Zellchirugie mittels plasmonischer Strukturen an lebenden Zellen mittels Femtosekunden-Laserpulsen
Final Report Abstract
Transfection, a method for transferring foreign genetic material into mammalian cells, is widely used by biologists. None of the current transfection techniques is entirely satisfactory and for each method efficiency strongly depends on the cell type and/or the vector used to deliver the gene of interest. As viral approaches face major safety and immunogenicity concerns for in vivo applications, non-viral methods are being investigated. However, current non-viral approaches present major disadvantages including low levels of transfection efficiency in vivo, lack of sustained expression, as well as tissue damage. Within the scope of this project, a new high throughput, non-viral and low toxicity nanotechnology based transfection technique of living cells. As a proof of principle, human cancer melanoma cells were used for the transfection experiments. The most important results presented, based on the use of near infrared femtosecond (fs) laser and out of resonance plasmonic gold nanoparticles include: - A very low toxicity (< 1%) of the gold nanostructures used. - A high cell optoporation efficiency (~70%) and cell viability (~80%). - A significantly higher (by a factor of 3) transfection efficiency as compared with the best standard transfection technique (Lipofection, optimized for the cell type used). - A high treatment speed, up to 40 mm2/minute, enabling to treat square centimeter regions in a few minutes. - An unambiguous proof that the NPs are not broken by the process by performing scanning electron and confocal fluorescence microscopy as well as in-situ spectroscopy. The last point is critically important, as if nanoparticles (NPs) were fragmented into small (< 10 nm) pieces, they may eventually interfere with DNA as intercalating particles. Microscopy and in situ spectroscopy results presented in the paper clearly indicates that the NPs stay intact because the NPs do not absorb very strongly the photon energy but rather amplify the scattered field to induce a highly localized process leading to the cell optoporation in the biological medium. Additionally, the spatial selectivity is an important advantage for possible in vivo applications. Only laser exposed cells covered with nanoparticles are treated and a bioconjugation of those particles adds a cellular selectivity, leading to a treatment limited to the cells of interest, even in mixed culture or tissue. Therefore, the proposed method shows promises as an alternative transfection technology that could be adapted to therapeutic tools in the clinic. Melanoma is one of the most aggressive human cancers and is the leading cause of skin cancer related mortalities, accounting for approximately 80% of deaths. Although melanoma can be cured by surgical resection in its early stages, it is unresponsive to treatment once it has progressed to the metastatic stage, leading to a poor prognosis. As an alternative new therapeutic strategy, transfection can be used to reduce the aggressiveness of melanoma cells by manipulating the tumorigenesis in the melanoma cells, which would prevent the metastatic spreading to distant organs. The approach, based on RNA interference technology, consist in efficiently delivering small B-RAF silencer siRNA molecules into melanoma cells. Migration assays using laser scrape wounding and laser transfection successfully showed the effect of reduced migration velocity compared to non-treated cells, hence a lower level of aggressiveness of the cells.
Publications
- “Plasmonic enhanced fs-laser optoporation of human melanoma cells”. Proc SPIE, 7925, 79250I (2011)
Judith Baumgart
