Development, characterization and antibacterial activity of polyether-co-amide matrix films incorporating choline-calixarene nanoassembly

Introduction Calix[n]arenes are a family of phenolic-based macrocycles of great interest for their synthetic versatility
and ability to self-assembly in nanostructured systems. In the search for novel antibacterial agents, we previously
demonstrated that the micellar calix[4]arene amphiphile (Chol-Calix), bearing hydrophilic choline moieties and long
hydrophobic aliphatic chains, is a nanocarrier for antibiotics (ofloxacin, tetracycline and chloramphenicol) and possesses
intrinsic antibacterial properties also against antibiotic-resistant bacteria. To extend the research, here we develop a novel
material by incorporating the Chol-Calix into a polyether-co-amide matrix (Pebax®2533) and study its physico-chemical
and antibacterial properties.
Materials and Methods Flexible films based on Pebax®2533 loaded with Chol-Calix were prepared by solution casting
method. The films were characterized for morphology, phase miscibility, thermal stability, gas transport, spectral
properties, and Chol-Calix release. The antibacterial activity of the films, neat Pebax® and Pebax® blends loaded with
Chol-Calix (0.5, 1, 5 wt%), was evaluated against Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 6538
at different time intervals (2, 4, 6, 8, 10, 24 h) by cell number evaluation and time kill plots construction. The effect on
biofilm formation was estimated by biomass measurements. MTT assay was employed to investigate the interference on
vitality of mouse embryonic fibroblast cells (NIH-3T3).
Results The thermal stability of the copolymer was not affected by the Chol-Calix incorporation, nevertheless it was
detected an increase of crystallinity, gas permeability and wettability of the blend films according to the additive
concentration. Leaching of Chol-Calix was tracked by release tests. Neat Pebax® and Pebax®-0.5 wt% Chol-Calix
showed no significant antibacterial activity against both E. coli and S. aureus. Pebax®-1 wt% Chol-Calix displayed good
antibacterial activity against S. aureus, with a reduction of 1.8 and 2.1 log CFU/mL observed at 10 and 24 h, respectively.
A clear effect was observed with Pebax®-5 wt % Chol-Calix that reduced the number of viable E. coli cells of 2.57 and
2.66 log CFU/mL at 10 and 24 h, respectively. A similar trend was observed for S. aureus with a reduction of 2 log
CFU/mL at 10 h that increased to 2.49 log CFU/mL at 24 h. Pebax®-5 wt % Chol-Calix showed biofilm biomass reduced
(~30%) as compared to the neat polymer. The films were non-cytotoxic as revealed by MTT assay.
Discussion and Conclusions The results indicate the Pebax®/Chol-Calix combination as a promising approach for the
development of novel biocompatible flexible antibacterial thin-films upgradable by loading antibacterial drugs in the
Chol-Calix nanocarrier.