Details
| Original language | English |
|---|---|
| Article number | 10 |
| Journal | Journal of Nanobiotechnology |
| Volume | 13 |
| Issue number | 1 |
| Publication status | Published - 3 Feb 2015 |
Abstract
Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.
Keywords
- Gene delivery, Laser transfection, Nanoparticles, Permeabilization mechanisms, Plasmonics, siRNA
ASJC Scopus subject areas
- Chemical Engineering(all)
- Bioengineering
- Medicine(all)
- Medicine (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Medicine
- Engineering(all)
- Biomedical Engineering
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
- Pharmacology, Toxicology and Pharmaceutics(all)
- Pharmaceutical Science
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In: Journal of Nanobiotechnology, Vol. 13, No. 1, 10, 03.02.2015.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine
AU - Schomaker, Markus
AU - Heinemann, Dag
AU - Kalies, Stefan
AU - Willenbrock, Saskia
AU - Wagner, Siegfried
AU - Nolte, Ingo
AU - Ripken, Tammo
AU - Murua Escobar, Hugo
AU - Meyer, Heiko
AU - Heisterkamp, Alexander
N1 - Funding information: The authors thank Regina Carlson for technical support in flow cytometry and the German Research Foundation DFG (within the Transregio 37 and the excellence cluster REBIRTH) for the financial support. We thank Ulrich Martin (Leibnitz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School) for providing the hES3 and hCBiPS2 cells.
PY - 2015/2/3
Y1 - 2015/2/3
N2 - Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.
AB - Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.
KW - Gene delivery
KW - Laser transfection
KW - Nanoparticles
KW - Permeabilization mechanisms
KW - Plasmonics
KW - siRNA
UR - http://www.scopus.com/inward/record.url?scp=84924364079&partnerID=8YFLogxK
U2 - 10.1186/s12951-014-0057-1
DO - 10.1186/s12951-014-0057-1
M3 - Article
C2 - 25645721
AN - SCOPUS:84924364079
VL - 13
JO - Journal of Nanobiotechnology
JF - Journal of Nanobiotechnology
SN - 1477-3155
IS - 1
M1 - 10
ER -