Cell internalization kinetics and surface charge accessibility of surface-modified PAMAM dendrimers.

Surface-modified PAMAM dendrimers have important applications in drug delivery, yet a gap remains about the role that surface functionalization plays on their cell internalization capacity. We examined the cell internalization kinetics of PAMAM dendrimers that were surface-modified with acetyl, folate and poly(ethylene glycol), as model functional groups differing in size, charge, and chemical functionality. Dendrimers with 25% functionalization were internalized by HEK cells, but with slower rates and lower maximum uptakes than the native dendrimer between 1-6 h of incubation. Dendrimers with 50% functionalization exhibited negligible internalization capacities at all incubation times. Molecular dynamics simulations revealed that the solvent accessibility of the cationic surface charges is a key factor affecting cell internalization, unlike the total charge, functionality or size of surface-modified PAMAM dendrimers. These findings provide valuable insights to assist the design of PAMAM-based systems for drug delivery applications.

Rational Discovery of Microtubule-Stabilizing Peptides.

Microtubule (MT) stabilization is an attractive pharmacological strategy to hamper the progress of neurodegenerative diseases. In this regard, seeking peptides with MT-stabilizing properties has awoken great interest. This work reports the rational discovery of two structurally related MT-stabilizing octapeptides using a combination of protein-peptide docking, conventional molecular dynamics, Gaussian accelerated molecular dynamics (GaMD), and tubulin polymerization assays. FASTA sequences for ∼1000 peptides were crafted from single and double mutants of davunetide (NAP) and docked against the Taxol (TX) site on an octameric MT model representing a portion of the MT wall. Docked peptides were rescored after MM minimization and binding free energy refinement through single-point MM/GBSA calculations. The 60 best-ranked peptides were subjected to 50 ns MD simulations on peptide-MT complexes at the terminal TX site in the octameric Tau-MT model resulting in 11 complexes with occupancies greater than 99% and peptide-protein binding free energies less than -40 kcal/mol. Selected peptides were then examined through 300 ns GaMD simulations in complexes containing two identical ligands at the terminal and intermediate TX sites in the Tau-MT model to account for the differential association of MT-binding peptides to different regions of the MT structure. Six candidates showed a favorable MT-binding potential based on the analysis of interaction frequencies and relative mobilities of the complex components, suggesting a pivotal role of Arg278, Gln281, and Arg369 residues for peptides recognition. Four candidates were predicted to preserve an adequate balance of longitudinal and lateral interactions between tubulin dimers in peptide-MT complexes such that MT-stabilizing effects could be expected. MT polymerization experiments confirmed that four peptides (HAPVSIHQ, NYPVSIHQ, NWPVSIWQ, HAPVSIIQ) exhibit MT-stabilizing activity in vitro with NWPVSIWQ (P43) and HAPVSIIQ (P52) being the most active. Tryptophan quenching assays verified that P43 and P52 bind to nonpolymeric tubulin, whereas viability experiments on HEK cells confirmed their safety to pursue future pharmacological studies. The results herein presented are valuable to making progress in the rational design of MT-stabilizing peptides.

Polyamidoamine-based nanovector for the efficient delivery of methotrexate to U87 glioma cells.

The purpose of this study was to design a polyamidoamine (PAMAM)-based nanovector for the efficient delivery of methotrexate to U87 glioma cells. To this end, 0-100% acetylated PAMAM dendrimers of the fourth generation were synthesized and evaluated using drug encapsulation measurements, molecular dynamics simulations, neurotoxicity assays and neuronal internalization experiments. The best system was tested as a nanovector for methotrexate delivery to U87 glioma cells. The authors found that 25% acetylated PAMAM dendrimers of the fourth-generation combine low intrinsic toxicity, large drug complexation capacity and efficient internalization into hippocampal neurons. Nanovector complexation enhances the cytotoxic response of methotrexate against U87 glioma cells compared with free drug solutions. In conclusion, 25% acetylated PAMAM dendrimers of the fourth-generation increase drug uptake by glioma cells and thereby act as efficient nanovectors for methotrexate delivery.