Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications

Abstract

Extrinsic probes have outstanding properties for intracellular labeling to visualize dynamic processes in and of living cells, both in vitro and in vivo. Since extrinsic probes are in many cases cell‐impermeable, different biochemical, and physical approaches have been used to break the cell membrane barrier for direct delivery into the cytoplasm. In this Review, these intracellular delivery strategies are discussed, briefly explaining the mechanisms and how they are used for live‐cell labeling applications. Methods that are discussed include three biochemical agents that are used for this purpose—purpose‐different nanocarriers, cell penetrating peptides and the pore‐foraming bacterial toxin streptolysin O. Most successful intracellular label delivery methods are, however, based on physical principles to permeabilize the membrane and include electroporation, laser‐induced photoporation, micro‐ and nanoinjection, nanoneedles or nanostraws, microfluidics, and nanomachines. The strengths and weaknesses of each strategy are discussed with a systematic comparison provided. Finally, the extrinsic probes that are reported for intracellular labeling so‐far are summarized, together with the delivery strategies that are used and their performance. This combined information should provide for a useful guide for choosing the most suitable delivery method for the desired probes.

Black phosphorus mediated photoporation: a broad absorption nanoplatform for intracellular delivery of macromolecules

“…Furthermore, AuNPs tend to break up into small fragments already after the first laser pulse, giving cause for the concern of genotoxicity. 34 Other reported sensitizers for photoporation like carbon dots, 35 graphene-based materials 19 and polymeric nanoparticles also have quite broad absorption spectra, but they quickly decay towards the NIR. Instead, BPQDs have an extended uniform absorption spectrum from the visible to the near infrared.…”

Microfluidic mechanoporation for cellular delivery and analysis

“…Also, most of the molecular probes used in experiments are either small-sized organic dyes or large antibodies. Subsequent research can, for example, emphasize on creating nanosensor probes, which can facilitate the study of intracellular microenvironment for applications in in vivo cell tracking [ 4 ]. An in vivo analysis is highly necessary to understand the physiological behavior of these mechanoporation devices and subsequently promote their clinical applications.…”

“…Intracellular delivery strategies are popularly classified as – carrier-mediated, and membrane disruption-based methods [ 1 , 4 , 5 ]. Carrier-based techniques can be either viral [ [6] , [7] , [8] ] or chemical [ 9 , 10 ] mediated, involving endocytosis or fusion processes to internalize the targeted cargo.…”

“…Carrier-based techniques can be either viral [ [6] , [7] , [8] ] or chemical [ 9 , 10 ] mediated, involving endocytosis or fusion processes to internalize the targeted cargo. Although effective for gene transfer and therapy, carrier based methods are specific to cell-types, and are often restricted by cytotoxicity, and unwanted immune, inflammatory responses, leading to lower cell viabilities [ 4 , 5 , 8 , 11 ]. On the other hand, membrane disruption methods involve the physical penetration or permeabilization of the cellular membrane to generate transient nanopores, which then permits the delivery of exogenous molecules through diffusion or fluid convection [ 1 , 4 , 5 , 11 , 12 ].…”

“…Although effective for gene transfer and therapy, carrier based methods are specific to cell-types, and are often restricted by cytotoxicity, and unwanted immune, inflammatory responses, leading to lower cell viabilities [ 4 , 5 , 8 , 11 ]. On the other hand, membrane disruption methods involve the physical penetration or permeabilization of the cellular membrane to generate transient nanopores, which then permits the delivery of exogenous molecules through diffusion or fluid convection [ 1 , 4 , 5 , 11 , 12 ]. The methods are advantageous because of minimal chances of inducing harsh cellular responses such as unwanted immunogenic or chemical reactions, thereby improving delivery efficiencies.…”