Supramolecular LEGO With Antifungal Properties

Researchers have reported synthesis of molecules that self-assemble into elongated nanostructures that rupture the cell walls of some fungi.No matter what you need to pierce — a dragon skin or a fungal membrane — you need a weapon, and a really small, nano-sized weapon would work best for fungi. Now imagine a “nano sword” that is attracted to fungi and ruptures the membrane all by itself!

This describes what likely is happening during the interaction of certain supramolecular, rodlike nano assemblies and fungi.

A collaborative team from IBM (U.S.A.), Yamagata University (Japan), the Institute of Bioengineering and Nanotechnology (Singapore), and Zhejiang University (China) has reported synthesis of amphiphilic molecules that undergo spontaneous self-assembly into elongated nanostructures that exhibit polymer properties and are toxic to fungi. The word amphiphilic describes their hydrophilic (water-loving, polar) and lipophilic (fat-loving) properties.

Here is how Kazuki Fukushima and his co-authors described their achievements on Dec. 9 in Nature Communications:

Here we present cationic small molecules that exhibit excellent microbial selectivity with minimal host toxicity. Unlike typical cationic polymers possessing molecular weight distributions, these compounds have an absolute molecular weight aiding in isolation and characterization. However, their specific molecular recognition motif (terephthalamide-bisurea) facilitates spontaneous supramolecular self-assembly manifesting in several polymer-like properties. Computational modelling of the terephthalamide-bisurea structures predicts zig-zag or bent arrangements […] Antifungal activity against drug-sensitive and drug-resistant strains with in vitro and in vivo biocompatibility is observed.

The scientists started with synthesis of the building blocks, cationic terephthalamide-bisurea compounds. Each building block has a zigzag shape, consisting of a rigid core (three benzene rings, connected by and rotated around peptide bonds), flanked by variable-length arms, either flexible (aliphatic) or rigid (aromatic), capped with cations (protonated aminogroups).

These zig-zag structures pack together into planar sheets stabilized by urea-urea hydrogen bonds and aromatic stacking. As the amide and urea groups are perpendicular, planar sheets further stack against one another, stabilized by hydrophobic interactions and amide-amide hydrogen bonds, generating a multilayer nanorod.

Think of building tall columns out of molecular Z-shaped LEGO blocks (aromatic stacking) and connecting a few together into thicker columns (hydrophobic and hydrogen bonding). The resulting nanostructures were 5-10nm in diameter and from hundreds of nanometers to several micrometers in length, as was observed by transmission electron microscopy (TEM).

The cationic surface of nanorods (zeta potential: 32–45 mV) would adhere to the cell wall of negatively charged fungi (zeta potential of Candida albicans: -4mV), causing membrane disruption and lysis. The scientists tested the building block molecules with different cationic arms, discovering that using more rigid and more hydrophobic arms resulted in higher antifungal activity.

Obtained cationic nanorods were efficient in killing C. albicans and C. neoformans. The efficiency was comparable to that of the antifungal drug fluconazole, but, unlike fluconazole, the nanorods did not promote drug-resistance in fungi when used at lower concentrations. Fluconasole-resistant strains were killed with same efficiency.

The nanorods were also shown to disrupt and lyse C. albicans biofilm on contact lenses. Fungal biofilm formation is a serious problem for implantable medical devices and typically biofilms demonstrate high resistance to any treatments. Finally, the cationic nanorods were active in vivo, in mice infected with fungal keratitis. A water solution of nanorods resulted in a reduction of fungal tissue invasion, as well as a reduction in the amount of viable fungi. The nanorod solution demonstrated excellent selectivity and mammalian cell biocompatibility.

A detailed characterization of the nanorods and their building blocks was undertaken to understand the mechanism of antifungal activity. The terephthalamide-bisurea compounds had a glass transition temperature (typical of polymers), as was confirmed by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The effect of the assembled structures on fungi was observed using scanning electron microscopy (SEM) and TEM before and after interaction with nanorods. Membrane rupture was associated with release of cytoplasm and generic material, confirmed by UV spectroscopy.

This work is a continuation of  previous research on self-assembly and properties of cationic polymers carried out by the same group of scientists. Publication in ACS Nano in 2012 described tri-block cationic polymers, consisting of a terephthalamide-bisurea core, a poly(lactide) interior block, and a cationic polycarbonate exterior block, and their self-assembly into rods and spheres. Although both shapes possessed broad-spectrum activity (against Gram-negative and Gram-positive bacteria), only the rodlike assemblies were effective against C. albicans. Inspired by the action of cationic antimicrobial peptides (see simulation video below) during the immune response, cationic nanostructures present an interesting and valid alternative to existing antimicrobial treatments.

Source: Supramolecular high-aspect ratio assemblies with strong antifungal activity, K. Fukushima K. et al., Nature Communications 4: 2861. doi:10.1038/ncomms3861, Dec. 9, 2013.
Source: Broad-spectrum antimicrobial supramolecular assemblies with distinctive size and shape. Fukushima K. et al, ACS Nano, Oct. 23, 2012;6(10):9191-9. doi: 10.1021/nn3035217.
Image by akunthita
Video Antimicrobial peptides by pinwheel2006, youtube