Unusual health threats emerging not only from the large immunological exclusion caused by the use of protective respiratory equipment during the COVID-19 (coronavirus disease 2019) pandemic but also the ever-increasing occurrence of bacterial resistance to antibiotics and virus mutations raise the need for new materials with antimicrobial properties. A very attractive option is the use of 3D printing for the inexpensive production of various environmentally friendly objects that possess excellent properties, especially in terms of strength and robustness.

The characteristics of 3D printed objects (i.e., adequate dimensional control, good surface finish and adaptability) make this technology interesting for various applications in fields such as healthcare, manufacturing, education, construction as well as home use (Tack et al., 2016; Roopavath and Kalaskar, 2017). This technology is based on high-temperature sintering of thermoplastic or polymer filaments and subsequent solidification of the printed product at room temperature. Polylactic acid (PLA) is one of the most commonly used polymers for 3D printing (Nagarajan et al., 2016). It is suitable for the production of various medical products due to its unique properties, such as smooth appearance, low toxicity, favorable mechanical properties (especially a low deformation effect) and high geometric resolution (Pajarito et al., 2019; Vicente et al., 2019). There are several published works on this topic (Lasprilla et al., 2012; Elmowafy et al., 2019; Singhvi et al., 2019), including our previous study dealing with PLA 3D printed safety masks during the COVID-19 pandemic (Vaňková et al., 2020) or other studies evaluating the use of PLA for 3D printing of tissue scaffolds (Zhang and King, 2020; Grigora et al., 2023). However, the 3D printed PLA objects can be easily colonized by various microorganisms due to cavities in their microstructure. Although disinfection of the 3D printed PLA objects with common chemicals is feasible (Vaňková et al., 2020), a self-disinfecting surface offers a low maintenance operation, high handling safety while omitting material degradation by chemicals or other conventional degradation techniques.

A functional antimicrobial layer added on the surface of PLA would represent a solution and further increase the usability of these products. One way to functionalize the surface of 3D printed PLA objects (Knetsch and Koole, 2011; Casalini et al., 2019; Elmowafy et al., 2019) is to coat them with a smooth metallic coating or metallic nanoparticles (NPs) with antimicrobial properties. Among metals, silver and especially its nanoparticles (AgNPs) have been often investigated for their possible medical use due to their excellent oligodynamic antimicrobial effect (Marambio-Jones and Hoek, 2010; Sotiriou and Pratsinis, 2010; Kalwar and Shan, 2018; Kumar et al., 2022). The oligodynamic effect (Prasher et al., 2018) occurs at low concentrations of heavy metals, where silver was identified as one of the most suitable for a variety of applications. AgNPs are capable of penetration into cells, and generate the reactive oxygen species and free radicals. For viruses (e.g., Coronaviridae), the ions can also bind to the spike glycoprotein, thus preventing its binding to host cells (Salleh et al., 2020). In the case of bacteria, AgNPs can penetrate the outer membrane and accumulate in the inner membrane, where the adhesion of NPs to the cell leads to destabilization and damage of the membrane, increasing its permeability, leading to leakage of cell contents and subsequent cell death (Bruna et al., 2021; Tripathi and Goshisht, 2022). There are a large number of studies describing the antimicrobial effect of AgNPs, e.g., when coated on polyurethane film (Prabhakar et al., 2011), stainless steel as implant materials (Furqon et al., 2021), paper (Srikhao et al., 2022), Eichhornia crassipes fiber (Palai et al., 2022), or silk surgical sutures (Syukri et al., 2020). However, despite the strong antimicrobial effects, AgNPs may have a cytotoxic effect against mammalian cells mainly by disrupting the membranes, affecting ATP production and DNA replication, altering gene expression, and oxidizing biological compartments in cells due to the mentioned production of reactive oxygen species (Fahmy et al., 2019). This effect occurs, especially when AgNPs are detached from the surface (Singh and Ramarao, 2012). To prevent it, the layer of AgNPs can be covered with a very thin protective film of various kinds (Kylián et al., 2017; Kratochvíl et al., 2018a). One of the precursors suitable for the preparation of such an overcoating is hexamethyldisiloxane (HMDSO), whose main advantage is that the physicochemical properties of the resulting film can be easily tuned over a wide range from hydrophobic to hydrophilic films (Kuzminova et al., 2014). This protective film can improve adhesion of the layer, while allowing the diffusion of Ag⁺ ions, but preventing direct contact between the AgNPs and the surrounding environment. The resulting coating should possess antimicrobial properties and, due to better fixation on the substrate, also low toxicity to human cells (Kratochvíl et al., 2018a).

