Controlled Delivery of MicroRNAs into Primary Cells Using Nanostraw Technology

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that play key roles in posttranscriptional gene regulation. Being involved in regulating virtually all cellular processes, from proliferation and differentiation to migration and apoptosis, they have emerged as important epigenetic players. While most interest has gone into which miRNAs are involved in specific cellular processes or pathologies, the dosage‐dependent effects of miRNAs remain vastly unexplored. Different doses of miRNAs can cause selective downregulation of target genes, in turn determining what signaling pathways and cellular responses are triggered. To explore this behavior, the effects of incremental miRNA dosage need to be studied; however, current delivery methods for miRNAs are unable to control how much miRNA enters a cell. Herein, an approach is presented based on a nanostraw–electroporation delivery platform that decouples the delivery from biological mechanisms (e.g., endocytosis) to enable precise control over the amount of miRNA delivered, along with demonstrating ratiometric intracellular delivery into primary dermal fibroblasts for miR‐181a and miR‐27a. In addition, it is shown that the nanostraw delivery platform allows efficient delivery of miRNAs into primary keratinocytes, opening new opportunities for successful miRNA delivery into this hard‐to‐transfect cell type.

Use of dielectrophoresis for directing T cells to microwells before nanostraw transfection: modelling and experiments

“…Whereas previous studies using nanostraws have used centrifugation or magnetic forces to direct cells to nanostraws, 3,5,17,23 the present method offers an alternative method to direct cells towards the substrate, not requiring any centrifugation step, cell concentration or cell modication. The method can be useful when fewer cells are available to work with, such as when working with circulating tumor cells and antigen specic T cells, for example.…”

Current research status of tumor cell biomarker detection

“…miRNA can be detected by translocation of the probe into the cell through the deformation of the cell’s own plasma membrane during endocytosis. Pop et al designed another miRNA detection method employing a nanostraw-based electroporation delivery platform 122 . As shown in Fig.…”

Recent Advances in Microscale Electroporation

“…Pretreating the surface can reduce the potential cytotoxicity of nanostructures to certain sensitive primary cell types. Pop and Almquist found that the nanostructures were harmful specifically when culturing primary basal keratinocytes on-chip without additional treatments . Cell viability significantly increased by adding a fetal bovine serum coating, which reduced membrane perturbations caused by the nanoscale protrusions.…”

“…Experiments with different dosages revealed that the delivery of frequent, low-concentration doses is preferable to a single large dose, as the continuous approach maintained a comparable response while requiring 22% less siRNA . RNAi has also been used with microscale EP to deliver miR-181a into cells to promote fibroblast proliferation . Additionally, microscale EP was effective in the delivery of cell penetrating peptides conjugated to peptide nucleic acids (CPP-PNAs) into macrophages infected with bacteria .…”

“…142 RNAi has also been used with microscale EP to deliver miR-181a into cells to promote fibroblast proliferation. 160 Additionally, microscale EP was effective in the delivery of cell penetrating peptides conjugated to peptide nucleic acids (CPP-PNAs) into macrophages infected with bacteria. 301 A PNA sequence was selected to selectively inhibit bacteria growth within the cell.…”