Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking.

We present a method that takes advantage of the fluorophore loading dependence of fluorescence nanoparticle tracking (fNTA) to determine the content of specific miRNA targets in extracellular vesicles (EVs) and their stoichiometry across the entire EV population. The method is based on an assay for detecting EV miRNA by hybridization to fluorescently labeled, miRNA-specific molecular beacons encapsulated in cationic lipoplex nanoparticles that fuse non-specifically with negatively charged EVs. To demonstrate the method, we carry out a stoichiometric analysis of miR-21 in EVs released from A549 lung cancer cells. We find approximately 2.3% of the A549 EVs have an average copy number of ~44 miR-21/A549 EV and contain at least a threshold number of 33 miR-21 copies/A549 EV required for fluorescence tracking. Potential applications of sizing, enumerating, and phenotyping EVs using this method include specifying dosages for therapeutic applications and identifying specific EV subpopulations in patient samples for diagnostic applications.

On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance.

Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurrence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more efficacious treatments. Here, we introduce a novel micro-/nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (>10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to ∼4-fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44-high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living-cell-interrogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.

Effect of surface modification on interfacial nanobubble morphology and contact line tension.

Past research has confirmed the existence of surface nanobubbles on various hydrophobic substrates (static contact angle >90°) when imaged in air-equilibrated water. Additionally, the use of solvent exchange techniques (based on the difference in saturation levels of air in various solvents) also introduced surface nanobubbles on hydrophilic substrates (static contact angle <90°). In this work, tapping mode atomic force microscopy was used to image interfacial nanobubbles formed on bulk polycarbonate (static contact angle of 81.1°), bromo-terminated silica (BTS; static contact angle of 85.5°), and fluoro-terminated silica (FTS; static contact angle of 105.3°) surfaces when immersed in air-equilibrated water without solvent exchange. Nanobubbles formed on the above three substrates were characterized on the basis of Laplace pressure, bubble density, and contact line tension. Results reported here show that (1) the Laplace pressures of all nanobubbles formed on both BTS and polycarbonate were an order of magnitude higher than those of FTS, (2) the nanobubble number density per unit area decreased with an increase in substrate contact angle, and (3) the contact line tension of the nanobubbles was calculated to be positive for both BTS and polycarbonate (lateral radius, Rs < 50 nm for all nanobubbles), and negative for FTS (Rs > 50 nm for all nanobubbles). The nanobubble morphology and distribution before and after using the solvent exchange method (ethanol-water), on the bulk polycarbonate substrate was also characterized. Analysis for these polycarbonate surface nanobubbles showed that both the Laplace pressure and nanobubble density reduced by ≈98% after ethanol-water exchange, accompanied by a flip in the magnitude of contact line tension from positive (0.19 nN) to negative (-0.11 nN).

Author Correction: Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-021-00725-w.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Nonendocytic delivery of lipoplex nanoparticles into living cells using nanochannel electroporation.

The delivery of biomolecules, including siRNAs (≈21 bp) and large plasmids (≈10 kbp), into living cells holds a great promise for therapeutic and research applications. Lipoplex nanoparticles are popular nanocarriers for gene delivery. In conventional transfection methods, the cellular uptake of lipoplex nanoparticels occurs through the endocytosis process. The entrapment of lipoplex nanoparticles into endocytic vesicle is a major barrier in achieving efficient gene silencing and expression. Here, a novel nanochannel electroporation (NEP) method is employed to facilitate the cellular uptake and release of siRNAs/DNAs from lipoplexes. First, it is demonstrated that in a NEP device, lipoplex nanoparticles can be injected directly into the cell cytoplasm within several seconds. Specifically, it is found that lipoplexes containing MCL-1 siRNA delivered by NEP can more efficiently down-regulate the expression of MCL-1 mRNA in A549 cancer cells than conventional transfection. Quantum dot-mediated Förster resonance energy transfer (QD-FRET) reveals that lipoplexes delivered via NEP can directly release siRNA in the cytoplasm without going through the endocytosis route, which unravels the responsible mechanism for efficient gene delivery. Furthermore, the advantage of combining NEP with lipoplex nanoparticles by the successful delivery of large plasmids (pCAG2LMKOSimO, 13 kbp) into CHO cells is demonstrated.