The preparation of various antimicrobial materials based on coating the substrate with a layer of AgNPs or their combination with other overcoatings has been described in a number of studies. An example could be the coating of papers with chitosan/agar-AgNPs (Balalakshmi et al., 2020), or polydopamine/chitosan/AgNPs coating of urinary catheter and titanium surfaces (Wang et al., 2020). Furthermore, similar studies denoting specific applications were published for antibacterial fabrics (Allehyani et al., 2022; Cheng et al., 2022) or paper for food packaging materials (He et al., 2021). In agriculture or food industry, the coating with hydroxypropyl methylcellulose with AgNPs (Vieira et al., 2020), polyvinylpyrrolidone based glycerosomes with AgNPs (Saravanakumar et al., 2020), and carboxymethyl cellulose and guar gum-based AgNPs (Hmmam et al., 2021) extended the shelf life of papaya, bell pepper, and mango, respectively. Other potential applications in medicine have been reported for the antibacterial surfaces of titanium implants (Thukkaram et al., 2020; Wei et al., 2022), urinary catheter (Yassin et al., 2019), or for the treatment of onychomycosis (Dantas et al., 2021). In addition, the antiviral activity of polyethyleneimine with AgNPs coated on fabric from a surgical mask, hygienic mask, and combined polypropylene-glass fibers from a HEPA H13 filter was also reported (Baselga et al., 2022).

The desired smooth metallic and metallic NP coatings can be prepared by various methods. Among of all possible coating methods, the vacuum method based on a magnetron sputtering and subsequent gas aggregation in the case of NP deposition was found to be more suitable than classical chemical methods, yielding layers of better purity due to the absence of solvents and stabilizing agents (Wegner et al., 2006).

Although this issue has been studied already (Kratochvíl et al., 2018a; Kumar et al., 2022), the actual ambient conditions for medical application of AgNPs have not been determined. For instance, the above mentioned protective masks (Vaňková et al., 2020) are surrounded by a dry environment from the outside and a rather humid or wet one from the inside (close to the body). While for applications as biological tissue scaffolds, the surrounding environment is wet, saline-like, and body temperature. Although most studies have focused on Ag, antimicrobial properties are also possessed by copper (Vincent et al., 2016), which is cheaper and naturally occurs in trace amounts in the human body, which is not the case with Ag (Liu et al., 2019). To our knowledge, this is the first study directly comparing the antimicrobial effects of smooth Ag or other metallic coatings and AgNPs and monitoring the potential antiviral activities of such materials at the same time. Since proper efficiency of metallic coatings requires wetting and the large variation in humidity and/or wetting that can be expected in a real-life environment, we tested both dry (containing only atmospheric moisture) and wet conditions.

The aim of this study is to address the above-mentioned gaps in knowledge regarding the potential bio-applications of Ag, AgNP, and Cu coatings, such as a direct comparison of their biological properties (cytotoxicity, antibacterial, and antiviral action) in one study and consideration of the actual ambient conditions under which they can be practically used. Therefore, we compared the cytotoxic, antibacterial, and antiviral effects of AgNP layers (with and without a plasma-polymerized hexamethyldisiloxane (ppHMDSO) protective film) with Ag or Cu metallic coatings. We used three ambient conditions that mimic medical use (dry and wet conditions and conditions that simulate the human body). Coated PLA surface morphology was visualized using scanning electron microscopy (SEM). Human lung epithelial cells A549 were used to test the cytotoxicity of smooth metallic and metallic NP coatings. The bacterium Pseudomonas aeruginosa PAO1 and human rhinovirus species A, both representatives of respiratory pathogens, were used to test the efficiency of the coatings.

The study of NPs, their properties and use as active surfaces on various materials for application in medicine and other fields has been a highly attractive research topic in the last decades (Stark et al., 2015; Anselmo and Mitragotri, 2016; Anu Mary Ealia and Saravanakumar, 2017; Casalini et al., 2019; Elmowafy et al., 2019; Sánchez-López et al., 2020). Nevertheless, to our best knowledge, this is the first study that directly compares the biological effect of smooth Cu and Ag coatings to layers of AgNPs with and without a protective polymer (ppHMDSO) coating under different ambient conditions (dry and wet conditions and conditions simulating human body environment).

In general, the toxicity of AgNPs is strongly related to the particle size (ranging between 1 and 100 nm). Due to their extensive surface area, AgNPs are able to release a large amount of Ag⁺ ions, affecting the cells in their surroundings (Yin et al., 2020) Moreover, NPs of up to 10 nm in diameter can enter cells, increase the permeability of cell membranes, cause intracellular oxidative stress and even interrupt the replication of DNA (Marambio-Jones and Hoek, 2010; Kalwar and Shan, 2018; Yin et al., 2020; Kumar et al., 2022). The effects described are completely independent of cell type, applicable to human cells as well as bacteria (Sotiriou and Pratsinis, 2010; Quinteros et al., 2016; Kalwar and Shan, 2018). The mechanism of action of AgNPs is the subject of other scientific studies. For example, bacteria Staphylococcus aureus and Escherichia coli were treated by AgNPs powder and visualized using field emission SEM which shows not only morphological changes of cell surface but also cell fragments formed through damage of cell membrane (Hwan et al., 2010). Ashmore et al. (2018) treated E. coli with AgNP displayed an irregularly shaped appearance with nanoparticles attached to the cell membrane and fewer damaged or lysed cells. Ag⁺ ions are also implicated in the inhibition of uptake of phosphorus, causing the release of mannitol, phosphate, and amino acids such as proline and glutamine outside the cell membrane through the production of reactive oxygen species. The search for the mechanism of action was therefore not the focus of our study, in which the main objective was to compare the antibacterial and antiviral activity of individual metallic coatings under different conditions.

