Optimization of Cell Surface Binding Enhances Efficiency and Specificity of Molecular Conjugate Gene Delivery

Abstract

Molecular conjugates, or polyplexes, are promising synthetic vectors for targeted, in vivo gene delivery, if their efficiency can be improved. Gaining mechanistic information on conjugate gene delivery can potentially yield significant improvements in transfer efficiency by revealing barriers to conjugate transfer from the cell surface to the nucleus. We have developed an experimental system that employs epidermal growth factor as the ligand to direct delivery of DNA encoding the green fluorescent protein to mouse fibroblasts. We report here that the initial step of delivery, binding of the conjugate to the cell surface, is a barrier to gene transfer. We examined the effects of conjugate charge, ligand crosslinker spacer length, and ligand valency on polyplex cell surface binding, internalization, and gene transfer. We find that delivery is both efficient and specific only within a relatively narrow window of conjugate charge, results that correlate with binding and internalization of radiolabeled conjugate. In addition, increasing the cross-linker length can improve binding affinity and delivery. Finally, there is a significant optimum in gene delivery as a function of ligand valency, due to saturation of receptor binding and internalization. Optimizing parameters that affect surface binding therefore improves the efficiency and specificity of molecular conjugate gene delivery.

A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery

“…Though targeting often increases transfection, some experiments show that uptake that is both specific and efficient only occurs at a narrow window of charge ratios or not at all. 25,26 To generate cell-specific targeting, we have looked at two complementary approaches for attaching targeting ligands to our polymeric gene delivery nanoparticles. The first approach is the covalent modification of the polymer side chain to allow for facile ligand incorporation.…”

An integrin-targeted non-viral vector for pulmonary gene therapy

“…The median luciferase activity with the LID vector was three times greater than with the LSD vector suggesting that the LID vector is acting, at least partly, via integrin-mediated mechanisms. Gene transfer by non-viral systems relies in part on their physical characteristics, 12 and both the LID and LSD complexes have similar positive charge and size characteristics, which may account for the residual activity of the LSD complex.…”

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells

“…To visualize the uptake and transfection in cell cultures, we used a rhodamine-labeled, GFP-encoding cDNA cross-linked to b-anti-PECAM by SA-polylysine 35 (Figure 7). Rhodamine and GFP fluorescence colocalized in REN/PECAM cells incubated with anti-PECAM/SA-polylysine/DNA (350 nm in diameter), yet not all DNA-labeled cells expressed GFP ( Figure 7B).…”

“…Poly-L-lysine (181 lysine residues) and SA were chemically cross-linked with N-succinimidyl 3-[2-pyridyldithio]proprionate as previously described. 35 Per 1 g DNA, 1.5 g SA-polylysine (electroneutral with DNA) was incubated on ice for 10 minutes, and then 6 g antibody was added. Polyplex synthesis in 1 M NaCl/20 mM Hepes buffer and 0.1 M NaCl/2 mM Hepes provided smaller (diameter, 350 Ϯ 28 nm) and larger (diameter, 4200 Ϯ 248 nm) polyplexes, respectively.…”

“…Available data show that optimal particle size threshold for intracellular uptake varies in different cell types. 7,12,[33][34][35][36] Although macrophages internalize large complexes of 1 m in diameter or larger, [37][38][39] little is known about how the size of complexes affects their uptake by other cell types. There are no studies on effects of size on endothelial internalization via constitutive surface adhesion molecules.…”