Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Journal of Nanobiotechnology

Alexander Heisterkamp³  Heiko Meyer⁴  Hugo Murua Escobar²  Tammo Ripken¹  Ingo Nolte⁵  Siegfried Wagner⁵  Saskia Willenbrock⁵  Stefan Kalies¹  Dag Heinemann¹  Markus Schomaker¹ 

[1] Department of Biomedical Optics, Laser Zentrum Hannover, Hollerithallee 8, Hannover, 30419, Germany;Department of Hematology, Oncology, and Palliative Medicine, University of Rostock, Ernst- Heydemann-Str. 6, Rostock, 18057, Germany;Institut für Quantenoptik Leibniz Universität Hannover Welfengarten 1, Hannover, 30167, Germany;Department of Cardiothoracic Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, 30625, Germany;Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, Hannover, 30559, Germany

关键词: Gene delivery;    siRNA;    Permeabilization mechanisms;    Nanoparticles;    Plasmonics;    Laser transfection;   

Others  :  1133167
DOI  :  10.1186/s12951-014-0057-1

 received in 2014-09-10, accepted in 2014-12-01,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Schomaker et al.; licensee BioMed Central.

【 图 表 】

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