591. Particle Tracking for Intracellular Trafficking of Octaarginine-Modified Liposome: Comparative Study with Adenovirus

CHEMICAL AND MOLECULAR CONJUGATES 590. The Effects of PNA-Based prePEGylation on the In Vitro and In Vivo Properties of DNA Polyplexes Peter G. Millili,1 Ulhas P. Naik,2 Millicent O. Sullivan.1 Chemical Engineering, University of Delaware, Newark, DE; 2 Biological Sciences, University of Delaware, Newark, DE.

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Gene therapy holds the potential to revolutionize disease treatment, but depends upon successful nucleic acid transport and intracellular delivery. Nanoparticle (NP) nucleic acid formulations are in principle ideal for gene therapy: NPs are small enough to be ingested by cells and to access molecular-scale transport mechanisms, but large enough to contain full-length genes as well as cellular and intracellular targeting moieties. Unfortunately, common non-viral packaging techniques often employ cytotoxic polycations that assemble with DNA into polydisperse NPs whose properties are not optimal for in vivo applications. Thus, we have developed an alternative approach for DNA packaging. Our approach involves the direct functionalization of DNA with poly(ethylene glycol) (PEG) via peptide nucleic acid (PNA) clamps. DNA prePEGylation enables the formation of homogeneous polyplex NPs with low cytotoxicities and the capacity for efcient gene transfer. In this work, DNA-PNApeptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). These complexes exhibit low polydispersity and average diameters ranging from 30 – 50 nm, with minimal dependence of complex size on N:P ratio (PEI amines to DNA phosphates). PEI-DNA interactions were altered by the derivitization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison with PEI-DNA complexes. The PEI-DP3 complexes have also been explored in transfection experiments in Chinese Hamster Ovary (CHO) cells. The prePEGylated complexes exhibited comparable transfection efficiencies but significantly reduced cytotoxicities relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. Because of the favorable properties of the PEI-DP3 complexes, ongoing work is focused on the exploration of the properties of these new particles for in vivo applications. We have added various cell targeting peptides, including the RGD peptide, to the surface of the PEI-DP3 NPs. We are exporing the in vivo biodistributions of these NPs in normal and tumor-bearing mice as a function of NP size and targeting peptide density. Preliminary ndings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.

591. Particle Tracking for Intracellular Trafcking of Octaarginine-Modied Liposome: Comparative Study with Adenovirus

Hidetaka Akita, Kaoru Enoto, Tomoya Masuda, Hiroyuki Mizuguchi, Tomomi Tani, Hideyoshi Harashima. Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo City, Hokkaido, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, Sapporo City, Osaka, Japan; Research Institute for Electronic Science, Hokkaido University, Sapporo City, Hokkaido, Japan.

We previously reported that octaarginine (R8)-modied liposomes (R8-Lip) are potent carriers for encapsulated plasmid DNA (pDNA) and have a high rate of gene transfection activity. The liposomes are taken up via macropinocytosis, a unique pathway that circumvents lysosomal degradation, with efficeint delivery to the nuclear periphery. However, little is known concerning their movement through the cytosol, where the diffusion of pDNA is strictly limited. In the present study, we investigated the mechanism of cytoplasmic Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

transport of R8-modied liposomes compared with that of adenovirus using a particle tracking technique. The transfection activity of pDNAencapsulating R8-Lip was drastically inhibited in the presence of a microtubule disruption reagent (nocodazole) with a higher degree (98.8%) compared with that of adenovirus (75.9%). Directional transports of R8-Lips along green orescence protein (GFP)-tagged microtubules was observed. Furthermore, these directional motions were abrogated in R8-Lips by the disruption of microtubules, while adenovirus continued to undergo random motion. It suggests that the nuclear access of R8-Lip dominantly involves microtubule-dependent transport, whereas an alternative mechanism (i.e. apparent diffusive motion) is also operative nuclear access of adenovirus. Quantum dot-labeled pDNA in R8-Lips shared the directional motion with rhodamine-labeled lipid envelopes, indicating that R8-Lips were subject to the microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip is directionally transported and associated with endosomes, whereas this occurs after endosomal escape in the case of adenovirus. The directional transport of endosomes when associated with R8-Lip is slower than that for adenovirus and endosomes that are devoid of R8-Lip, suggesting that the loading of nanoparticles affects the speed of vesicular transport, or that macropinosomes may be transported more slowly than early and/or late endosomes. Collectively, vesicular transport plays a key role in the cytoplasmic transport in R8-Lips.

592. Advanced In Vitro Models Combined with Mathematical Representations Yield New Quantitative Understanding of Synthetic Gene Delivery System Performance

Michelle C. Lowe,1 Todd D. Giorgio.1,2 Department of Biomedical Engineering, Vanderbilt University, Nashville, TN; 2Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN. 1

In vivo performance of synthetic gene delivery systems is poorly predicted by corresponding results of in vitro studies conducted in cell culture models. Cell culture systems poorly reproduce critical in vivo characteristics, including the effects of cardiovascular uid forces and gene delivery complex transport in three dimensional tissues. This study uses a mathematical model to quantitatively uncouple two early events in synthetic gene delivery under simulated cardiovascular system uid forces: transport of delivery complexes to the cell from the bulk uid and association of delivery complexes with the cell surface. Model inputs are obtained from synthetic gene delivery experiments performed previously under controlled convective ow in a parallel plate ow chamber. Lipoplexes [1] and His-pLK or PEI [2] polyplexes served as gene carriers to ECV-304 and human lung microvascular endothelial cells, respectively. Results for delivery complex diameters ranging from 100-450 nm and uid shear stresses ranging from 0.06-9.7 dyn/cm2 support an increase in transport efciency (which quanties gene complex delivery to the cell surface) with increasing delivery complex size and a decrease in transport efciency with increasing shear stress. The collisional efciency (which quanties irreversible gene complex binding to the cell surface) decreased as shear stress increased, presumably reecting the interaction of complex-cell association characteristics and uid force exposure. Interestingly, collisional efciencies of lipoplexes were six orders of magnitude greater than for polyplexes when compared under similar uid transport conditions that reect the human vasculature. The addition of serum resulted in negative zeta potentials for the polyplexes, in contrast to the positive zeta potentials of the serum-free lipoplexes. Based on this result, zeta potential is hypothesized to be a likely modulator of collisional efciency in ow. The collisional efciency of polyplexes increased as the uid medium was changed from 10% serum (polyplex diameter 210 nm) to 100% serum (polyplex diameter 80 nm). Thus, under owing conditions, polyplex S229