|
1.IntroductionThere are many situations in medicine and biology when it is desirable to introduce a macromolecule into the cytoplasm of mammalian cells. One important application is gene therapy, where it is necessary to deliver genes or synthetic oligonucleotides into the cell. A number of methods involving chemical, viral, or physical approaches have been developed to transfer DNA and other macromolecules into mammalian cells, although each method has limitations.1 Also, the transfer of antibodies into cells has interesting applications such as the staining of intracellular structures in living cells. With the constant progress in laser technology and laser applications many scientists turn their eyes to pulsed laser systems, using them for biotechnology. Currently three laser-based methods for gene transfer have been reported: microinjection, laser-induced stress waves, and selective cell targeting with light-absorbing particles. Microinjection was developed2, 3 in the 1980s, but because this method uses focused microbeams that requires targeting each cell individually, this approach is both slow and limited to cell culture. It has not yet become a commonly used technique. There have been many reports on the application of laser-induced stress waves (LISWs) to drug delivery,4, 5, 6, 7 but this method was capable of delivering only small drug molecules. Terakawa recently reported the successfully delivery of plasmid DNA to mammalian cells by application of nanosecond pulsed laser-generated stress waves and an elevated temperature.8 Other studies have shown that laser-irradiated micro- and nanoparticles can selectively destroy cells or proteins.9, 10, 11 Pitsillides 12 and Umebayashi 13 reported an introduction of exogenous materials into the cytoplasm of living cells by irradiating gold nanoparticles or latex microparticles, respectively. Gold nanoparticles, which are extensively used as immunoconjugates in electron microscopy, strongly absorb visible light. At their absorption peak around , the absorption cross section can exceed the geometrical cross section, which enables an efficient heating of the particles by pulsed lasers to more than . To achieve thermal confinement to a radius of less than in water, the pulse duration should be9 shorter than . Compared to microparticles, the smaller size of gold nanoparticles makes them more suitable for tissuelike samples or in vivo applications. Pitsillides 12 were able to show the up-take of a dextran to T lymphocytes, which were isolated from fresh blood, with particle diameter. However, slightly larger particles with diameters either showed no effect or nearly complete cell killing. In that study, the experimental conditions were not varied in a systematic way and no numbers were given for the fraction of permeabilized cells. Additionally, rather long pulses that are commonly not available with -switched lasers were used. Therefore, the first report on nanoparticle-mediated cell permeabilization leaves open the question of how generally this method can be used to permeabilize cells and what efficacy can be achieved. Based on that work, we systematically studied the ability of the nanoparticles to inflict the plasma membrane permeability without causing cell death in the two established cell lines L428 and Karpas 299 with different particle sizes, pulse energies, and particle densities. The L428 line is a long-term tissue culture line of Reed-Sternberg cells that was originally established from primary cells of a patient with Hodgkin’s disease.14 The cells are positive for CD30, a member of the tumour necrosis factor receptor (TNFR) superfamily, which is found on Hodgkin/Reed-Sternberg (H/RS) cells, as well as on activated T and B lymphocytes. The human large-cell anaplastic lymphoma cell line Karpas 299 was established from blast cells in the peripheral blood of a -old white man.15 Karpas 299 is positive for CD30 and CD25, which is the interleukin-2 receptor alpha chain. CD25 is expressed by early progenitors of T and B lineage as well as by activated mature T and B lymphocytes. The different structures of the both antigens might result in differing efficacies with which macromolecules are transferred into the cells. The degree of membrane permeabilization and the viability of cells were determined by flow cytometry, which measured the uptake of a fluorescing dextran (FITC-D) and propidium iodide (PI) to test permeabilization and cell death, respectively. 