The recently developed laser-induced cell transfection mediated by Au nanoparticles is a promising alternative to the well-established lipid-based transfection or to electroporation. Optoporation is based on the laser plasmonic heating of nanoparticles located near the cell membrane. However, the uncontrollable cell damage from intense laser pulses and from random attachment of nanoparticles may be crucial for transfection. We present a novel plasmonic optoporation technique that uses Au nanostar layers immobilized in culture microplate wells. HeLa cells were grown directly on Au nanostar layers, after which they were subjected to continuous-wave 808 nm laser irradiation. An Au monolayer density ~15 μg/cm and an absorbed energy of about 15 to 30 J were found to be optimal for optoporation. Propidium iodide molecules were used as model penetrating agent. The transfection efficiency evaluated using fluorescence microscopy for HeLa cells transfected with pGFP under optimized optoporation conditions (95% ± 5%) was similar to the efficiency of TurboFect. The technique's efficiency (295 ± 10 relative light units, RLU), demonstrated by transfecting HeLa cells with the pCMV-GLuc 2 control plasmid, was greater than that obtained by transfection of HeLa cells with the TurboFect agent (220 ± 10 RLU). The cell viability in plasmonic optoporation (92% ± 7%), too, was greater than that in transfection with TurboFect (75% ± 7%).

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Laser optoporation systems are now increasingly used for intracellular delivery. However, data on the response of cells to radiation-induced nondamaging changes in the integrity of the membrane lipid bilayer remain limited. Traditionally, confocal laser scanning microscopy and electron microscopy are used for such studies, but they have limitations for in situ experiments. The modern capabilities of atomic force microscopy (AFM) combine the resolution of electron microscopy and the possibility of noninvasive lifetime imaging of cells in vitro. Herein we used long-term AFM mapping integrated with fluorescence microscopy imaging for investigation of the whole cell cycle from irradiation time point to the total recovery to the intact cell state. For the first time we performed a comprehensive study of long-term posteffects of continuous laser and pulsed laser on the mechanical properties and the membrane recovery of HeLa cells grown on the Au nanoparticle layers of various morphologies. The set of nonpenetrating agents with various sizes ranging from 1 to 1.5 nm for propidium iodide (PI) up to 6–8 nm for 40 kDa FITC-labeled dextran was used to control the delivery efficacy. The main parameters recorded with AFM scanning of cells are Young’s modulus (YM) and the cell surface topography. We revealed that self-healing of HeLa cell from the moment of irradiation to complete restoration of the membrane integrity is lasting 22–30 h when using a continuous-wave source and 2–5 h when using a pulsed laser, respectively. The estimated time elapse was in good correspondence with the relative change in YM during the entire experiment. Our findings demonstrate the capability of AFM coupled with fluorescent microscopy for further in situ investigations of the morphological and functional state of the cells exposed to the influence of other external conditions.

Promising biomedical applications of hybrid materials composed of gold nanoparticles and nucleic acids have attracted strong interest from the nanobiotechnological community. The particular interest is owing to the robust and easy-to-make synthetic approaches, to the versatile optical and catalytic properties of gold nanoparticles combined with the molecular recognition and programmable properties of nucleic acids. The significant progress is made in the development of DNA–gold nanostructures and their applications, such as molecular recognition, cell and tissues bioimaging, targeted delivery of therapeutic agents, etc. This review is focused on the critical discussion of the recent applications of the gold nanoparticles–nucleic acids hybrids. The effect of particle size, surface, charge and thermal properties on the interactions with functional nucleic acids is discussed. For each of the above topics, the basic principles, recent advances, and current challenges are discussed. Emphasis is placed on the systematization of data over the theranostic systems on the basis of the gold nanoparticles–nucleic acids hybrids. Specifically, we start our discussion with observation of the recent data on interaction of various gold nanoparticles with nucleic acids. Further we describe existing gene delivery systems, nucleic acids detection, and bioimaging technologies. Finally, we describe the phenomenon of the polymerase chain reaction improvement by gold nanoparticles additives and its potential underlying mechanisms. Lastly, we provide a short summary of reported data and outline the challenges and perspectives.

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