Preparation and Characterization of Lipoplexes and Polyplexes

There is relatively little physical understanding of why certain preparation conditions of nonviral gene vectors generate better gene expression than others [68]. This deficiency has hindered the development of nonviral gene delivery vectors, since relatively few reports quantitatively evaluate DNA complex structure and composition, especially as it relates to gene delivery efficiency. Some structural and physical characteristics, including shape, size, and zeta potential, have been studied for various synthetic DNA delivery systems and have, in some instances, been related to transfection efficiency.

Shape. Lin et al. demonstrated a correlation between lipoplex structure and transfection efficiency [102]. Liposomes that formed hexagonal structures were more efficient in gene delivery than those that formed a lamellar, owing to improved fusion with mouse cell membranes (both endosomal and plasma membranes), whereas lamellar structures remained stable inside the cell. This correlation of structure and transfection efficiency was used to explain the increased efficiency of DOTAP:DOPE liposomes (hexagonal structures) compared to DOTAP:DOPC liposomes (lamellar structures) [102].

Polyplexes, made with cationic polymers such as PEI and PLL, have been reported to be both spherical and toroidal in shape [103]. The distinction between the two may be due to the visualization technique and sample preparation procedures [103]. Gebhart and Kabanov found that some polymers formed large aggregates when complexed with DNA, one of which (25K PEI) had high transfection efficiency, while the others did not [99]. Tang et al. studied PEI, dendrimers, and PLL and found that the type of polymer plays a role in aggregation and that models predicting electrostatic stability do not adequately describe the aggregation of polyplexes [103].

Size. Vector size has a marked effect on transport across several barriers to gene delivery in the lung, including mucus lining the airways and intracellular barriers. The ultimate size of the vector depends on a variety of parameters, including DNA and condensing agent concentrations, solvent choice and concentration, and the specific choice of condensing agent and its molecular weight [104,105]. The size of DNA condensates is nearly independent of plasmid size when the plasmid is between 400 and 50,000 bp for a variety of agents capable of condensing DNA, including cationic lipids, cationic polymers, and multivalent cations [104,106,107]. The cationic polymer, PEI, has been reported to condense and efficiently deliver yeast artificial chromosome (YAC) DNA that was up to 2.3megabase (Mb) pairs [108]. PEI/DNA polyplexes measured using transmission electron microscopy (TEM) and atomic force microscopy (AFM) have been reported to be between 20-100nm in diameter [75,109], while measurements using dynamic light scattering (DLS) reported polyplex sizes between 40 and 750nm [103,110,111].

Interestingly, larger PEI/DNA complexes were more effective transfection agents than smaller particles over a range of PEI molecular weights in one study

[111]. A possible explanation was that the "proton sponge" effect, as discussed later, may be more efficient with larger complexes. Larger particles may also sediment to interact with cells more readily under in vitro culture conditions

[112]. Enhanced transfection with larger vectors was also seen with lipid-DNA lipoplexes in CHO cells [113]. However, the opposite was found with linear PEI (L-PEI) in vivo. Smaller complexes led to higher levels of gene expression in adult and newborn mice, which correlated to their diffusivity through tissue [100]. In this study, L-PEI DNA complexes were shown to cross the endothelial cell barrier following intravenous administration and preferentially transfect pulmonary cells [92]. Cationic lipids, on the other hand, show some expression in pulmonary cells following intravenous administration but preferentially transfected endothelial cells [51,53,114,115], perhaps due to their reduced stability compared to polyplexes.

Surface Charge. DNA polyplexes with a positive surface charge have been found to translocate preferentially to the nucleus of cells [116-118]. One hypothesis is that high charge density endows the carriers with properties similar to nuclear localization sequences that are made up of cationic amino acids [119]. Another explanation may be that, following endocytosis, particles are delivered to the perinuclear region of cells as part of the normal cellular processing of endosomes [118]. Negatively charged complexes have more unbound phosphate groups (from DNA) than positive groups (from condensing species), such that the DNA is not fully condensed and may not be fully protected from degradation. Negatively charged complexes also are not taken up by cells as efficiently as neutral or positively charged complexes. As a result, negatively charged vectors provide lower transfection efficiency in general. Finally, as discussed more fully later, small neutral particles diffuse through the mucus barrier most rapidly [120], which may be a prerequisite to high transfection efficiency in the lung.

Density. Density of synthetic gene carriers [Eq. (1)] was recently correlated with transfection efficiency for both lipoplexes [121,122] and polyplexes [123,124], where Rg is the mean-square radius of gyration. Carrier density for nearly equal-sized vectors is a measure of the amount of DNA packed into each carrier. Therefore, higher-density vectors deliver more DNA per carrier particle that successfully reaches the cell nucleus. Also, high-density vectors may provide improved protection of DNA from degradation.

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Recently, we systematically varied PEI/DNA preparation parameters, including PEI molecular weight, N/P ratio, solution pH, and ionic strength, in order to determine their effect on vector size, molar mass, and density [124]. The molar mass of PEI/DNA polyplexes made with 25-kDa molecular weight PEI varied nonmonotonically with the N/P ratio, with a maximum value at N/P = 6. The maximum polyplex molar mass occurred at an N/P ratio near the transition between negatively and positively charged complexes, possibly due to reduced charge repulsion. Since these polyplexes typically had similar diameters but vastly different molar masses, their densities varied depending on N/P ratio and PEI molecular weight. Polyplexes made with and N/P ratio of 6 contained 60 times more DNA per complex than those made with N/P = 10. PEI/DNA complex molar mass changed by an additional order of magnitude over the range of PEI molecular weights studied when N/P = 6 and varied by two orders of magnitude when N/P = 10, but the geometric size and charge remained approximately the same [125]. The densest PEI/DNA polyplexes were those of PEI molecular weight and N/P ratio reported in the literature to have the highest transfection efficiencies [126].

Stability. Current protocols for nonviral delivery systems call for the preparation of the vector at the bedside, due to their aggregation over time [127-129]. Specifically, aggregation of some lipoplex formulations stored as liquids reduced their transfection efficiency [130,131]. Electron microscopy was used to show that PEI/DNA complexes did not aggregate, but vectors made with poly-L-lysine and dendrimers did in one study [103].

Recently, van Zanten and coworkers showed that PLL/DNA and PEI/DNA polyplexes maintain their size over long periods of time but decrease in molar mass quickly [121-125], suggesting that liquid storage of these polyplexes may reduce their efficacy.

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