2.Materials and Methods2.1.ApparatusFor all experiments sample irradiation was performed with a -switched frequency doubled Nd:YAG laser (Surelite I, Continuum, California, USA), which generates pulses at , in wells with a diameter of , which were custom-made in a slide of optical glass (Hellma). Each of the 18 wells took a sample volume of . The laser gave stable output energy with 10% standard deviation and a bell-shaped intensity distribution. The laser beam had a diameter of approximately and the samples were irradiated with an unfocused beam. The radiant exposure in the center of the beam was for pulse energy and scaled linearly with the pulse energy. Either single pulses or multiple pulses at a frequency of were used. Permeabilization of the plasma membrane and cell death were probed by fluorescence staining with FITC-D (molecular weight of , Molecular Probes) or PI, respectively. Fluorescence was quantified by a flow cytometer (FACSCAN, Beckman Coulter), in which the fluorescence of PI and FITC-D was excited by an argon ion laser emitting at . Green fluorescence (for FITC-D) and red fluorescence (for PI) were collected using bandpass and longpass filters, respectively. Fluorescence data were stored and processed with WinMDI 2.8 software. A total of 5000 cells were examined in each sample. Results were confirmed by fluorescence microscopy (OLYMPUS BH2-RFL-T2), and the fluorescence images were recorded using a charge-coupled device (CCD) camera. 2.2.Cell PreparationHodgkin’s disease cell line L428 and the human lymphoma cell line Karpas 299 were kindly provided by Prof. Horst Dürkop, Institute for Pathology, Charité, Campus Benjamin Franklin, Berlin. Cells were routinely grown in suspension culture in a RPMI (Roswell Park Memorial Institute) 1640 with Hepes and L-Glutamine medium supplemented with 10% fetal calf serum, antibiotic/antimycotic solution (all cell culture media PAA Laboratories, Pasching, Austria) in a humidified incubator (5% , 95% air). Cells at logarithmic growth phase were spun down at for at and then resuspended in phosphate-buffered saline (PBS) with different cell densities ( and ). Immunogold particles, which were made from colloidal gold with diameters of 15 and (British Biocell International) and the antibodies BerH2 against CD30 (Ref. 16) and ACT1 against CD25 (Ref. 17), were then added to the cell suspension at certain ratios of gold nanoparticles to number of cells. The mixture of cells and particles was incubated for at . Immediately prior to irradiation, FITC-D was added to test membrane permeabilization. The amount of gold bound to the cells was estimated by absorption measurements at (Lambda 14, Perkin Elmer, Überlingen, Germany). The mixture of cells, nanoparticles, and FITC-D was then transferred to an 18-well slide. At least after irradiation, during which the 18-well slide was kept in an incubator, the cells were washed with PBS and resuspended in PBS containing PI to assay cell death. Subsequently, the fluorescence of the cells was measured by flow cytometry. 3.ResultsIn this study, we used two cell lines (L428 and Karpas 299) and four different types of conjugates made from two different-sized gold nanoparticles (15 and ) and the two antibodies BerH2 and ACT1, which recognize CD30 and CD25 positive cells, respectively. Both cell lines were targeted by gold conjugate with the BerH2 antibodies. Additionally, ACT1 gold conjugates were used with Karpas 299 cells, which binds both antibodies. Therefore the influence of the cell type and the target receptor could be separated. Different numbers of pulses and pulse energies were used to find the optimal conditions for permeabilization of the plasma membrane. By comparing two different cell lines, the universality of this approach was confirmed. 