How do nanopillars really kill bacteria?

With the rapid increase in hospital infections and antibiotic resistance, we urgently need improved methods to reduce bacteria growth and proliferation on surfaces. Inspired by the nano-protrusions on cicada and dragonfly wings, nanostructures have been bio mimicked onto materials to kill bacteria without harmful chemicals. However, scientists are only beginning to unravel the mysteries of how they work and the mechanisms of how nanopillars kill bacteria is still unclear.
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This study presents two goals: 1) To bio mimic and investigate the antibacterial difference of polar versus non-polar nanopillar polymers. 2) Provide an explanation for nanopillar mediated bacterial death to enhance the antibacterial activity of nanomaterials.
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Our investigation provides a hybrid approach that will advance solutions for antibiotic resistance and biofilm formation on polymers in medical and industrial settings.  In addition, providing a greater understanding of this very unexplored phenomenon, this study will catalyze the design of more effective antibacterial biomaterials.

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This diagram represents the proposed biophysical model of bacterial death, that gravitational and mechanical force deform the bacterial membrane, causing bacteria death. However, this force would no longer exist if the entire system (bacterium and nanopillar) was immersed in a liquid medium. Most of these studies overlook the role of surface charge on bacteria adhesion and death,  especially on nanostructured surfaces. Since electrostatic charge influences the polarity of the bacterium to function optimally, it is an extremely important physicochemical parameter to investigate.
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The insect-inspired nanopillars were imprinted on the polymers via hot embossing, an effective nanoimprinting process. Polymethyl methacrylate (PMMA) and polycarbonate (PC) are polymers chosen in this experiment as they are commonly used for medical devices or everyday items because of their transparency, strength, and environmentally friendly properties. Cyclic olefin copolymer (COC) and high-density polyethylene (HPDE) were also chosen for their chemical inertness properties while also having many common applications. The NT-MDT Raman Microscope was used to characterize the differences in molecular features of the polymer once they have been imprinted with nanopillars. Raman spectroscopy analysis showed a difference in the bond behavior of polymers with and without nanopillars. Imprinting nanopillars increased the functional groups sticking out on the surface of polar polymers.

The nano-pillared polar cationic polymers resulted in a significant reduction in bacteria biofilm formation, while the nanopillar non-polar polymer had similar amounts of biofilm formation as a regular flat polymer. Image shows: Antibiofilm activity of the polymer films against Pseudomonas (PAo1). Flat control surface on the bottom, nanopillar surface on top. (a) COC nanopillar and flat surfaces, PAo1 appeared to form similar amounts of biofilm. (b) PMMA nanopillar and flat surfaces, PAo1 appeared to form larger biofilms on flat surfaces. (c)PC nanopillar and flat surfaces, PC significantly reduced biofilm formation. 

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In our study, flat charged surfaces, such as PC and PMMA, resulted in profuse bacteria growth (highest CFU/ uL), even when compared to chemically inert surfaces, such as COC and HDPE which didn’t inhibit biofilm formation. This may have resulted from greater initial adhesion of bacteria to the surfaces due to the electrostatic interactions between the slightly positive charge of the flat PC and PMMA polymers and the overall negative charge of Pseudomonas. The CFU and SEM results show that PC and PMMA nanopillars kill more bacteria than inert nanopillar films, since both PMMA and PC have a slight positive charge. 
These results explain how polar nanopillars and cicada wing nanopillars kill bacteria, supporting the hypothesis that electrostatic interactions between a bacterium and a positively charged nanostructured surface can cause bacterial adhesion and turgor pressure, leading to cell death. ​

  (a) This is a representation of the zeta potential of bacteria, which represents the  net surface charge of bacteria. Taken from (b) This demonstrates bacteria adhesion onto surfaces and the bacterial membrane pushing against the nanopillars, increasing turgor pressure until the cell cannot take the pressure any longer.

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Challenges: Imprinting Nanopillar surfaces on polymers with different properties (glass transition temp, polarity, melting point, surface chemistry, density, modulus)