Using the epigenetic code to promote the unpackaging and transcriptional activation of DNA polyplexes for gene delivery.

Nonviral gene delivery has seen limited clinical application due in part to the inefficiency with which most nonviral vehicles navigate the intracellular gene delivery pathway. One key problem is the inability of most DNA-packaging materials to release DNA and enable its efficient transcription. Thus, our aim was to develop gene delivery polyplexes capable of initiating their own transcription upon arrival in the nucleus. We created nuclease-resistant polyplexes with plasmid DNA (pDNA) and post-translationally modified histone 3 (H3K4Me3) tail peptides known to signal transcriptional activation on chromosomal DNA. When the H3K4Me3-pDNA polyplexes were directly microinjected into the nuclei of NIH/3T3 mouse fibroblasts, protein expression occurred earlier and in a greater fraction of cells than when polyethylenimine-pDNA polyplexes were microinjected. The rate of protein expression initiated by the H3K4Me3-pDNA polyplexes was also significantly accelerated in comparison with the rate initiated by non-trimethylated H3-pDNA polyplexes. These differences in protein expression rates were quantified by the development of a noncompartmentalized cellular kinetics model. These results highlight the importance of polyplex unpackaging as a gene delivery barrier, and demonstrate for the first time that the epigenetic code can be utilized in nonviral gene delivery.

Polyplexes traffic through caveolae to the Golgi and endoplasmic reticulum en route to the nucleus.

The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI-pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi-ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles.

Histone H3 tail peptides and poly(ethylenimine) have synergistic effects for gene delivery.

This goal of this work was to explore histone H3 tail peptides containing transcriptionally activating modifications for their potential as gene delivery materials. We have found that these H3 tail peptides, in combination with the cationic polymer poly(ethylenimine) (PEI), can effectively bind and protect plasmid DNA. The H3/PEI hybrid polyplexes were found to transfect a substantially larger number of CHO-K1 cells in vitro compared to both polyplexes that were formed with only the H3 peptides and those that were formed with only PEI at the same total charge ratio; however, transfection was similarly high for polyplexes both with and without transcriptionally activating modifications. Transfections with the endolysosomal inhibitors chloroquine and bafilomycin A1 indicated that the H3/PEI hybrid polyplexes exhibited slower uptake and a reduced dependence on endocytic pathways that trafficked to the lysosome, indicating a potentially enhanced reliance on caveolar uptake for efficient gene transfer. In addition, whereas PEI polyplexes typically exhibit a cytotoxic effect, the H3/PEI hybrid polyplexes did not compromise cell viability. In total, the current studies provide new evidence for the potential role for histone-based materials as effective gene transfer agents, and support for the importance of subcellular trafficking for nonviral gene delivery.

Effect of pharmaceuticals on thermoreversible gelation of PEO-PPO-PEO copolymers.

Pluronic F127, a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), has generated considerable interest as a drug delivery vehicle due to its ability to gel at physiological temperatures. This work examines the gelation behavior of Pluronic F127 in the presence of a series of hydrophobic pharmaceuticals, to determine whether there is any correlation between gelation and physicochemical parameters of drug solutes. The study includes the local anesthetics dibucaine, lidocaine, and tetracaine; the pharmaceutical additives methyl paraben, ethyl paraben, and propyl paraben; the anti-cancer agents paclitaxel and baccatin III; and the anti-inflammatory agent sulindac. The results indicate that the presence of local anesthetics and pharmaceutical additives allows F127 solutions to form gels at lower copolymer concentrations; local anesthetics and pharmaceutical additives also shift gelation down to a lower gelation temperature. This behavior is strongly dependent on drug solubility; poorly soluble drugs (paclitaxel, baccatin III, sulindac) do not change the lower gelation temperature or minimum F127 concentration for gelation. An equation relating the decrease in gelation temperature to drug solubility is presented, and the equation fits the data well. The results have significant positive implications on the toxicity and economic issues related to use of Pluronic F127 in drug delivery.

The effect of pharmaceuticals on the nanoscale structure of PEO-PPO-PEO micelles.

We present results on the effects of various hydrophobic drugs and additives on the micellar structure of Pluronic F127 solutions. Small-angle neutron scattering experiments on 5wt% F127 solutions were used to measure micelle core size (R(1)), micelle corona size (R(2)), intermicellar interaction distance (R(int)), polydispersity (sigma), and aggregation number (N(agg)); dynamic light scattering was used to measure critical micelle concentration (CMC); and ultraviolet spectroscopy was used to measure drug solubility and apparent micelle-water partition coefficient (K(mw)). The core and corona size were found to generally increase in the presence of the drugs, as did R(int). Both sigma and N(agg) were found to decrease in the presence of most of the drugs, and the CMC was found to vary considerably with no clear correlation. A design of experiments (DOE) approach was used to analyze the results and build empirical correlations. All of the parameters from the SANS experiments were found to depend strongly on drug solubility, with a weak dependence on K(mw) in most cases. The aggregation number, however, was found to depend strongly on both K(mw) and solubility. The correlations can be used to roughly predict the structural parameters of F127 micelles for other hydrophobic drugs.