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 >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.

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.

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Femtosecond laser photodisruption of the ocular lens yields a self-limited lesion with bubbles that resolve with time. In living animal eyes, no cataract formation was found with no loss of lens function or induced light scatter after 3 months. These results suggest that use of a low-energy femtosecond laser might be safe when modifying the lens for presbyopia correction.

Optical stimulation of the cochlea with laser light has been suggested as an alternative to conventional treatment of sensorineural hearing loss with cochlear implants. The underlying mechanisms are controversially discussed: The stimulation can either be based on a direct excitation of neurons, or it is a result of an optoacoustic pressure wave acting on the basilar membrane. Animal studies comparing the intra-cochlear optical stimulation of hearing and deafened guinea pigs have indicated that the stimulation requires intact hair cells. Therefore, optoacoustic stimulation seems to be the underlying mechanism. The present study investigates optoacoustic characteristics using pulsed laser stimulation for in vivo experiments on hearing guinea pigs and pressure measurements in water. As a result, in vivo as well as pressure measurements showed corresponding signal shapes. The amplitude of the signal for both measurements depended on the absorption coefficient and on the maximum of the first time-derivative of laser pulse power (velocity of heat deposition). In conclusion, the pressure measurements directly demonstrated that laser light generates acoustic waves, with amplitudes suitable for stimulating the (partially) intact cochlea. These findings corroborate optoacoustic as the basic mechanism of optical intra-cochlear stimulation.