Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

Abstract

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.

Figure 1

Normalized transfection efficiency (A) and percentage of cells transfected (B) of targeted and untargeted block copolymer formulations at various polymer:DNA ratios. Transfections of EGFP were performed in 10% serum and analyzed 48 hrs later using flow cytometry. An optimized formulation of PEI at a polymer:DNA ratio of 2:1 was used and data is reported as mean fluorescence intensity normalized to that of the PEI transfected cells. Error bars represent SEM of 5 replicate experiments (*** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant). These results are in agreement with previously published reports of transfection efficiency of this block copolymer system (26).

Figure 2

Uptake of polyplexes formed by targeted and untargeted block copolymers against PEI. FITC-labeled polymers were complexed with EGFP plasmids and added to DU145 cells in 10% serum. Median cell associated fluorescence was measured at 30 min, 1h, and 4h as indicated in panel A (w/w = 25:1 for block copolymers). Panel B shows the effect of varying the polymer:DNA ratio of the block copolymers. Fluorescence was measured at 4 h in panel B. PEI was used at an optimized w/w ratio of 2:1. Error bars represent SEM of 5 replicate experiments (*** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant).

Figure 3

Panel A is a schematic of the high-throughput calcein assay used to quantify endosomal escape. When calcein alone is taken up by cells, it remains in the endosomal compartments and thus a punctuate pattern of fluorescence in the cytoplasm is visible. With the addition of an endosomal escape agent, e.g. a polymer or polyplex, the calcein is released into the cytosol yielding a uniform pattern of fluorescence. The images in panel A are representative of these morphologies and taken at 20x. The images have been intensity normalized for this figure in order to more easily visualize the punctuate vs diffuse pattern of fluorescence. Nuclei are shown in blue, calcein as green. Panel B is a grayscale image of the calcein channel of DU145 cells treated with G5-PEG-WIFP polyplexes. The Cellomics algorithm has differentiated cells with endosomal escape (blue circle overlay) vs. those without (orange circle overlay). In panel C, the concentration of PEI is increased as is the time in which the cells were exposed to the calcein/escape agent. Panel D shows the percentage of escaped cells from treatments with various polyplex formulations in OPTI-MEM with 10% serum. PAMAM based polyplexes were used at a 25:1 w/w ratio. PEI was used at 2:1 w/w ratio. Error bars are SEM of 3 replicates (*** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant).

Figure 4

Confocal fluorescence micrographs of cells transfected with targeted/untargeted block copolymers as well as PEI. Transfection took place in 10% serum and micrographs were taken on fixed cells 24h after polyplexes were added. Scale bars are 5 microns. In the merged channel, yellow indicates colocalization of polymer (green) and DNA (red).

Scheme 1

Synthesis of PAMAM-G5-PEG-WIFP conjugates. The targeting peptide is first reacted with a bifunctional linker before being precipitated and added to reduced G5 PAMAM dendrimer.