Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound‐Propelled Nanomotors

Abstract

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.

Rapid Detection of AIB1 in Breast Cancer Cells Based on Aptamer‐Functionalized Nanomotors

“…Representative tracking lines of individual bare and modified motors were also compared (Figure B, right side). As displayed by the bar plot shown in Figure B, a small speed decrease was found using modified AuNWs due to the FAM‐AIB1 aptamer cargo . Despite this small change in the nanomotors propulsion, the movement of the FAM‐AIB1‐apt modified GO/AuNWs provide sufficient thrust for cell membrane penetration and subsequent intracellular propulsion.…”

“…The corresponding bar plot displaying the fluorescence intensities (Figure C) indicates ∼4 times higher fluorescence intensity under the US actuation compared to static conditions. These results confirm the important role of the US propulsion in facilitating the internalization of nanomotors inside the cells . On the other hand, negligible fluorescence was observed in control groups based on untreated MCF‐7 cells, non‐cancerous HFF‐1 cells (not overexpressing AIB1) treated with FAM‐AIB1‐apt modified GO/AuNWs, and using MCF‐7 cells treated with GO/AuNWs functionalized with nonspecific FAM‐labelled aptamers (FAM‐Ricin‐apt used as a model; see Supporting information).…”

“…[16] Later USpropelled nano/micromotors gained more interest for a wide range of biomedical applications including drug loading [17] and delivery. [18] In the past 5 years US-propelled nanomotors have successfully demonstrated rapid intracellular detection of miRNA, [19] as well as accelerated intracellular delivery of siRNA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 for gene silencing, [20] Cas9/sgRNA, [21] apoptotic enzymes [22] or oxygen. [23] The fast propulsion of these nanomotors under the acoustic field allows them to easily penetrate cell membranes and deliver the carried payloads in a faster way compared to passive nanocarriers without causing significant toxicity.…”

“…These results confirm the important role of the US propulsion in facilitating the internalization of nanomotors inside the cells. [19][20][21] On the other hand, negligible fluorescence was observed in control groups based on untreated MCF-7 cells, non-cancerous HFF-1 cells (not overexpressing AIB1) treated with FAM-AIB1-apt modified GO/AuNWs, and using MCF-7 cells treated with GO/AuNWs functionalized with nonspecific FAMlabelled aptamers (FAM-Ricin-apt used as a model; [28] see Supporting information). These control experiments further confirmed the specificity and stability of the AIB1 aptamer and the importance of the US propulsion, which accelerates the nanomotor cell internalization without causing cell disruption or significant toxic effects.…”

An NIR‐Responsive DNA‐Mediated Nanotetrahedron Enhances the Clearance of Senescent Cells

“…Due to excellent in vivo stability, significant progress has been made in the self‐assembly of discrete nanoparticles with a DNA frame 8–13. These well‐defined nanoassemblies could be used as an effective nanocarrier for the delivery of small molecules or some proteins into target areas 14–16. Given the significant advantage of remote spatial/temporal control and non‐invasiveness, light‐responsive systems have attracted significant attention, particularly with regards to near‐infrared (NIR) light; such systems have significant potential for biomedical applications 17–21…”