Back in 2012, Professor Elena Ivanova and her colleagues hit a roadblock. They were trying to design surfaces with physical features that would repel bacteria but weren’t having much luck. Bacteria kept attaching to the nanoscale surfaces they were investigating – even to those that were nanoscopically smooth. So, they decided to change tack, drawing inspiration from surfaces that are naturally bacteria-free. First, they considered lotus leaves, which have a unique nanopatterned surface that traps air bubbles. This makes them super hydrophobic – a property that prevents bacteria from attaching. But when they attempted to recreate the lotus leaf topography on titanium, it didn’t hold air bubbles well enough to have the desired effect. Next, they tried cicada wings, another super hydrophobic surface. The team immersed a piece of cicada wing in a solution with bacterial cells overnight, expecting that no cells would attach. To their surprise, there was bacteria on the surface the next day – but they were dead.
“The morphology of the cells was compromised,” Ivanova, who undertook the research while at Swinburne University of Technology but is now distinguished professor at Royal Melbourne Institute of Technology (RMIT) University, recalls. “The wings of the insect remain clean, but not through the repelling of bacterial cells, through efficiently killing them on contact.”
It was a finding that would shape the direction of the field. Now, labs and start-ups around the world are hard at work developing surface topographies that can kill and repel bacteria through physical contact. These nanopatterned surfaces offer an alternative to traditional coatings such as silver nanoparticles, which kill bacteria by releasing chemicals. While these coatings are effective, there’s a risk that microbes will develop a resistance to them over time.
“Since then, what we see in the field is a huge race to create synthetic analogues of what nature has to offer,” says lecturer in nanomanufacturing at University College London Martyna Michalska.
Killing on contact
We’re still learning about how these surfaces kill bacteria, Michalska explains. But we know that the mechanical force on the bacteria when they interact with the surface is key. There are different ways that bacteria can become damaged and die. For one, cells can be stretched between nanopillars, Ivanova explains. “It’s like a balloon. If you start pinching a balloon, it won’t break on the tip of your fingers, it will break in between your fingers.” These can be long and slim, she adds, causing the membrane to bend and stretch. “The membrane elasticity will reach its limit, and it will also break.” Cells can also be cut or pierced on features that are very sharp, such as sharp tips or thin nanosheets that are like blades.
“As long as it’s a protruding surface,” says professor of biomedical materials at the University of Bristol Bo Su, “that’s what causes the impedance of bacteria”. Su and his colleagues identified another killing mechanism at play here: oxidative stress. When the bacteria hit the patterned surface and deform, they generate reactive molecules called ROS that damage cells, Su explains. “They’re actually killing themselves.”
Researchers are currently exploring which different patterns and features could kill bacteria more effectively, and in different ways. Yet there are important parameters we’re aware of, such as the height, diameter and spacing of nanopillars, says Ivanova. “Those three parameters are critical for achieving the maximum effect.” There are also groups, including Michalska’s, investigating whether bacteria can impale themselves or whether there are other forces, such as capillary action, that are causing this.
Because bacteria come in different shapes and sizes, the efficacy of different surfaces would depend on which species are present. For instance, you could expect cicada wing-like patterned surfaces to be effective around Gram-negative bacteria, Ivanova explains. As well as having a thinner cell wall than their Gram-positive counterparts, “Gram-negative bacteria are easier to kill because they have more surface area to interact with the pillars”.
Ivanova, Michalska and Su all agree that chemical coatings, such as silver, are likely to outperform a patterned surface in general. Yet while the efficacy of nanopatterns may vary, Su reports that these surfaces can still perform quite well. “Our killing [rate], normally, is from 90% to 60% or 50%, depending on the time scale.”
A clean surface
Nanopatterned surfaces can also work to prevent bacteria from adhering in the first place, a property known as antifouling. The patterns that we see in nature, such as on insect wings but also plant leaves and eyes, have the ability to control surface interactions with light, liquid and cells – including bacteria, Michalska explains. The joint effect of the pattern and surface chemistry “engineer the wetting properties of the surface”, she says.
Here, surface chemistry could be due to the inherent properties of the material being used or something that’s added to functionalise the surface. While the surface pattern – by trapping tiny pockets of air that block liquid from penetrating through – can physically make it difficult for microbes to stick onto it.
Though there could be a situation where the chemistry of the material changes due to shifts in the environment around the implant, Michalska notes, which might then interfere with its antifouling properties. How these surfaces may behave in the body over time is still something the field needs to investigate, she says. “It should be earlier, rather than later that we start looking into these long-term effects. Because we just don’t know.”
In addition, Su has observed that when the bacteria are physically deformed on the nanopattern, such as being impaled or pinned down, there’s no biofilm formation – when microbes attach to a surface and stick together, forming a community and introducing risk of infection. “If you want to prevent the infection, you want to prevent biofilm formation,” he says. “When you have [bacterial cells] pinned down, it’s not easy for them to divide or differentiate. So that actually delays or reduces the biofilm formation.”
Enhanced effects
Yet this nanopattern approach is not without shortcomings. For one, due to limitations in nanofabrication techniques, it can be difficult to create a pattern to the precise specifications you want. Plus, one pattern is never going to work for all bacterial species, says Michalska. “That’s why people are looking at augmenting that mechanical bactericidal activity.”
One way to do that is by functionalising the surface – adding chemical functional groups that alter its properties. For instance, Su and his team functionalised one of their nanopillar surfaces with an enzyme. “We know that enzymes are more lethal to Gram-positive bacteria,” he says. “And we noticed that this did actually kill more Gram-positive bacteria.”
You may also need to alter the surface properties based on the function of the implant itself. Su gives an example: you might have an implant that needs to promote growth of the host cell while keeping microbes at bay. Designing a surface that can do both is a challenge Su and his team have been working on over the past few years.
There may be additional requirements depending on where in the body the implant is going to be applied as well, adds Michalska. She hopes that in future, there will be more focus within the field on how surfaces could meet all these different criteria at once. “Right now, we’re still quite stuck in looking at ‘How do I increase the bactericidal activity of these surfaces?’”
Plus, having an additional antimicrobial mechanism up your sleeve can be useful if the nanopattern gets scratched or damaged. After all, the properties brought on by the physical pattern will only stay in effect if the pattern stays intact. Damage could happen if the implant was subject to screwing or hammering, for instance, while the features of the pattern might be altered if the surface becomes covered in microbial debris. “If one mechanism fails, you can still rely on the other,” says Michalska.
Worth the wait
It’s going to be a while before we see these surfaces investigated in clinical trials, let alone in medical devices on the market. But in the meantime, there’s been interest in non-medical applications, such as air or water filtration. These efforts could help us to better understand the antibacterial mechanisms at play, says Su. What about viruses and fungi? There’s been less research on this front, but we may see more movement in the coming years. Some nanopatterned surfaces might be effective on viruses, says Ivanova, but they’re particularly tough to kill because they’re so small. But, she adds, as nanofabrication becomes more advanced, we may soon be able to design surfaces with even tinier features. With fungi, Ivanova and her colleagues showed that a surface inspired by dragonfly wings caused rupturing of fungal spores. Improvements in nanofabrication technology will really help move the needle here, Ivanova emphasises. “The key is the precision at nanoscale, that’s the main challenge.” And our capabilities have already come a long way, she says. “Ten years ago, we couldn’t even dream that it would be possible to [create nanopatterns] with this precision.”
