Metal-seeded lithium niobate nanoparticles as hybrid nanozymes for mimicking bioactivity

Nanozymes are nanomaterials that mimic natural enzymes. Recently, they have been positioned as a new emerging technology that has attracted particular attention over the last decade. Nanozymes, compared to natural and artificial enzymes, offer great value in terms of price and, especially, allow multi-functionalization due to their tunability in shape, size, type of material, type of coating or even the modulation of their physical properties through external stimuli such as light. These qualities make them attractive to be used in many different environmental and bio-related applications. Nanozyme-based sensors are one of the most appealing applications. The combination of Nanozymes with antibodies and antigens allows immunological analysis, especially in enzyme-based detection techniques such as enzyme-linked immunosorbent assay (ELISA). Aptamers, i.e. single-stranded DNA or RNA molecules that bind specifically to a target, are also attractive for the functionalization of Nanozymes as they reduce costs and diversify the types of targets that can be detected. Moreover, they have shown outstanding properties in one of the most critical calls of the World Health Organisation (WHO): point-of-care devices. Point-of-care Nanozyme-based sensors fulfill the criteria required by WHO of affordable, fast, sensitive, specific, user-friendly, equipment-free, robust and delivered to the end users. Nanozyme therapeutic applications are useful in many areas. For instance, some Nanozymes increase the production of reactive oxygen species (ROS) and thus induce oxidative stress, which is beneficial in antimicrobial applications or tumour cell treatment. On the contrary, other Nanozymes relieve the overexpression of ROS, reducing oxidative stress. Their use has been reported in anti-inflammatory processes or to decrease tumour micro-environment hypoxia. The latter application is not only beneficial to avoid healthy-cell apoptosis but also to ensure the correct functioning of external therapies requiring oxygen, such as photodynamic therapy or radiotherapy. Related to this, light interaction can be used to stimulate Nanozyme performance in therapeutics. Among Nanozymes, noble metal-based Nanozymes have been widely used, and their synthesis methods and ease of functionalization and modification make them attractive candidates. Likewise, their Nanozyme-mimic properties have been established. They have shown different enzyme mimics as peroxidases, oxidases, catalases and superoxide dismutases, which are tunable depending on intrinsic factors such as composition or shape and extrinsic factors such as light or pH. Catalytic activity is highly dependent on the size, and the best performances have been shown for metal nanoparticles with diameters below 5 nm. However, this can provoke an opposite effect: their aggregation and the loss of their catalytic activity. To overcome this, their deposition over metal oxides has been widely used in classic oxidoreduction catalysis. Additionally, their combination with metal oxide nanoparticles has resulted in a synergetic effect: properties of both materials are combined with an extra benefit, an increased catalytic performance due to charge transfer processes, band structure stabilization and defect creation. Among metal oxides, LiNbO3 has not yet been investigated in combination with metal nanoparticles as Nanozyme. Our group already investigated the synthesis of LiNbO3/Au hybrid nanoparticles composed of LiNbO3 nanoparticles (LN NPs), AuSeeds (Au nanoparticles below 5 nm diameter) using branched polyethyleneimine as a chemical link. Based on this, this work aims to prove the applicability of LiNbO3/metal nanoparticles as Nanozymes by extending the synthesis to other materials. We want to confirm the presence of a synergetic effect arising from the material combination. Additionally, we aim to prove their applicability in ROS-related applications. Chapter 1 presents the basic concepts of Nanozymes and the advantages of using metal-based ones. We also introduce the theory behind the catalytic synergetic effect between metal-metal oxide nanomaterials and their application in enzyme mimics. Then, we present the state-of-the-art of metal-based Nanozymes in ROS boosting and scavenging therapeutic applications with a special focus on metal-metal oxide hybrid Nanozymes and light interaction. Finally, the objectives of the thesis are outlined. Chapter 2 overviews the different characterization techniques and the principles behind them. The techniques for the characterization of nanoparticles are transmission electron microscopy (TEM), including the variations with scanning TEM (STEM), high-resolution TEM (HRTEM), energy dispersive X-ray spectroscopy (EDS or EDX). Other characterization techniques are dynamic light scattering (DLS), laser doppler velocimetry and electrophoresis (zetammetry), UV-vis spectroscopy and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The techniques for Nanozyme activity characterization and mechanistic elucidation are UV-vis spectroscopy, fluorescent spectroscopy and X-ray and UV photoelectron spectroscopy (XPS/UPS). Finally, Nanozymes and their building blocks interaction with microbial growth is studied through optical density measurements. Chapter 3 presents the synthesis and characterization of the potential hybrid Nanozymes. Initially, we detail the synthesis protocols of the starting building blocks of the hybrid nanoparticles, i.e., LN NPs, LN@BPEI (LiNbO3 NPs coated with branched-polyethyleneimine) and metal seeds (Au, Pt and Ag). Subsequently, the synthesis of the hybrid nanoparticles is presented. The characterization of the building blocks and the hybrid nanoparticles is discussed in terms of size and colloidal properties. Chapter 4 presents the optimization of the enzyme mimics of the hybrid nanoparticles. Parameters such as substrate, pH and temperature are varied and adjusted. Their Nanozyme peroxidase activity is established in the optimized conditions and compared to the separated counterparts. Subsequently, we verified if the Nanozymes and the metal Seeds follow the Michaelis-Menten natural enzyme kinetic model. Finally, mechanistic elucidation is carried out through band structure studies, supplementary catalytic studies, and ROS-traps tests. Chapter 5 focuses on microbial growth studies in the presence of Nanozymes and oxidative stress induced through hydrogen peroxide (H2O2) and UV light. Bacteria growth is initially studied in the presence of Nanozymes, followed by the introduction of an optimized quantity of H2O2 and UV light irradiation. Finally, the effect of combining the two oxidative stress sources with the Nanozymes is studied. The manuscript ends with the conclusions and future perspectives of this work.