Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types

Abstract

Efficient delivery of compounds and macromolecules into living cells is essential in many fields including basic research, applied drug discovery, and clinical gene therapy. Unfortunately, current delivery methods, such as cationic lipids and electroporation, are limited by the types of macromolecules and cells that can be employed, poor efficiency, and/or cell toxicity. To address these issues, novel methods were developed based on laser-mediated delivery of macromolecules into cells through optoinjection. An automated high-throughput instrument, the laser-enabled analysis and processing (LEAP) system, was utilized to elucidate and optimize several parameters that influence optoinjection efficiency and toxicity. Techniques employing direct cell irradiation (i.e., targeted to specific cell coordinates) and grid-based irradiation (i.e., without locating individual cells) were both successfully developed. With both techniques, it was determined that multiple, sequential low radiant exposures produced more favorable results than a single high radiant exposure. Various substances were efficiently optoinjected--including ions, small molecules, dextrans, siRNAs (small interfering RNAs), plasmids, proteins, and semiconductor nanocrystals--into numerous cell types. Notably, cells refractory to traditional delivery methods were efficiently optoinjected with lower toxicity. We establish the broad utility of optoinjection, and furthermore, are the first to demonstrate its implementation in an automated, high-throughput manner.

Application of dynamic diffractive optics for enhanced femtosecond laser based cell transfection

“…Their operation is usually based on the creation of a shock wave [11,12] which limits the selectivity of the method, although single cell nanosecond transfection experiments have been reported [13]. The lack of selectivity is compensated with a higher throughput and injection efficiency in some cases [14]. Compared to fs poration, the beam focusing and precise positioning is less of an issue.…”

“…An automated poration system based on a nanosecond laser has recently been used to optoinject macromolecules and transfect genes [14]. Although this automated system provided satisfactory efficiencies and throughput it did not offer the possibility of targeted selective treatment.…”

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

“…As a result, there is an increasing interest to use pulsed laser microbeams for precise cellular manipulation, including laserinduced cell lysis [1], cell microdissection and catapulting [2][3][4][5], cell collection, expansion, and purification [6][7][8], cellular microsurgery [9][10][11], and cell membrane permeabilization for the delivery of membrane-impermeant molecules into cells [12 15], The processes of laser-induced optoinjection and optoporation offer the ability to load cells with a variety of biomolecules on short time scales (milliseconds to seconds) through optically produced cell membrane permeabilization [12,14,15], Despite the innovative utilization of laser microbeams in cell biology and biotechnology, only recently have studies provided insight regarding the mechanisms that mediate the interactions of highly focused pulsed laser beams with cells [16][17][18][19][20][21][22], A better understanding of these processes will prove critical to the continued development of laser microbeams for both research and practical applications. In previous studies, we provided a detailed characterization of the physics involved in the interaction of highly-focused nanosecond laser microbeams with cells [19,20], However, it is important to relate these physical effects to the biological response of the cells.…”

Electrical Impedance Monitoring of Photothermal Porated Mammalian Cells

“…Methods include electroporation, sonoporation, optoporation, microinjection, and other chemical and biological approaches. [1][2][3][4][5][6][7][8][9][10] The size of delivered cargo can vary by several orders of magnitude, from small molecules such as DNA, molecule probes, quantum dots, proteins, and microbeads up to micrometersized intracellular pathogens, such as bacteria. 11 Technologies that allow large-size cargo delivery into live cells are highly sought but rare.…”