Star-Like Oligo-Arginyl-Maltotriosyl Derivatives as Novel Cell-Penetrating Enhancers for the Intracellular Delivery of Colloidal Therapeutic Systems

A novel nonpeptide, multiarmed oligo-arginyl derivative was engineered as a cell-penetration enhancer for the delivery of bioactive macromolecules and colloidal drug systems. Hepta-arginyl-maltotriosylamido-N-acetyl-dodecanoyl acid (Arg(7)-Malt-NAcC12 acid) was synthesized through a carefully designed multistep chemical protocol, as follows: (1) maltotriose derivatization with 12-amino-dodecanoic acid and acetylation of the free amino group; (2) esterification of the maltotriosyl hydroxyl groups with 2-bromo-isobutyryl bromide; and (3) synthesis of star like oligomer bearing multiple copies of arginine moieties under atom transfer radical polymerization (ATRP) conditions. The intermediates and final product were characterized by H-1 NMR, IR, mass spectrometry, colorimetric assays, and elemental analysis. Cytotoxicity studies on the final polymeric material showed that this novel cell penetrating enhancer does not have significant toxic effects on MCF-7 and MC3T3-E1 cell lines. The IC50 was greater than 100 mu M with both cell lines, while the polyethylenimine with similar average molecular mass (M-n) that was used as a reference showed an IC50 of 30 and 40 mu M, for MCF-7 and MC3T3-E1, respectively. The biological properties of the novel bioconjugate were investigated using a fluorescein labeled bovine serum albumin (FITC-BSA) as a hydrophilic cargo model. MCF-7 and MC3T3-E1 cells were incubated for 60 min with the Arg(7)-Malt-NAcC12-conjugated FITC-BSA [(Arg(7)-Malt-NAcC12)(2)-FITC-BSA] or FITC-BSA, and the intracellular fluorescence level was analyzed by spectrofluorimetric analysis of cell lysate, cytofluorimetry, and confocal microscopy. The fluorescence of the lysate of MCF-7 and MC3T3-E1 cells that were incubated with (Arg(7)-Malt-NAcC12)(2)-FITC-BSA at 37 degrees C was approximately 4.5 times higher than the fluorescence obtained with cells incubated with FITC-BSA. At 4 degrees C, the cell uptake of (Arg(7)-Malt-NAcC12)(2)-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg(7)-Malt-NAcC12)(2)-FITC-BSA treatment, over 80% of MCF-7 cells and over 95% of MC3T3-E1 cells displayed enhanced fluorescence. Confocal investigations showed punctuated fluorescence within the cytosol in both cell lines, indicating that (Arg(7)-Malt-NAcC12)(2)-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.