3.1.Permeabilization of L428 and Karpas 299 with BerH2 Gold ConjugateIn the first experiment, single pulses were used and the influence of the pulse energy on permeabilization efficacy was tested in both cell lines with BerH2 conjugates. The cell density was . In the dot plot (Fig. 1 ) three subpopulations of the cells were seen as follows: intact viable cells exhibited no FITC-D and no PI fluorescence; transiently permeabilized cells containing only FITC-D but no PI; and permanently damaged or dead cells, which were positive for FITC-D and/or PI that was added at least after irradiation. Figure 1 shows the results for the Karpas 299 cell line with BerH2 gold conjugates. From the dot plots, the fluorescence thresholds for FITC-D were chosen in such a way that in the control experiment less than 5% were positive for FITC-D. The threshold for the PI fluorescence lay half between the average fluorescence of viable and damaged cells. With these thresholds, the fractions of permeabilized and dead cells were calculated as shown in Fig. 2 for different pulse energies. Both the fraction of transiently permeabilized cells (lower right square) and damaged or dead cells (upper right square) increased with pulse energy. As expected from the lower absorption and the stronger effect of heat diffusion, particles required more energy than particles for permeabilization or cell killing. The effect of multiple pulses was measured with conjugates at single pulse energy of (Fig. 3 ). No significant dependence on the number of applied pulses was found when more than five pulses were applied. FITC-D and PI fluorescence increased with the number of pulses only for the range of one to five pulses. 3.2.Permeabilization of Karpas 299 Cell with ACT1 Gold ConjugatesBecause Karpas 299 cells were easier to permeabilize and could be targeted by both antibodies, further experiments concentrated on this cell line. To determine the optimal conditions for the transfer of FITC-D into the cells, energy and pulse numbers were changed simultaneously. The cell density was , and the ratio of gold particles to cells remained unchanged. The results are shown for 15- and particles in Fig. 4 . Both the number of permeabilized cells and dead cells first increased with the pulse energy and number of applied pulses. More permeabilized than dead cells were observed at appropriate conditions. When the energy and the number of the pulses exceeded a certain value, the fraction of permeabilized cells decreased and the fraction of dead cells increased strongly. The particles were more efficient for permeabilization than the particles, which was in agreement with the experiments using BerH2 immunogold particles. 3.3.Influence of the Ratio of the Gold Concentration During Incubation on the Membrane PermeabilizationTo investigate the effect of the numbers of particles bound to the cells on the transient cell membrane permeabilization, we used different concentrations of particles and cells during the incubation. With an increased ratio of particles to cells, the percentage of dead cells increased, whereas the fraction of successfully permeabilized and resealed cells depended on the gold concentration in a form that resembled a normal distribution (Fig. 5 ). 3.4.Time for the Resealing of the Cell MembraneTo study the time at which the cell membrane reseals, Karpas 299 cells were irradiated and the PI was added at different times after the irradiation (Fig. 6 ). As expected, nearly all cells are PI positive when this comparatively small molecule is added directly after irradiation. As the time at which PI was added was increased, the fraction of PI-positive cells decreased and the number of FITC-positive cells increased. After , the cell membrane was completely closed for PI uptake. 4.Conclusion and DiscussionThe presented study shows that the combination of gold-antibody conjugate, which binds to the cell membrane, with laser light irradiation can be used to increase the membrane permeability. A molecule, which normally does not cross the cell membrane, can be transferred into the cells. This study with two cell lines investigated the effects of different energy levels, numbers of laser pulses, particle diameters, and cell:particle ratios on transient plasma membrane permeabilization to determine optimal conditions (Table 1 ). Table 1The best efficacies for the transfer of 10kDal FITC-D into Karpas 299 and L428 cells with the corresponding irradiation parameters.
