Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs.

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG5K-succinyl-ACDCRGDCFCG-COOH (DSPE-PEG5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of alphavbeta3 and alphavbeta5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.

Toward targeted oral vaccine delivery systems: selection of lectin mimetics from combinatorial libraries.

PURPOSE: Various lectins bind specifically to oligosaccharides on intestinal cells. Exploiting this specificity, Ulex europaeus agglutinin I (UEA1) has been used as a ligand for targeted oral vaccine delivery to M cells (antigen-presenting cells) in follicle-associated epithelium. In this study we characterized compounds identified from mixture-based positional scanning synthetic combinatorial libraries, which mimic UEA1 and, thus, may have properties applicable to targeted drug delivery. METHODS: Two UEA1 mimetics were synthesized and their activity was verified on live cells. The ability of the lead compound, a tetragalloyl D-Lysine amide construct (4-copy gallic acid construct), to deliver dye-loaded polystyrene particles to M cells was assessed in an in situ mouse gut loop model. RESULTS: The 4-copy gallic acid construct inhibited UEA1 binding to Caco-2 cell membranes with an IC50 of 3 microM, a 650- to 5000-fold increase over the natural UEA1 substrate alpha-L-fucose. The biotin-labeled derivative of this construct demonstrated comparable binding activity as verified on live cells by fluorescence-activated cell sorting. Preclinical studies confirmed its ability to mediate M cell-specific delivery of streptavidin-coated particles in vivo. CONCLUSIONS: Polyphenolic compounds, D-Lysine scaffolds with multiple galloyl groups, can mimic functional activities of UEA1. Properties of such molecules, including low molecular weight, stability, ease of synthesis and low cost, highlight their potential for application in targeted vaccine delivery.