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

Markus Schomaker,Ingo Nolte,Dag Heinemann,Alexander Heisterkamp,Saskia Willenbrock,Heiko Meyer,Siegfried Wagner,Hugo Escobar,Stefan Kalies,Tammo Ripken

doi:10.1186/s12951-014-0057-1

Abstract

BackgroundIn 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.ResultsThe 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.ConclusionThis 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.

Highlights

In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies
The results indicate that both, near field and heating effects contribute to the mechanism of nanoparticle mediated membrane permeabilization in the fs regime
The AuNP luminescence was still visible after washing, indicating that the particles remained adhered to the cell membrane

Summary

Introduction

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. In order to address these challenges methodicaly, a variety of optical transfection techniques have been developed based on pulsed as well as continuously emitting lasers [9,10,11,12,13] None of these techniques fulfills the requirements of an efficient and low-toxic transfection method combined with high throughput. Gold nanoparticle (AuNP) labeled cells are irradiated with a weakly focused laser beam This method allows targeting many cells simultaneously, ensuring high throughput while maintaining a high spatial selectivity. This physical method using resonant laser pulses is very promising for the manipulation of a variety of cell types

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Conclusion