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 -