Novel Monodisperse PEGtide Dendrons: Design, Fabrication, and Evaluation of Mannose Receptor-Mediated Macrophage Targeting

Novel PEGtide dendrons of generations 1 through 5 (G1.0-5.0) containing alternating discrete poly(ethylene glycol) (dPEG) and amino acid/peptide moieties were designed and developed. To demonstrate their targeting utility as nanocarriers, PEGtide dendrons functionalized with mannose residues were developed and evaluated for macrophage targeting. PEGtide dendrons were synthesized using 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocols. The N-alpha-Fmoc-N-epsilon-(5-carboxyfluorescein)-L-lysine (Fmoc-Lys(5-FAM)-OH) and monodisperse Fmoc-dPEG(6)-OH were sequentially coupled to Fmoc-beta-Ala-resin to obtain the resin-bound intermediate Fmoc-dPEG(6)-Lys(5-FAM)-beta-Ala (1). G1.0 dendrons were obtained by sequentially coupling Fmoc-Lys(Fmoc)-OH, Fmoc-beta-Ala-OH, and Fmoc-dPEG(6)-OH to 1. Dendrons of higher generation, G2.0-5.0, were obtained by repeating the coupling cycles used for the synthesis of G1.0. Dendrons containing eight mannose residues (G3.0-mannose(8)) were developed for mannose receptor (MR) mediated macrophage targeting by conjugating alpha-D-mannopyranosylphenyl isothiocyanate to G3.0 dendrons. In the present study PEGtide dendrons up to G5.0 were synthesized. The molecular weights of the dendrons determined by MALDI-TOF were in agreement with calculated values. The hydrodynamic diameters measured using dynamic light scattering (DLS) ranged from 1 to 8 nm. Cell viability in the presence of G3.0 and G3.0-mannose(8) was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and was found to be statistically indistinguishable from that of untreated cells. G3.0-mannose(8) exhibited 12-fold higher uptake than unmodified G3.0 control dendrons in MR-expressing J774.E murine macrophage-like cells. Uptake was nearly completely inhibited in the presence of 10 mg/mL mannan, a mannose analogue and known MR substrate. Confocal microscopy studies demonstrated the presence of significant intracellular punctate fluorescence colocalized with a fluid endocytosis marker with little surface fluorescence in cells incubated with G3.0-mannose(8). No significant cell-associated fluorescence was observed in cells incubated with G3.0 dendrons that did not contain the targeting ligand mannose. The current studies suggest that PEGtide dendrons could be useful as nanocarriers in drug delivery and imaging applications.