title: Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine.
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.
  
  
  CONCLUSIONS
  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.
authors: Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, 30419, Hannover, Germany. m.schomaker@lzh.de.
authors: Markus
authors: M
authors: Schomaker
authors: Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, 30419, Hannover, Germany. d.heinemann@lzh.de.
authors: Dag
authors: D
authors: Heinemann
authors: Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, 30419, Hannover, Germany. s.kalies@lzh.de.
authors: Stefan
authors: S
authors: Kalies
authors: Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559, Hannover, Germany. saskia.willenbrock@tiho-hannover.de.
authors: Saskia
authors: S
authors: Willenbrock
authors: Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559, Hannover, Germany. siegfried.wagner@tiho-hannover.de.
authors: Siegfried
authors: S
authors: Wagner
authors: Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559, Hannover, Germany. ingo.nolte@tiho-hannover.de.
authors: Ingo
authors: I
authors: Nolte
authors: Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, 30419, Hannover, Germany. t.ripken@lzh.de.
authors: Tammo
authors: T
authors: Ripken
authors: Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559, Hannover, Germany. hugo.murua.escobar@med.uni-rostock.de.|Department of Hematology, Oncology, and Palliative Medicine, University of Rostock, Ernst- Heydemann-Str. 6, 18057, Rostock, Germany. hugo.murua.escobar@med.uni-rostock.de.
authors: Hugo
authors: H
authors: Murua Escobar
authors: Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, 30419, Hannover, Germany. h.meyer@lzh.de.|Department of Cardiothoracic Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany. h.meyer@lzh.de.
authors: Heiko
authors: H
authors: Meyer
authors: Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, 30419, Hannover, Germany. heisterkamp@iqo.uni-hannover.de.|Institut für Quantenoptik Leibniz Universität Hannover Welfengarten 1, 30167, Hannover, Germany. heisterkamp@iqo.uni-hannover.de.
authors: Alexander
authors: A
authors: Heisterkamp
mesh_terms: D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D002470:Cell Survival; D004285:Dogs; D004867:Equipment Design; D055785:Gene Knockdown Techniques; D006046:Gold; D025743:HMGA2 Protein / Q000235:genetics; D007834:Lasers; D008855:Microscopy, Electron, Scanning; D062346:Molecular Medicine / Q000295:instrumentation / Q000379:methods*; D053758:Nanoparticles; D034741:RNA, Small Interfering; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
chemical_list: D025743:HMGA2 Protein; D034741:RNA, Small Interfering; D006046:Gold
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