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

Abstract

Genetically manipulating immune cells using siRNAs is important for both basic immunological studies and therapeutic applications. However, the 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, which is able to deliver siRNA to a variety of cells including primary immune cells. We provide here the protocols for the synthesis of this dendrimer and its siRNA delivery into immune cells such as primary T-and B-cells, natural killer cells, macrophages, and primary microglia. It is noteworthy that the dendrimer synthesis is straightforward, and the siRNA delivery protocol is easy, requiring simple mixing of 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 outperforms largely the standard technique of electroporation, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis which requires 3 weeks, the primary immune cell preparation which takes 3-10 days depending on the tissue source and cell type, the dendrimermediated siRNA delivery and subsequent functional assay which takes additional 3-6 days.

From Bench to the Clinic: The Path to Translation of Nanotechnology-Enabled mRNA SARS-CoV-2 Vaccines

“…Polymersomes, self-assembled vesicles composed of amphiphilic polymers, offer several advantages over traditional liposomes in terms of structural stability; nevertheless, lipid/polymer hybrid vesicles allow a greater control and adaptability of physicochemical properties to any desired functionality or application [ 58 ]. Dendrimer NPs, composed of three-dimensional branched synthetic polymers that form interior and exterior layers ideal for molecular conjugation and encapsulation, have been employed in nanovaccination approaches for DNA or RNA delivery [ 59 – 61 ]. Polymeric NPs have been regarded as excellent candidates to deliver molecules due to their biodegradability, water solubility, biocompatibility, stability, and ability to perform targeted delivery.…”

Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery

“…The transformation of the progenitors into fully active DCs was performed over a 10-day time frame. BMDMs were also generated from bone marrow extracted from C57BL/6 mice as previously described (22). Briefly, the erythrocytes were removed by the RBC lysis buffer, and the remaining cells were cultured in a complete DMEM with 20% L929 (Sigma, #85011425) in conditioned medium (source of macrophage colony-stimulating factor) for 7 days.…”

Tuning the immunostimulation properties of cationic lipid nanocarriers for nucleic acid delivery

“…BMDMs were also generated from bone marrow extracted from C57BL/6 mice as previously described (Chen et al, 2021). Briefly, the erythrocytes were removed by the RBC lysis buffer, and the remaining cells were cultured in a complete DMEM with 20% L929 (Sigma, #85011425) in conditioned medium (source of macrophage colony-stimulating factor) for 7 days.…”