pH-responsive lipid core micelles for tumour targeting.

A new acid-sensitive drug-delivery nanocarrier has been developed for tumour targeting. The self-assembling co-polymer stearoyl-PEG-poly-sulfadimethoxine methacrylate (stearoyl-PEG-polySDM) was prepared to obtain micelles with responsive behaviour in the physiopathologic pH range. Stearoyl-PEG-polySDM was synthesised using a multi-step procedure that includes pH-sensitive sulfadimethoxine methacrylate polymerisation by AGET-ATRP at the amino terminal side of stearoyl-PEG-NH2. Chemical analysis showed that the stearoyl-PEG-polySDM co-polymer contained a mean of seven methacryloyl sulfadimethoxines per molecule. Potentiometric and turbidimetric analyses showed that stearoyl-PEG-polySDM has an apparent pKa of 7.2 and a cloud point at pH 7.0. In water at pH 7.4, the co-polymer assembled spontaneously into 13.2±3.1 nm micelles with a critical micelle concentration (CMC) of 36 µM. Cell-culture studies showed that the material was more biocompatible with respect to the control Brij-700®. The paclitaxel loading capacity of the micelles was 3.25±0.25% (w/w, %). The colloidal formulations were stable at pH 7.4 for several hours, while at pH 6.5, they rapidly rearranged and aggregated. Fluorescence spectroscopic and cytofluorimetric studies showed that the incubation of MCF-7 tumour cells with fluorescein-labelled stearoyl-PEG-polySDM at pH 6.5 resulted in massive time-dependent cell association, while the incubation at pH 7.4 showed significantly lower cell interaction. Confocal microscopy confirmed that at pH 6.5, the micelles are taken up by cells and that the fluorescein-labelled stearoyl-PEG-polySDM is distributed into the cytosol. At pH 6.5, paclitaxel-loaded stearoyl-PEG-polySDM micelles had a higher cytotoxic effect than the micelles incubated at pH 7.4. The former displayed similar cytotoxic activity to free paclitaxel.

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-NAcC(12) 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 (1)H 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 IC(50) was greater than 100 µM with both cell lines, while the polyethylenimine with similar average molecular mass (M(n)) that was used as a reference showed an IC(50) of 30 and 40 µM, 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-NAcC(12)-conjugated FITC-BSA [(Arg(7)-Malt-NAcC(12))(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-NAcC(12))(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 (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg(7)-Malt-NAcC(12))(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-NAcC(12))(2)-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.