Single Cell Kinetics of Intracellular, Nonviral, Nucleic Acid Delivery Vehicle Acidification and Trafficking

Abstract

Mechanistic understanding of the intracellular trafficking of nonviral nucleic acid delivery vehicles remains elusive. A live, single cell-based assay is described here that is used to investigate and quantitate the spatiotemporal, intracellular pH microenvironment of polymeric-based nucleic acid delivery vehicles. Polycations such as polyethylenimine (PEI), poly-l-lysine (PLL), beta-cyclodextrin-containing polymers lacking or possessing imidazole termini (CDP or CDP-imid), and cyclodextrin-grafted PEI (CD-PEI) are used to deliver an oligonucleotide containing a single fluorophore with two emission lines that can be employed to measure the pH. Delivery vehicles were also sterically stabilized by addition of poly(ethylene glycol) (PEG) and investigated. The intracellular trafficking data obtained via this new methodology show that vectors such as PEI and CDP-imid can buffer the endocytic vesicles while PLL and CDP do not. Additionally, the PEGylated vectors reveal the same buffering capacity as their unstabilized variants. Here, the live cell, spatiotemporal mapping of these behaviors is demonstrated and, when combined with cell uptake and luciferase expression data, shows that there is not a correlation between buffering capacity and gene expression.

Polymers in Small-Interfering RNA Delivery

“…Therefore, in addition to low immunogenicity such approaches provide multitude of opportunities to incorporate various functionalities to address variable requirements in a range of therapeutic applications. However, one of the major obstacles encountered by CDP is their inability to escape endosomal pathway (Gonzalez et al, 1999;Kulkarni et al, 2005). CDPs modified with imidazole groups demonstrated enhanced intracellular delivery overcoming the degradative endosomal entrapments.…”

“…CDPs modified with imidazole groups demonstrated enhanced intracellular delivery overcoming the degradative endosomal entrapments. The imidazole-containing CDP (CDPim) is known to buffer the pH inside endosomal vesicles, giving rise to increased intracellular delivery compared with unmodified CDPs (Kulkarni et al, 2005).…”

Can non-viral technologies knockdown the barriers to siRNA delivery and achieve the next generation of cancer therapeutics?

“…The structure-activity relationship of beta-cyclodextrin-based polycations demonstrated that improvement of the hydrophilicity of the spacer unit could reduce cytotoxicity, probably resulting from increased hydration around the CD monomers and higher flexibility. However, initial data indicated that β-CD-based polyplexes did not exhibit buffering capacity in the endosomal environment unless further modified by an imidazole-distal moiety (Kulkarni et al, 2005). In order to avoid aggregation in vivo, as seen with other cationic systems, a PEG group attached via adamantane (a hydrophobic molecule) was incorporated into the beta-CD-containing polycations through the formation of an inclusion complex.…”

Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA

“…Interestingly, the delivery of siRNA via the cyclodextrin polycation system developed in our laboratory at the California Institute of Technology did not produce immune stimulation in mice, even when the siRNA contained a highly stimulatory base sequence motif (19). Possible reasons for the lack of immune stimulation include endosomal buffering by this delivery system (20,21) [inhibitors of endosomal acidification have been shown to block immune activation (16)] and lack of uptake by specific immunocompetent cell populations (this system does not enter CD34 ϩ progenitor cells; J.D.H. and J. J. Rossi, unpublished data).…”