Efficient decoration of nanoparticles intended for intracellular drug targeting with targeting residues, as revealed by a new indirect analytical approach.

In our previous studies, we developed a nanodrug delivery system (nano-DDS) based on poly(lactic-co-glycolic acid) PLGA nanoparticles encapsulating antigenic peptide and fluorescent marker and 3-stage approach for its decoration with peptide targeting residues. The objectives of this study were (a) to develop methods for quantitative analysis of efficiency of individual conjugation steps and (b) to determine, based on these methods, the efficiency of our 3-stage approach of nano-DDS decoration. We prepared antigenic peptide-loaded PLGA-based nano-DDSs and sequentially decorated them with specific residues using carbodiimide and Click (azide-alkyne Huisgen cycloaddition using copper(I) catalysis) reactions. The extent of cargo encapsulation and release kinetics were analyzed using HPLC-based and colorimetric analytical methods. The efficiency of residue conjugation to the nano-DDSs was analyzed using FTIR spectroscopy and by quantifying the unreacted residues in the reaction mixture (i.e., by indirect analysis of reaction efficiencies). We revealed that copper, the catalyst of the Click reactions, formed complexes with unreacted targeting residues and interfered with the analysis of their conjugation efficiency. We used penicillamine (a chelator) to disrupt these complexes, and to recover the unreacted residues. Quantitative analysis revealed that 28,800-34,000 targeting residues (corresponding to 11-13 nm(2) surface area per residue) had been conjugated to a single nano-DDS using our 3-stage decoration approach, which is much higher than previously reported conjugation efficiencies. We conclude that the applied analytical tools allow quantitative analysis of nano-DDSs and the efficiency of their conjugation with targeting residues. The 3-stage decoration approach resulted in dense conjugation of nano-DDSs with targeting residues. The present decoration and analytical approaches can be effectively applied to other types of delivery systems and other targeting residues.

Toxicity assessment of extracts from infusion sets in cEND brain endothelial cells.

In vitro safety assessment of disposable medical devices, including infusion sets, is usually performed using L-929 mouse keratinocytes. However, cells of different origin (endothelial, lymphoid and myeloid cells) are also exposed to infusion sets' extractables during their clinical use. We studied whether the cEND mouse brain endothelial cells can be suitable for in vitro safety assessment of infusion sets. We analyzed infusion sets from different manufacturers that varied in design and storage time. cEND cells were incubated with extracts of individual parts of the infusion sets (tube, cup, latex), and relative toxicities were analyzed using MTT test, DCFH-DA-based analysis of reactive oxygen species formation, apoptosis and cell cycle analyses. We identified a pattern of yellowing of the infusion sets upon storage and revealed that it originated from the latex part. Extracts of the individual parts of the infusion sets, primarily of the latex, were toxic to the cEND cells leading to induction of apoptosis and cell death. We conclude that infusion sets release extractables that can be toxic to the endothelial cells of the patients that receive infusion. We suggest to use cEND cells for in vitro safety assessment of infusion sets and other medical devices that release extractables to the bloodstream.