Rapid and facile quantitation of polyplex endocytic trafficking

Abstract

Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3'-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors.

Boronic Acid Copolymers for Direct Loading and Acid-Triggered Release of Bis-T-23 in Cultured Podocytes

“…As early as 20–30 min, fluorescently labeled PBA copolymers were mostly internalized (Figure A) and colocalized with LysoTracker, a marker of lysosomes (Figure B). This is consistent with other studies reporting that polycationic materials rapidly traffic into acidic compartments as early as 5 min, with greater trafficking into more acidic late endosomes and lysosomes observed 10–20 min later. , Bis-T-23 promotes dynamin oligomerization, which causes actin polymerization and cross-linking and focal adhesion maturation. ,− By image analysis, while Bis-T-23 significantly increased F-actin content in cultured podocytes, polymer-drug conjugates did not (Figure S6), likely due to the low dynamic range of this method. Therefore, we sought to develop a more high-throughput method of evaluating Bis-T-23 delivery in cultured podocytes.…”

“…This is consistent with other studies reporting that polycationic materials rapidly traffic into acidic compartments as early as 5 min, with greater trafficking into more acidic late endosomes and lysosomes observed 10–20 min later. 27, 28 Bis-T-23 promotes dynamin oligomerization, which causes actin polymerization and crosslinking and focal adhesion maturation. 7, 29–31 By image analysis, while Bis-T-23 significantly increased F-actin content in cultured podocytes, polymer-drug conjugates did not (Figure S6), likely due to the low dynamic range of this method.…”

Intracellular Trafficking and Endosomal Release of Oligonucleotides: What We Know and What We Don’t

“…A recent report used both fluorescent microscopy and a novel organelle tagging technique to follow polyplexes through the uptake and trafficking process [48]. This study showed rapid passage through EE and LE compartments and accumulation in lysosomes.…”

Quantitative colocalization analysis of DNA delivery by PEI‐mediated cationic polymers in mammalian cells

“…Thus, these results support the use of these technologies as a complementary tool for bioprocess characterization and diagnosis at nanometric range (Ping‐Jie et al ., ; Reitan et al ., ; Pike et al ., ). For instance, Manders' overlap coefficient has been recently used to track the presence of siRNA/PEI complexes in endocytic vesicles (Lazebnik & Pack ). Compared to other quantitative techniques, quantitative colocalization is time consuming and requires technical expertise.…”