In both cell lines and with both particle diameters, a transfer of FITC-D into the cells was possible. Efficiencies of more than 26% were reached for all combinations at optimized irradiation parameters, except for nanoparticles and L428 cells. An increase of pulse energy, number of pulses, or loading of the cells with gold particles beyond the optimal parameters invariably caused cell death. Small particles required higher pulse energies due to smaller absorption and higher loss of energy by heat conduction during the laser pulse. Permeabilization was easier with the Karpas 299 cell line than with the L428 cell line. Finally, experiments with ACT1-gold conjugate provided better results. In contrast to Pitsillides, 12 who were not able to use particles successfully, we showed that both 30 and gold particles could achieve membrane permeabilization, and the particle even gave better results. Under optimal conditions 68% of the cells were loaded with FITC-D. Therefore, we conclude that no dramatic dependence on particle size exists when experimental conditions are adjusted correctly. From a synopsis of all our experimental results, we conclude that permeabilization by the nanoparticle system is connected with an increase of cell death. Deviation from the optimal pulse energy or particle concentration by 50% reduces the transfer efficacy for FITC-D significantly and damages the cells to a large extent. Permeabilization seems to be a sublethal effect of the nanoparticles on the cells and a careful optimization of all parameter seems to be necessary. Beside the different cell system and the three times longer pulse width, the deviations of these results from the work of Pitsillides 12 could also be caused by aggregation of gold particles and the temporal structure of the laser pulses, which both influence the reproducibility of the experiments. Aggregation of particle effectively enlarges the particle size, which increases the biological effect of the particles, as can be seen from our experiments with 15- and -sized particles and from temperature calculations.18 Aggregation may have occurred when the antibodies were conjugated to the particles, although we tried our best to optimize the protocols. It may also result because of a nonisotropic distribution of the receptors to which the particles bind on the cell surface. Due to the statistical interference of the longitudinal modes in the -swiched Nd:YAG laser, which was used for irradiation, the laser pulse consists of a series of very short spikes, which change in a statistical fashion. This effect is common to all -switched lasers that are not single longitudinal mode. The spikes cause statistical temperature variation on the surface of the gold particles, which may to influence our results, especially with single pulses. There are three possible reasons for the disparity in ACT1 or BerH2 gold conjugate performances. First, the number of particles bound to the cells is different. The amount of the antigen CD30 on the Karpas 29919, 20 is as almost twice that on the L428, which was confirmed by absorption measurements. Second, different distribution of the receptors on the cell surface, and finally, a difference in structure of the receptor (CD25 and CD30) or position of binding site of the antibody21 could be responsible. The mechanisms of the nanoparticle-based techniques for cell permeabilization is still unclear. In any case, a high local temperature is created in and around the particles during membrane permeabilization. Estimates of the temperatures11, 12, 18 for our experimental conditions give a temperature increase of when particles are irradiated with . The particles are heated to . These temperatures are high enough to evaporate water in a layer around the particles, which creates a rapidly expanding bubble. Such bubbles with submicrometer diameters around gold nanoparticles were experimentally observed under laser irradiation.22, 23 Additionally, the gold melts24, 25 and breaks up into smaller particles. Particle fragmentation was observed at a radiant exposure starting from for particles.26, 27 Since under nanosecond irradiation the particle temperature scales with the square of the particle diameter, at fixed radiant exposure, the melting temperature will not be reached for particles below a certain diameter. Therefore, after a certain number of pulses, no further effect is expected when all particles are fragmented. This effect explains the comparably weak dependence of permeabilization and cell death on the number of pulses. Cavitation