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 30 min 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.
|Number of pages||9|
|Journal||Journal of Controlled Release|
|State||Published - Feb 10 2017|
Bibliographical noteFunding Information:
This work was partially supported by the American Heart Association Predoctoral Fellowship (11PRE5960002) (ML), National Science Foundation (BES 06-02636) (DWP) and National Institutes of Health GM085222 (DWP). In addition, we thank Sandy Mattick at the Cell Culture Media Facility at the University of Illinois for help with preparation of cell media. Confocal fluorescence microscopy was performed at the Institute of Genomic Biology Core Facilities, University of Illinois, with technical help from Mayandi Sivaguru.
- Gene delivery
- Intracellular trafficking
ASJC Scopus subject areas
- Pharmaceutical Science