A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

Abstract

There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.

High-throughput and high-content bioassay enables tuning of polyester nanoparticles for cellular uptake, endosomal escape, and systemic in vivo delivery of mRNA

“…Plasmid eGFP-N1(Addgene 2491) was purchased from Elim Biopharmaceuticals (Hayward, CA) and amplified by Aldevron (Fargo, ND). Cy5-labeled plasmid DNA was synthesized following a method reported by Wilson et al ( 47 ). 5-Methoxyuridine–modified CleanCap eGFP mRNA (L-7201), fLuc mRNA (L-7202), and Cy5-labeled mRNA (L-7702) were purchased from TriLink Biotechnologies (San Diego, CA).…”

Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing

“…R6,7,8_64 nanoparticles (10 w/w) were prepared as described above using premixed Cy3-labeled siRNA and Cy5-labeled plasmid DNA at a 1 w/w ratio of nucleic acids. Cy5-labeled plasmid DNA was prepared as previously described , and mixed at a 4 w/w ratio with unlabeled eGFP-N1 plasmid DNA. Nanoparticles were then diluted into media and added to cells at a total nucleic acid dose of 1000 ng/well and incubated for 2 h. Prior to imaging, cells were stained with Hoechst 33342 at a 1:5000 dilution for nuclei visualization.…”

The role of assembly parameters on polyplex poly(beta‐amino ester) nanoparticle transfections

“…Composite buffers mimicking calcium phosphate transfection conditions were also utilized (Kingston, Chen, & Rose, ). Uptake experiments were performed with 20% of the plasmid DNA labeled with Cy5 as previously described (Wilson, Routkevitch, et al, ).…”