Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Abstract

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.

Modulation of cardiomyocyte activity using pulsed laser irradiated gold nanoparticles

“…In all tested cells, reactions were only observed in the presence of gold nanoparticles (see Fig. 8) According to former studies, laser manipulation with the applied radiant exposures and irradiation times leads to heating of the gold nanoparticles by several hundred K [5,21,31]. The thermal process evokes a transient cell membrane permeabilization, probably through the photothermal formation of plasmonic nanobubbles [7,32].…”

“…Sedimentation leads to unspecific attachment of the particles to the cell membrane. As validated in former studies, a concentration of 0.5 µg/cm 2 corresponds to about ten to thirty particles per cell [5,6,21]. A laser beam with a previously determined spot diameter of 80 µm irradiated the cells, which were placed on an epifluorescence microscope, from the top.…”

“…These include techniques for the sensitive detection of molecules [1,2] and methods for cell treatment or manipulation, such as photothermal therapy [1,3,4] or cell transfection [5][6][7][8]. Yet, it has to be determined if the same principle would be applicable to a direct modification of molecule or cell function.…”

“…The nanoparticles attach unspecifically to the membrane through sedimentation. Irradiation of the particles with a laser system operating at 532 nm and a pulse length of 850 ps led to heating of the particles of several hundred K [5]. Based on our previous work, which analyzed different cell parameters including calcium exchange during gold nanoparticle mediated laser manipulation, we aimed to achieve cardiac activity [21].…”

Ultrasound-Mediated Surface Engineering of Theranostic Magnetic Nanoparticles: An Effective One-Pot Functionalization Process Using Mixed Polymers for siRNA Delivery

“…In addition, due to their large molecular weights (MW, of ∼13 kDa), negative charges and hydrophilic nature, siRNA molecules are unable to cross cell membranes by passive diffusion mechanisms. These limitations have led to the development of a wide range of nanocarriers, including cyclodextrin polymers, [20][21][22][23] lipid-based carriers, [24,25] dendrimers, [25,26] neutral or cationic polymers, [27,28] conjugated-siRNAs [29,30] and various nanoparticle formulations [31][32][33]. The polycationic branched polyethyleneimine polymer (branched PEI, 25 kDa-b-PEI 25 ) is one of the most common and efficient transfecting agents for the electrostatic capture and delivery of nucleic acids and was therefore chosen for this versatile NPs surface engineering.…”