Assembling patchy plasmonic nanoparticles with aggregation-dependent antibacterial activity

https://doi.org/10.1016/j.jcis.2020.07.006Get rights and content

Abstract

We realise an antibacterial nanomaterial based on the self-limited assembly of patchy plasmonic colloids, obtained by adsorption of lysozyme to gold nanoparticles. The possibility of selecting the size of the assemblies within several hundred nanometres allows for tuning their optical response in a wide range of frequencies from visible to near infrared. We also demonstrate an aggregation-dependent modulation of the catalytic activity, which results in an enhancement of the antibacterial performances for assemblies of the proper size. The gained overall control on structure, optical properties and biological activity of such nanomaterial paves the way for the development of novel antibacterial nanozymes with promising applications in treating multi drug resistant bacteria.

Introduction

The ability of handling materials at the nanoscale allows for developing multi-functional systems with highly programmable properties for a wide range of applications including biotechnology and nanomedicine [1], [2], [3]. Of particular relevance are novel artificial nanomaterials with enzyme-like properties, namely nanozymes, that demonstrated an intrinsic antibacterial activity [4], [5], [6] as well as the capability of enhancing or triggering the action of other agents [5]. Such systems are proving to be very effective as self-therapeutic systems in treating multi drug resistant bacteria [7], [8].

The antibacterial activity of nanozymes based on inorganic materials typically relies on the release of metal ions or reactive oxygen species [9], [10], [11], which interfere with different biological processes including cell metabolism and alter cell membrane stability. For these reasons, these species could have toxic effects also on the cells of the host organism and induce undesired side effects [12], [13], [14]. Moreover, it has been recently demonstrated that different bacteria species can develop resistance to silver nanoparticles [15]. These drawbacks can be easily overcome by employing as active component of the nanozyme biomolecules such as proteins, which combine intrinsic biocompatibility with highly selective and specific recognition properties, nearly impossible to achieve using synthetic materials [9].

Among the different scaffolds that could be employed for assembling multi-functional materials, gold nanoparticles (AuNPs) offer several advantages. They are inert and stable under most environmental conditions and exhibit low toxicity [14]. In addition, they allow for easy manipulation and surface conjugation, by both covalent and noncovalent interactions, with functional agents [16], [17], [18]. On top of this, AuNPs provide further antibacterial mechanisms, arising from their versatile optical and photothermal properties [13]. These are determined by the excitation of collective electronic oscillations at the metal surface, namely the localised surface plasmons, whose resonance frequency can be tailored by the nanoparticles size, shape and spatial organisation, as well as by the dielectric properties of the surrounding media [18], [19], [20]. The efficient conversion of the absorbed light into heat allows for designing photothermal vectors capable to burst bacteria [21]. Moreover, the possibility of tuning the optical properties in a wide range of frequencies, spreading from visible to infrared, opens for the in vivo application of these plasmonic devices, taking advantage of the transparency of blood and tissues in near infrared (NIR) [22].

A key issue in designing versatile nanoparticle-based materials is to reach a strict control on the assembly process underlying the spatial arrangement of AuNPs. An effective strategy adopted for controlling the organisation in solution relies on interfacing AuNPs with biomolecules, whose programmable intermolecular interactions provide the opportunity of assembling hybrid systems with the desired structure and functionality [23], [24], [25]. In addition, their responsiveness to external stimuli such as temperature, pH and incident light supplies further degrees of freedom in controlling the properties and the assembly of the whole system [26]. Thus, in order to take full advantage of such potentialities, it is essential to gain a fine control not only on the guided assembly, but also on the effects prompted by the environmental conditions. In this respect, several studies report on the protein-induced aggregation of AuNPs [27], [28], [29], [30], but a comprehensive explanation of all the mechanisms involved is still lacking. In particular, how the adsorption of molecules affects the colloids surface properties and triggers the aggregates formation is far from being fully elucidated.

In this work, we developed a novel plasmonic nanozyme with tunable antibacterial properties based on the assembly of patchy AuNPs. Colloids with inhomogeneous surface charge were obtained by adsorption of lysozyme to anionic AuNPs. The arising charge-patch interactions allow for driving their self-limited aggregation into stable clusters with selected finite size [31], [32]. We chose lysozyme (Lyz), an antimicrobial enzyme with size of 3 nm, for its stable globular folding and positive net charge at physiological pH due to the high isoelectric point at pH 11.3.

We performed a thorough analysis of the assembly of Lyz-decorated AuNPs (Lyz-AuNPs) as a function of all the experimental parameters involved in the process to gain a close control on the fabrication of Lyz-AuNPs assemblies. We therefore focused on the antibacterial function of the system, in terms of both plasmonic and catalytic properties, in relation to the colloidal assembly with the aim of highlighting their strict integration and interplay and realising a nanozyme with a high level of tunability.

Section snippets

Materials

Citrate-stabilised AuNPs with nominal size of 100 nm and 60 nm were provided by Ted Pella Inc. The concentrations of the stock solutions were 9.3 pM and 43 pM, respectively. Chicken egg white lysozyme powder (purity ≥ 90%) and (3-Aminopropyl)triethoxysilane (APTES, purity ≥ 98.5%) were provided by Sigma-Aldrich. Sodium citrate buffers at pH 6.5 and pH 4.0 were provided by Merk Millipore. The components of the system were characterised by Dynamic Light Scattering (DLS) measurements, in terms of

Theoretical background

It is well known (see for example ref. [32] and the literature cited therein) that a non-uniformly distributed electric charge at the surface of colloidal particles in aqueous solution results in an inter-particle potential that, even if the net charges on the two particles have the same sign, may show a significant attractive component. Intuitively such attraction originates from the interplay of short range, local interactions between oppositely charged patches on the approaching particles

Results and discussion

The assembly of citrate-stabilised AuNPs upon mixing with a lysozyme solution was studied with the aim of gaining a fine control on the different features (optical response and catalytic activity) involved in the antibacterial activity of the resulting system. A scheme of the assembling strategy adopted and of the investigation performed is reported in Fig. 1. The aggregation in solution is the pivotal mechanism, thus a detailed characterisation of the process was performed as a function of the

Conclusions

We realised a plasmonic active nanozyme with antibacterial properties based on the controlled aggregation of lysozyme decorated gold nanoparticles (Lyz-AuNPs). We demonstrated the aggregation-dependent modulation of the antibacterial activity of the nanomaterial and highlighted the possibility of acting on the assembly process to reach the tunability of both the optical response and catalytic activity.

In this respect, we exploited the key role of charge-patch interactions in generating

CRediT authorship contribution statement

Francesco Brasili: Investigation, Methodology, Conceptualization, Formal analysis, Visualization, Writing - Original draft. Angela Capocefalo: Investigation, Methodology, Conceptualization, Formal analysis, Visualization, Writing-Original draft, Funding acquisition. Damiano Palmieri: Investigation, Formal analysis. Francesco Capitani: Investigation. Ester Chiessi: Software, Visualization, Writing - Review & Editing. Gaio Paradossi: Resources, Methodology. Federico Bordi: Supervision, Resources,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Authors acknowledge the Physics Department of Sapienza University of Rome for providing access to the Sapienza Nanoscience & Nanotechnology Laboratories (SNN-Lab) of the Research Center on Nanotechnology Applied to Engineering of Sapienza University (CNIS) for AFM and SEM measurements. Authors acknowledge SOLEIL for providing synchrotron radiation facilities under proposal n. 20181452 at the SMIS beamline and under proposal n. 20180833 at the SWING beamline. Authors thank Thomas Bizien for

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