Bacterial Envelope Damage Inflicted by Bioinspired Nanostructures Grown in a Hydrogel

Abstract

Surface-associated bacterial communities, known as biofilms, are responsible for a broad spectrum of infections in humans. Recent studies have indicated that surfaces containing nanoscale protrusions, like those in dragonfly wings, create a hostile niche for bacterial colonization and biofilm growth. This functionality has been mimicked on metals and semiconductors by creating nanopillars and other high aspect ratio nanostructures at the interface of these materials. However, bactericidal topographies have not been reported on clinically relevant hydrogels and highly compliant polymers, mostly because of the complexity of fabricating nanopatterns in hydrogels with precise control of the size that can also resist aqueous immersion. Here, we report the fabrication of bioinspired bactericidal nanostructures in bacterial cellulose (BC) hydrogels using low-energy ion beam irradiation. By challenging the currently accepted view, we show that the nanostructures grown in BC affect preferentially stiff membranes like those of the Gram-positive bacteria Bacillus subtilis in a time-dependent manner and, to a lesser extent, the more deformable and softer membrane of Escherichia coli. Moreover, the nanostructures in BC did not affect the viability of murine preosteoblasts. Using single-cell analysis, we demonstrate that indeed B. subtilis requires less force than E. coli to be penetrated by nanoprobes with dimensions comparable to those of the nanostructured BC, providing the first direct experimental evidence validating a mechanical model of membrane rupture via a tension-induced mechanism within the activation energy theory. Our findings bridge the gap between mechano-bactericidal surfaces and low-dimensional materials, including single-walled carbon nanotubes and graphene nanosheets, in which a higher bactericidal activity toward Gram-positive bacteria has been extensively reported. Our results also demonstrate the ability to confer bactericidal properties to a hydrogel by only altering its topography at the nanoscale and contribute to a better understanding of the bacterial mechanobiology, which is fundamental for the rational design bactericidal topographies.

Effects of Nanopillar Size and Spacing on Mechanical Perturbation and Bactericidal Killing Efficiency

“…It is worth mentioning that certain studies could not be included or were intentionally omitted. This includes Mainwaring, et al [55] and Michalska, et al [14], which did not clearly specify nanopillar tip radius, and Hazell, et al [56] and Arias, et al [4], which involved nanopattern geometries that were significantly disordered.…”

“…The bactericidal efficacy of nanopatterning is not necessarily restricted to any one material or any one cell species. Nanopatterned surfaces of polymer [4,5], metal [6][7][8][9], and metalloid [10,11] material have all been demonstrated to kill several species of both gram-negative and gram-positive bacteria-in some cases even with antimicrobial resistant strains [8,9]. The ability of nanopatterning to confer a broad-spectrum killing effect on diverse materials has implied that the resulting surfaces operate on a mechanism that is predominantly physical.…”

“…Consistent with this mechanism, the killing efficiency of nanopatterned surfaces is known to be size sensitive; however, a clear design strategy remains elusive. Several experimental studies have established that killing efficiency can be modulated by only varying nanopattern dimensions, such as nanopillar radius, spacing and height [4,5,10,11,14,16,[19][20][21][22][23]. Such studies have been typically conducted by evaluating the killing efficiencies of different nanopattern designs from, either, (i) different cicada wing species [19,21,23], (ii) different exposure times with reactive ion etching [10,11,14], or (iii) different write patterns with electron beam lithography [16].…”

Ion Beam Nanopatterning of Biomaterial Surfaces

“…With few exceptions [128,129,137], the studies discussed in this review utilized ioninduced ripple patterns, presumably because ripple patterns are the most universal and can be generated on almost any surface. However, from a fundamental point of view, comparing the effects of different pattern types may be a rather interesting future endeavor.…”

“…Most remarkably, these high-aspect-ratio nanostructures did not collapse or change their shape upon heat sterilization and immersion in aqueous environments, rendering them suitable for a wide range of in vitro and possibly in vivo applications. In a follow-up study from the same lab, different BC types were compared, namely commercial BC (CoBC) and lab-grown BC (LgBC), which had different ribbon widths [137]. These differences resulted in smaller nanostructure dimensions for LgBC, despite maintaining a constant aspect ratio of abou 5.…”