Star-like oligo-arginyl-maltotriosyl derivatives as novel cell-penetrating enhancers for the intracellular delivery of colloidal therapeutic systems

A novel non-peptide, multi-armed 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 (Arg7-Malt-NAcC12 acid) was synthesized through a carefully designed multi-step 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-bromoisobutyryl 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 1H 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 M with both cell lines, while the polyethylenimine with similar average molecular mass (Mn) that was used as a reference showed an IC50 of 30 and 40 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 Arg7-Malt-NAcC12-conjugated FITC-BSA [(Arg7-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 (Arg7-Malt-NAcC12)2-FITC-BSA at 37°C was approximately 4.5 times higher than the fluorescence obtained with cells incubated with FITC-BSA. At 4°C, the cell uptake of (Arg7-Malt-NAcC12)2-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that after (Arg7-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 (Arg7-Malt-NAcC12)2-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.