Multilayer Ratiometric Fluorescent Nanomachines for Imaging mRNA in Live Cells

Small Methods. 2021 Apr;5(4):e2001047. doi: 10.1002/smtd.202001047. Epub 2020 Dec 18.

Abstract

Detection of mRNA expression in live cells during treatment is a challenging task, despite its importance in tumor biology and potential therapeutic leads. Here a multilayer ratiometric fluorescent nanomachine for live-cell perturbation and imaging of mRNA at single cell resolution is reported. The nanomachines fabricated by microfluidic approaches consist of fluorescent polymeric cores and multiple lipid layers, which can efficiently deliver siRNA and molecular beacons (MBs) to cytosol and then release the cargo in a sequential way. The siRNA molecules released from the outer lipid layers lead to silencing of multidrug resistance 1 (MDR1) gene, and the MBs from the middle lipid layers detect the presence of MDR1 mRNA. The fluorescent ratio of MBs to fluorescent polymeric cores positively correlates with the expression level of MDR1 mRNA in MCF-7/ADR cells during siRNA treatment. The nanomachines provide comparable results with traditional qPCR for quantifying mRNA, showing great potential for modulation and imaging of intratumoral mRNA in vitro and in vivo.

Keywords: live-cell imaging; molecular beacons; nanomachine; siRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • Cell Line, Tumor
  • Drug Resistance, Neoplasm
  • Humans
  • MCF-7 Cells
  • Optical Imaging / methods*
  • RNA, Messenger / chemistry*
  • RNA, Messenger / isolation & purification*
  • RNA, Small Interfering

Substances

  • ABCB1 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • RNA, Messenger
  • RNA, Small Interfering