ASSESSMENT OF THE BACTERICIDAL EFFECT OF BIOMIMICKED NANOPILLARS OF CICADA WINGS ON TITANIUM IMPLANTS Hesam Shahali M.Phil., BSc Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Mechanical, Medical and Process Engineering Science and Engineering Faculty Queensland University of Technology 2020 Keywords Bactericidal surface, nanopillars, natural bactericidal surface, surface characteristics, antibacterial nanostructure, cytocompatibility, biocompatibility, titanium, electron beam lithography, helium ion microscopy, scanning electron microscopy, atomic force microscopy. Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants i Abstract Bacteria colonization and biofilm formation are the major causes of infection in implants. The annual cost of implant-associated infections in the US is 150-200 million USD and 7-11 million Euro in the UK. It is therefore vital to eliminate the bacterial attachment and biofilm formation from the surface of implants. Currently, chemical- based detergents and traditional antibacterial/antibiotic coatings are used to produce antibacterial surfaces. These products are, however, not always effective since biofilm can still form on the implant surfaces. Moreover, there is an additional problem of long-term efficiency. Nanopillars on cicada wings have recently drawn the interest of scholars due to their bactericidal, self-cleaning and superhydrophobic characteristics. This research aimed to (i) systematically characterise and assess the bactericidal and cytocompatible characteristics of the insect wings of three cicada species, (ii) mimic the wings’ nanopillars architecture on titanium substrates using electron beam lithography and (iii) perform a simulation analysis to find the most optimum fabrication method. The nano topography of three Australian cicadas (Psaltoda claripennis (PC), Aleeta curvicosta (AC) and Palapsalta eyrei (PE)) were characterised using scanning electron microscopy (SEM), helium ion microscopy (HIM), atomic force measurement (AFM) and transmission electron microscopy (TEM). Chemical characteristics of nanopillar surface were investigated using X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (FTIR). Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria were selected for the antibacterial study and human osteoblast cell lines were used to study the biocompatibility of the insect wing. Bactericidal efficiency and biocompatibility were evaluated through the plate count method and AlamarBlueTM assay, respectively. The nanopillars on cicada wings were mimicked on titanium substrates using Electron Beam Lithography (EBL) and the process variables were optimised to achieve the closest nanopillar architecture to the cicada wings. The wings of all the tested cicada species possessed unique nanopillars architecture on the vein and membrane. The geometry of nanopillars (e.g. diameter, height, centre to centre distance, density, and aspect ratio) differed among the species as well as among the membrane and veins. The aspect ratio and density of nanopillars were considerably higher on membranes than on veins. Microscopy analysis of ii Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants bacteria attachment showed that both bacteria were killed on nanopillars. Nanopillars of cicada wings significantly reduced P. aeruginosa colonies after 18 hrs compared to the control surface. A considerable reduction in S. aureus was found after 2 and 4 hrs compared to the control surface. Species PE and AC produced the highest bactericidal effect after 18 hrs with values of 7.3 × 105 and 1.43 × 106 (CFU/mL) against P. aeruginosa and S. aureus, respectively. All three cicada species produced cytocompatibility in response to human osteoblasts. The human osteoblast cell morphology remained undamaged, demonstrating the biocompatibility of the insect wing surfaces. In the second stage, EBL was employed to mimic the cicada nanopillars on titanium substrates through a systematic design and modelling approach. Monte Carlo simulation was used to optimize the beam energy and pattern design (dot and circle) prior to the experimental study. EBL process variables including write field, pitch, and more importantly, exposure factor (EF) were optimized to fabricate titanium nanopillars close to those of the PE species as the most effective antibacterial surface. Three groups of titanium nanopillar arrays were fabricated: (i) Based on the simulation of a circle-pattern diameter of 70 nm and centre to centre distance of 160 nm, the fabricated nanopillar array had a base diameter of 94.4 nm, top diameter 12.6 nm, centre to centre distance 165.8 nm, height of 115.6 nm and aspect ratio 2.16. (ii) Based on the simulation of a circle pattern diameter of 120 nm and centre to centre distance of 200 nm, the fabricated nanopillar array had a base diameter of 148.6 nm, spike diameter of 21.05 nm, centre to centre distance of 200.3 nm, height of 221.6 nm and aspect ratio 2.32. (iii) Based on the simulation of a circle pattern diameter of 200 nm and centre to centre distance of 320 nm, the fabricated nanopillar array had a base diameter of 214 nm, spike diameter of 48.9 nm, centre to centre distance of 324.9 nm, height of 288 nm and an aspect ratio of 2.19. While the three fabricated groups of titanium nanopillar surfaces damaged the membrane of P. aeruginosa and S. aureus, the first group produced the best bacteria killing performance in the same manner as PE cicada wings. The titanium nanopillars were biocompatible with human osteoblasts, enhancing cell anchorage and proliferation. To conclude, the assessment of bactericidal activity and the biocompatibility of natural cicada nanopillars and the versatile design of biomimicked titanium nanopillars will enable biomedical engineers to identify an ideal solution for design and manufacture of biomedical devices, such as orthopaedic implants. Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants iii List of Publications Journal articles • Hesam Shahali, Jafar Hasan, Asha Mathews, Hongxia Wang, Cheng Yan, Tuquabo Tesfamichael, Prasad KDV Yarlagadda, Multi-biofunctional properties of three species of cicada wings and biomimetic fabrication of nanopatterned titanium pillars, 2019, Journal of Materials Chemistry B, 7(8), 1300-1310 (DOI: 10.1039/C8TB03295E). • Hesam Shahali, Jafar Hasan, Hongxia Wang, Tuquabo Tesfamichael, Cheng Yan, Prasad KDV Yarlagadda, Evaluation of particle beam lithography for fabrication of metallic nano-structures, 2019, Procedia Manufacturing, 30, 261-267 (DOI: 10.1016/j.promfg.2019.02.038). • Hesam Shahali, Alka Jaggessar, Prasad KDV Yarlagadda, Recent Advances in Manufacturing and Surface Modification of Titanium Orthopaedic Applications, 2017, Procedia Engineering, 174, 1067-1076 (DOI: 10.1016/j.proeng.2017.01.259). • Alka Jaggessar, Hesam Shahali, Asha Mathew, Prasad KDV Yarlagadda, Bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants, 2017, Journal of Nanobiotechnology, 15(1), 64 (DOI: 10.1186/s12951-017-0306-1). • Shahali, Hesam, Hasan, Jafar, Cheng, Han-Hao, Ramakrishna, Seeram, Yarlagadda, Prasad KDV, A systematic approach towards biomimicry of nanopatterned cicada wings on titanium using Electron Beam Lithography (under review in Nanotechnology) Conference paper • Hesam Shahali, Alka Jaggessar, Prasad KDV Yarlagadda, Recent Advances in Manufacturing and Surface Modification of Titanium Orthopaedic Applications, 2016 Global Congress on Manufacturing and Management, China. iv Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants • Hesam Shahali, Jafar Hasan, Hongxia Wang, Tuquabo Tesfamichael, Cheng Yan, Prasad KDV Yarlagadda, Evaluation of particle beam lithography for fabrication of metallic nanostructures, 14th Global Congress on Manufacturing and Management, Brisbane, Australia (GCMM-2018). Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants v Table of Content Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii List of Publications .................................................................................................................. iv Table of Content ...................................................................................................................... vi List of Figures .......................................................................................................................... x List of Tables ......................................................................................................................... xxi List of Abbreviations ........................................................................................................... xxiii Statement of Original Authorship ........................................................................................ xxv Acknowledgements ............................................................................................................. xxvi Chapter 1: Introduction .....................................................................................