4. ANÁLISIS E INTERPRETACIÓN DE LOS RESULTADOS 60
4.1. DISCUSIÓN 84
As already observed in experiments to optimize the incubation time of cells with complexes for magnetofection (figure 71 and 72), five minutes incubation of cells with magnetic particle containing lipoplexes in the presence of a magnetic field (magnetofection) resulted in significantly higher transgene expression than 4 hours incubation with the same lipoplexes without magnetic particles (corresponding conventional or standard transfection). But is magnetofection (in which cells are usually exposed to paramagnetic vectors plus a magnetic field for 30 min or less) always more efficient than the corresponding standard transfection (in which cells are usually incubated with nonmagnetic vectors for 2 to 4 hours)?
To compare the gene transfer efficiency of standard transfections and magnetofections comprehensively (fig. 73), two different cell types (NIH 3T3 and CHO-K1) were incubated for 4 hours with four different standard vectors (PEI-DNA, DNA /PEI / inactivated adenovirus, GenePorter-DNA and Lipofectamine-DNA) and for comparison for 10 minutes with the same vectors but plus magnetic particles and in the presence of a magnetic field (magnetofections). The results showed that with all vector types except GenePorter, the magnetofection method leads to significantly higher gene expression (up to 971-fold) in both cell lines than the corresponding standard transfection. The explanation for the enhanced gene transfer efficiencies with magnetofection is that magnetic sedimentation enables in a short
period of time (here 10 min) more vector-cell contacts than the standard transfection with relatively long incubation times (here 4 hours). This assumption was proven to be true by Gersting et al. (Gersting et al., 2004) who compared magnetofection and standard transfection in regard to adhesion patterns of fluorescently labeled gene transfer complexes on airway epithelial (16HBE) cells by fluorescence microscopy. In contrast to the 3 to 5-fold enhancement in figure 72, magnetofection with GenePorter complexes in figure 73 did not lead to a significant enhancement. This difference may be due to slight variations in transfection parameters such as incubation times during vector preparation, cell density and passage number at the time of transfection. In general, it is assumed that high concentrations of GenePorter-DNA vectors on cellular surfaces lead to saturation of uptake processes or even toxicity and therefore the higher number of vector-cell contacts achieved through magnetic sedimentation do not enhance gene transfer dramatically.
In the experiments mentioned above, the nucleic acid transfer efficiencies of magnetofection and the corresponding standard transfection were only compared at one DNA dose each. The next interesting question is how these two methods compare at different DNA doses. Therefore dose-response profiles of magnetofection and standard transfection with Lipofectamine-DNA complexes in two cell lines (NIH 3T3 and CHO-K1) were established and compared (figure 74 and 75). Over the range of DNA doses tested (from 0.0125 to 0.1 µg DNA/well), with equal DNA doses magnetofection showed always significantly higher gene expression than the corresponding standard transfection. Additionally, magnetofections with lower DNA doses can be more efficient than standard lipofection with much higher doses. For example in CHO-K1 cells (figure 75), magnetofection with an incubation time of 10 minutes achieved with 0.0125, 0.025 and 0.05 µg DNA/well 6.3, 43.2 and 336.8-fold higher gene expression than the standard transfection with 0.1 µg DNA/well and an incubation time of 4 hours. Obviously, even if higher DNA doses and longer incubation times significantly increase the the number of vector-cell contacts by Brownian motion and sedimentation in standard transfections, in some cases with magnetic sedimentation with low DNA doses and shorter incubation times still more vectors get in contact with the cells.
In addition to the experiments presented in this thesis, a number of colleagues from our institute and from other groups compared the efficiency of magnetofection and standard transfection as well. Gersting et al. found that in airway epithelial cells (16HBE cell line and primary cells) magnetofection was, with an incubation time of 15 min, more or at least equally efficient in gene transfer than standard PEI-polyfection with a 4 h incubation time. Further, magnetofection improved the DNA dose-response relationship significantly (Gersting
et al., 2004). Improved transfection efficiencies and DNA dose-response profiles through magnetofection were also observed with the lipofection reagent Metafectene in NIH 3T3 cells (Plank et al., 2003a), with various cationic lipids and PEI in primary human umbilical vein endothelial cells (HUVECs) (Krotz et al., 2003b) and with the lipofection reagent DMRIE in CT26 cells (Plank et al., 2003c). In the latter experiment, Plank et al. further demonstrated that not only the overall transgene expression but also the percentage of transfected cells can be enhanced by magnetofection. In a further publication of Krötz et al. (Krotz et al., 2003a), it was shown that magnetofection with various lipid vectors and PEI does not only improve the transfection efficiencies and dose-response relationships with plasmid DNA but also with antisense-ODNs in HUVEC cells. In these experiments Krötz and coworkers also found that magnetofection with its shorter incubation time is less toxic and therefore a useful tool for physiological examinations in sensitive primary cells. But it has to be mentioned that in contrast to magnetofection with FUGENE plus plasmid DNA (Krotz et al., 2003b), magnetofection with FUGENE plus antisense-ODNs was less efficient than the standard FUGENE transfection. Among the many comparisons performed, this was the only case where magnetofection led to a decrease in transfection efficiency. Additionally to DNA, magnetofection also increased the efficiency of transfections of siRNA. Plank et al. (Plank et al., 2003a) demonstrated that efficient knock down of stable eGFP expression in HT1080 cells with linear PEI and synthetic siRNA was only achieved through magnetofection. This result also indicates the potential of magnetofection for nucleic acid transfer into cells which are difficult to transfect with standard methods. The experiments described so far concerned only nonviral nucleic acid vectors, but magnetofection also improved the transduction efficiencies of adenoviruses (Scherer et al., 2002a), retroviruses (Haim et al., 2005; Scherer et al., 2002a) and measles viruses (Kadota et al., 2005).
All the experiments discussed in this chapter compared magnetofection with the corresponding standard transfection or transduction but it would also be interesting to perform side by side comparisons with other physical methods like e.g. centrifugation, convective flow towards the target cells, biolistic methods or electroporation.
In summary, usually magnetofection is significantly more efficient than standard transfection or transduction, but there are rare cases in which magnetofection is only equally or even less efficient. The often improved nucleic acid dose-response profiles and reduced incubation times with vectors make magnetofection a less material and time consuming method which could be especially useful for automated high throughput screening of genes and of therapeutically useful sequences.