Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down
Creators: name: Heinemann, Dag
name: Schomaker, Markus
name: Kalies, Stefan
name: Schieck, Maximilian
name: Carlson, Regina
name: Murua Escobar, Hugo
name: Ripken, Tammo
name: Meyer, Heiko
name: Heisterkamp, Alexander
Publisher: name: San Francisco, California, US : PLOS
Year: 2013
Subjects: subject: enhanced green fluorescent protein
subject: gold nanoparticle
subject: small interfering RNA
subject: animal cell
subject: article
subject: cell line
subject: cell viability
subject: controlled study
subject: density
subject: fluorescence
subject: gene delivery system
subject: gene silencing
subject: genetic transfection
subject: laser transfection
subject: neodymium YAG laser
subject: nonhuman
subject: pleomorphic adenoma
subject: protein depletion
subject: scanning electron microscopy
subject: tumor cell
subject: velocity
subject: Western blotting
subject: Animals
subject: Cell Line
subject: Cell Survival
subject: Dogs
subject: Gene Knockdown Techniques
subject: Gold
subject: Lasers
subject: Metal Nanoparticles
subject: RNA Interference
subject: RNA, Small Interfering
subject: Transfection
subject: Dewey Decimal Classification::500 | Naturwissenschaften
subject: Dewey Decimal Classification::600 | Technik::610 | Medizin, Gesundheit

description: Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ~8 s per well of a 96 well plate. The optimal particle density was determined to be ~6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of &gt;90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.