bubbles were also proposed by Pitsillides as the mechanism for cellular effects caused by laser-irradiated nanoparticles.12 Although bubble formation does certainly occur under our irradiation conditions and the spatial extend of the bubbles23 is expected to be larger than the volume that is directly heated by the particle,12 we cannot rule out the possibility that a direct damage to the targeted protein also contributes to membrane permeabilization or cell killing. The effects of laser-irradiated gold particles on the cell membranes seem to be more drastically than in the laser induced stress waves (LSWs) experiment. Membrane permeabilization caused by LSWs recovered completely28 within after LSW. With nanoparticles, the permeablilization for PI persisted up after light irradiation (Fig. 6). Therefore the mechanism involved in the permeabilizition and recovery of the plasma membrane caused by LSW may be different from those involved in our experiment. One advantage of the current approach over LSW is that the effects of the irradiation are mainly localized to cells to which the particles are bound, thereby enabling a selective permeabilization. In summary, this paper describes a technique that can be used to transfer relatively small exogenous molecules into cells. Immunogold particles were bound to membrane proteins and introduced into living cells by irradiation with nanosecond pulses transiently permeabilizing the plasma membranes for exogenous macromolecules. The described technique shows many advantages including high efficiency (68% permeabilized cells and 27% dead cells). Furthermore, our recent experiments show that larger molecules such as fluorescent antibodies can be transferred into L428 and Karpas 299 cells by this method, labeling internal structures. Future experiments are under way to improve the procedure for a transfer of antibodies and the transfection with genetic material. AcknowledgmentsWe thank Heyke Diddens, Barbara Flucke, Margit Kernbach, and Astrid Rodewald for their help during experiments. This work is supported by the National Nature Science Foundation of China (Grant No. 60178034 and No. 60378018) and the German Ministry for Education and Research (13N8461). ReferencesN.-S. Yang,
“Gene transfer into mammalian somatic cells,”
Crit. Rev. Biotechnol., 12 335
–356
(1992). 0738-8551 Google Scholar
S. Kurata and
Y. Ikawa,
“Novel method for substance injection into the cell by laser beam—a study of the injection volume,”
Cell Struct. Funct, 11 205
–207
(1986). 0386-7196 Google Scholar
S. Kurata,
M. Tsukakoshi,
T. Kasuya, and
Y. Ikawa,
“The laser method for efficient introduction of foreign DNA into cultured cells,”
Exp. Cell Res., 162 372
–378
(1986). https://doi.org/10.1016/0014-4827(86)90342-3 0014-4827 Google Scholar
T. Kodama,
M. R. Hamblin, and
A. G. Doukas,
“Cytoplasmic molecular delivery with shock waves: importance of impulse,”
Biophys. J., 79 1821
–1832
(2000). 0006-3495 Google Scholar
G. Jagadeesh,
K. Takayama,
A. Takahashi,
J. Kawagishi,
J. Cole, and
K. P. J. Reddy,
“Micro-particle delivery using laser ablation,”
785
–788
(2001). Google Scholar
M. Ogura,
S. Sato,
M. Kuroki,
H. Wakisaka,
S. Kawauchi,
M. Ishihara,
M. Kikuchi,
M. Yoshioka,
H. Ashida, and
M. Obara,
“Transdermal delivery of photosensitizer by the laser-induced stress wave in combination with skin heating,”
Jpn. J. Appl. Phys., Part 2, 41 L814
–L816
(2002). https://doi.org/10.1143/JJAP.41.L814 0021-4922 Google Scholar
T. Kodama,
A. G. Doukas, and
M. R. Hamblin,
“Delivery of ribosome-inactivating protein toxin into cancer cells with shock waves,”
Cancer Lett., 189 69
–75
(2003). 0304-3835 Google Scholar
M. Terakawa,
M. Ogura,
S. Sato,
H. Wakisaka,
H. Ashida,
M. Uenoyama,
Y. Masaki, and
M. Obara,
“Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave,”
Opt. Lett., 29 1227
–1229
(2004). https://doi.org/10.1364/OL.29.001227 0146-9592 Google Scholar
R. R. Anderson and
J. A. Parrish,
“Selective photothermolysis: Precise microsurgery by selective absorption of pulsed radiation,”
Science, 220 524
–527
(1983). 0036-8075 Google Scholar
D. Leszczynski,
C. M. Pitsillides,
R. K. Pastila,
R. R. Anderson, and
C. P. Lin,
“Laser-beam-triggered microcavitation: a novel method for selective cell destruction,”
Radiat. Res., 156 399
–407
(2001). 0033-7587 Google Scholar
