrm{mJ}∕{\mathrm{cm}}^2$ ; (c) $53.1\mathrm{mJ}∕{\mathrm{cm}}^2$ ; and (d) $88.5\mathrm{mJ}∕{\mathrm{cm}}^2$ . Addition of PI and flow cytometer analysis was performed $12\mathrm{h}$ after irradiation.

4.

Discussion and Conclusion

Laser-irradiated nanoparticles that are bound to the cell membrane can transiently permeabilize the membrane, allowing macromolecules that normally do not cross the cell membrane to enter the cells. In an earlier study,² we could show a transfer of $10\text{-}\mathrm{kD}$ dextran by the use of antibody gold conjugates, which were irradiated by a nanosecond pulsed laser. In this work we investigated with different lasers and cell lines the effects of different irradiation modes and pulse durations on transient plasma membrane permeabilization to determine the optimal parameters for a targeted transfer of antibodies into cells.

Our experiments showed that for single pulse irradiation with a broad beam, the membrane permeabilization and death of cells will strongly depend on the number of pulses. After the optimal number of pulses (approximately five), the number of dead cells is increasing and the number of permeabilized cells decreasing. On the other hand, with the scanning mode, once a certain number of pulses occurs, further increase of pulse number will have almost no effect on the cells, even if it is as much as four times the initial quantity. When the same sample was irradiated with different pulses in the scanning mode with a longer interval (e.g., one hour), the dead cells increased and the permeabilized cells decreased dramatically. For nanosecond irradiation, both cell lines incubated with both gold particle diameters in both irradiation modes (single pulse mode or scanning mode) could effectively increase the membrane permeabilization. In comparison, the picosecond laser can also induce membrane permeabilization, but the degree of permeabilization is slightly lower, especially for the $15\text{-}\mathrm{nm}$ conjugates.

From all our systemic experimental results, we conclude that all laser parameters, such as pulse duration, irradiation mode, irradiation frequency, and irradiation manner, play important roles in the permeabilization efficiency and death of cells. To determine the corresponding mechanism, it was useful to estimate the magnitude of the transient temperature rise to assess the possibility of the damage.

Estimations of the temperature increase in the particles during irradiation were carried out based on a heat transfer model of a uniformly heated homogeneous sphere embedded in an infinite homogeneous medium developed by Goldenberg and Tranter.¹⁹ For the sake of simplicity, we assumed that the laser intensity was constant during the pulse duration and that the thermal properties of gold and water were independent of temperature. ^{20, 21} The possibility of a phase change in the gold and surrounding water was also not taken into account. The temperature rises and decays at the surface of a $30\text{-}\mathrm{nm}$ particle and surrounding water for $\tau =6\mathrm{ns}$ and $\tau =35\text{-}\mathrm{ps}$ pulse with $1\text{-}\mathrm{mJ}∕{\mathrm{cm}}^2$ radiant exposure are shown in Fig. 6 . The temperature increases for nanosecond irradiances that gave good permeabilization results are around $1000\mathrm{K}$ , whereas picosecond irradiation resulted in two to three times higher temperature increases. Figure 6 a shows the laser-pulse-generated temperature profile, which falls to half its maximum value $10\mathrm{nm}$ from the gold particle surface. For picosecond irradiation, the heat diffusion is even lower [Fig. 6b]; here the temperature profile falls to one quarter of its maximum value at $10\text{-}\mathrm{nm}$ distance. These high and localized peak temperatures are necessary for the development of submicrometer cavitation bubbles around the gold nanoparticles. These bubbles are most likely the reason for a mechanical disruption of cell membrane integrity.

Fig. 6

Temperature change with time at the surface, at 5- and $10\text{-}\mathrm{nm}$ distance from the surface, when $30\text{-}\mathrm{nm}$ gold particles were irradiated by (a) $6\mathrm{ns}$ and (b) $35\text{-}\mathrm{ps}$ laser pulses with $1\mathrm{mJ}∕{\mathrm{cm}}^2$ .

Such bubbles with a submicrometer diameter around the gold nanoparticles were experimentally observed under laser irradiation.^{22, 23} Additionally, the gold melts^{24, 25} and breaks up into smaller particles. Particle fragmentation was observed at a radiant exposure starting at $80\mathrm{mJ}∕{\mathrm{cm}}^2$ for $40\text{-}\mathrm{nm}$ particles.^{26, 27, 28} Because the particle temperature is scaled with the square of the particle diameter under nanosecond irradiation, particles below a certain diameter will not reach the melting temperature. Therefore, after a certain number of pulses, no further effect is expected when all particles are fragmented.

Our experiments showed that different laser parameters can be used to transfer comparable small dextrans into the cells. An adjustment of the laser parameters also allowed antibodies that are more than ten times the size of dextran accumulating into the cells. An especially interesting application of a targeted internalization of antibodies is the chromophore-assisted light inactivation (CALI) of proteins.²⁹ In this approach for functional studies of proteins, irradiated dye antibody conjugates are used to selectively inactivate binding partners of the antibodies. CALI is the only method that gives full temporal and spatial control over the protein inactivation and played for example, a crucial role in functional analysis of the proliferation marker Ki-67.³⁰ Until now, targeting of proteins inside of cells has been hampered by the difficult and often time-consuming transfer of the dye-conjugates into cells, which is most often done by microinjection. The work presented in this study demonstrates that laser irradiation of nanoparticles shows promise to the automation of CALI.

In summary, we show that after adjustment of different irradiation parameters like irradiation energy and mode, a reliable membrane permeabilization is possible, and we show the use of this method to facilitate entry of antibodies into living cells. The fact that permeabilization is possible with different sized particles, different energies, and with nano- as well as picosecond irradiation will allow the use of many different laser systems. In addition to the previous benefits of this technique, it is possible to target entry of macromolecules into specific cells.