Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells

Nat Protoc. 2021 Jan;16(1):327-351. doi: 10.1038/s41596-020-00418-9. Epub 2020 Dec 4.

Abstract

Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3-10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3-6 d.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / metabolism*
  • Cell Line
  • Cells, Cultured
  • Click Chemistry
  • Dendrimers / chemical synthesis
  • Dendrimers / chemistry*
  • Humans
  • Mice, Inbred C57BL
  • RNA Interference*
  • RNA, Small Interfering / administration & dosage*
  • RNA, Small Interfering / genetics
  • T-Lymphocytes / metabolism*

Substances

  • Dendrimers
  • RNA, Small Interfering