G. Huettmann,
J. Serbin,
B. Radt,
B. I. Lange, and
R. Birngruber,
“Model system for investigating laser-induced subcellular microeffects,”
Proc. SPIE, 4257 398
–409
(2001). https://doi.org/10.1117/12.434739 0277-786X Google Scholar
C. M. Pitsillides,
E. K. Joe,
X. Wei,
R. R. Anderson, and
C. P. Lin,
“Selective cell targeting with light-absorbing microparticles and nanoparticles,”
Biophys. J., 84 4023
–4032
(2003). 0006-3495 Google Scholar
Y. Umebayashi,
Y. Miyamoto,
M. Wakita,
A. Kobaryashi, and
T. Nishisaka,
“Elevation of plasma membrane permeability on laser irradiation of extracellular latex particles,”
J. Biochem. (Tokyo), 134 219
–224
(2003). 0021-924X Google Scholar
M. Schaadt,
V. Diehl,
H. Stein,
C. Fonatsch, and
H. H. Kirchner,
“Two neoplastic cell lines with unique features derived from Hodgkin’s disease,”
Int. J. Cancer, 26 723
–731
(1980). 0020-7136 Google Scholar
P. Fischer,
E. Nacheva,
D. Y. Mason,
P. D. Sherrington,
C. Hoyle,
F. G. Hayhoe, and
A. Karpas,
“A Ki-1 (CD30)-positive human cell line (Karpas 299) established from a high-grade non-Hodgkin’s lymphoma, showing a 2;5 translocation and rearrangement of the T-cell receptor beta-chain gene,”
Blood, 72 234
–240
(1988). 0006-4971 Google Scholar
R. Schwarting,
J. Gerdes,
H. Durkop,
B. Falini,
S. Pileri, and
H. Stein,
“Ber-H2: a new anti Ki-1 (CD30) monoclonal antibody directed at s formol-resistant epitope,”
Blood, 74 1678
–1689
(1989). 0006-4971 Google Scholar
R. Schwarting,
J. Gerdes,
J. A. Ziegler, and
H. Stein,
“Immunoprecipitation of the interleukin-2 receptor from hodgkin’s disease derived cell lines by monoclonal antibodies,”
Hematol. Oncol., 5 57
–64
(1987). 0278-0232 Google Scholar
G. Hüttmann and
R. Birngruber,
“On the possibility of high-precision photothermal microeffects and the measurement of fast thermal denaturation of proteins,”
IEEE J. Sel. Top. Quantum Electron., 5 954
–962
(1999). https://doi.org/10.1109/2944.796317 1077-260X Google Scholar
A. F. Wahl,
K. Klussman,
J. D. Thompson,
J. H. Chen,
L. V. Francisc,
G. Risdon,
D. F. Chace,
C. B. Siegall, and
J. A. Francisco,
“The Anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin’s disease,”
Cancer Res., 62 3736
–3742
(2002). 0008-5472 Google Scholar
H. J. Gruss,
N. Boiani,
D. E. Williams,
R. J. Armitage,
C. A. Smith, and
R. G. Goodwin,
“Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines,”
Blood, 83 2045
–2056
(1994). 0006-4971 Google Scholar
L. Dong,
M. Huelsmeyer,
H. Duerkop,
H. P. Hansen,
J. Schneider-Mergener,
A. Ziegler, and
B. Uchsnska-Ziegler,
“Human CD30: structural implications from epitope mapping and modelling studies,”
J. Mol. Recognit., 16 28
–36
(2003). https://doi.org/10.1002/jmr.605 0952-3499 Google Scholar
V. P. Zharov and
V. Galitovsky,
“Photothermal detection of local thermal effects during selective nanophotothermolysis,”
Appl. Phys. Lett., 83
(24), 4897
–4899
(2003). https://doi.org/10.1063/1.1632546 0003-6951 Google Scholar
A. Plech,
V. Kotaidis,
M. Lorenc, and
M. Wulff,
“Thermal dynamics in laser excited metal nanoparticles,”
Chem. Phys. Lett., 401 565
–569
(2005). https://doi.org/10.1016/j.cplett.2004.11.072 0009-2614 Google Scholar
S. Link,
C. Burda,
M. M. B. B. Nikoobakht, and
M. A. El-Sayed,
“Laser photothermal melting and fragmentation of gold nanorods: energy and laser pulse-width dependence,”
J. Phys. Chem. A, 103 1165
–1170
(1999). https://doi.org/10.1021/jp983141k 1089-5639 Google Scholar
A. Plech,
V. Kotaidis,
S. Grésillon,
C. Dahmen, and
G. v. Plessen,
“Laser-induced heating and melting of gold nanoparticles studied by time-resolved x-ray scattering,”
Phys. Rev. B, 70 195423
(2004). https://doi.org/10.1103/PhysRevB.70.195423 0163-1829 Google Scholar
H. Kurita,
A. Takami, and
S. Koda,
“Size reduction of gold particles in aqueous solution by pulsed laser irradiation,”
Appl. Phys. Lett., 72 789
–791
(1998). https://doi.org/10.1063/1.120894 0003-6951 Google Scholar
A. Takami,
H. Kurita, and
S. Koda,
“Laser-induced size reduction of noble metal particles,”
J. Phys. Chem. B, 103 1226
–1232
(1999). https://doi.org/10.1021/jp983503o 1089-5647 Google Scholar
S. Lee,
T. Anderson,
H. Zhang,
T. J. Flotte, and
A. G. Doukas,
“Alteration of cell membrane by stress waves in vitro,”
Ultrasound Med. Biol., 22
(9), 1285
–1293
(1996). https://doi.org/10.1016/S0301-5629(96)00149-4 0301-5629 Google Scholar
|