The Ag layers adhesion to the polymeric substrates is in general poor, but it is improved by enhanced substrate roughness and successful direct deposition of Ag thin film on PLA by magnetron sputtering was already reported (Da Silva et al., 2023). The advantage of AgNPs compared to a smooth Ag layer is their enormous active surface. The layer of AgNPs (approx. size of 25 nm, Figure 1A) visualized by SEM appeared very porous with partially sintered NPs, which is typical for these highly pure AgNP films, previously described by Ahadi et al. (2022). However, such coatings have poor mechanical stability, even worse than smooth Ag layers, since the NPs are held together only by Van der Waals forces and can be easily wiped off the substrates. Therefore, we tried to improve this property by coating the AgNPs with a thin ppHMDSO film anchoring the NPs on the substrates without significant changes in the coating structure. This layer can improve the anchoring of AgNPs on the PLA surface, as the nanocomposites in general are known to exhibit enhanced hardness and improved mechanical properties (Jedrzejowski et al., 2004). Indeed, based on the SEM images (Figures 1A,B), the surface morphology of AgNPs and AgNPs-ppHMDSO is basically the same. The polymeric film, which forms the top layer of the entire structure, can also affect the wettability of the coating and the release of Ag⁺ ions, thus changing the antibacterial properties of this surface, as described in a review by Kratochvíl et al. (2018a). The optimal balance between biological effects and biocompatible properties can be adjusted by both the proper film thickness and the chemical composition. In our study, the surface chemical composition of ppHMDSO samples, investigated by the XPS analysis (66 at% C, 18 at% O and 16 at% Si), is close to the values reported by Keller et al. (2007). In the case of smooth Ag layer, granular growth was observed by SEM due to the low wettability of the PLA substrate. Such behavior was described in more detail in previous work of our colleagues Kratochvíl et al. (2015).

In cytotoxicity studies, the Ag-based coatings seemed to act as a very poor support for cell growth (“on carrier,” Figure 2A). Therefore, cells were more likely to attach around it, leading to a relatively high metabolic activity in the “around carrier” dataset (Figure 2B). According to Gliga et al. (2014), 25 nm AgNPs should not be cytotoxic to lung epithelial cells, which is in agreement with our results using AgNPs coating (Figures 2A,B). A cytotoxicity study of Ag-based dressings on human fibroblast showed a similar cell behavior when in contact with material releasing Ag⁺ ions (Burd et al., 2007). A detailed study of the toxicity of AgNPs covalently stabilized by a small peptide that includes visualization of THP-1-derived human macrophages exposed to AgNPs using transmission electron microscopy or confocal Raman microscopy was published by Haase et al. (2011). The layer of AgNPs used in our study showed fluctuating cytotoxicity, but overcoating with ppHMDSO reduced this effect.

As mentioned above, an additional effect of very small NPs (up to 10 nm) is the induction of oxidative stress inside cells and affecting of DNA replication (Sotiriou and Pratsinis, 2010; Kalwar and Shan, 2018). The AgNPs investigated in this study are relatively large (25 nm), therefore, their direct induction of oxidative stress is rather unlikely, and the main effect on P. aeruginosa cells is probably caused by the release of Ag⁺ ions (Sotiriou and Pratsinis, 2010; Knetsch and Koole, 2011). Accordingly, our results showed that the layer of AgNPs possessed a stronger antibacterial effect (Figure 3) than the smooth Ag layer, likely due to the larger surface area and thus increased release of Ag⁺ ions. In support of the above-described notion that in dry conditions, release of ions is reduced and air humidity (RH about 40%) is not sufficient for considerable antibacterial activity of Ag-based coatings, we observed a generally weaker antibacterial effect under dry conditions. The antibacterial effects were very similar under conditions simulating the human body and wet conditions, with the exception of the smooth Ag layer, which had a much stronger antibacterial effect in saline than in wet conditions. This could be due to increased Ag⁺ ions release facilitated by a higher processing temperature and the stirring of the sample in the saline.

Regarding the antiviral impact of metallic coatings, our tests showed that both rhinovirus infectivity and genome integrity were reduced by all analyzed active layers (Figure 4). These results point toward a complex damage inflicted on the virus particles, which is consistent with previous studies using AgNPs (Salleh et al., 2020), Ag and Cu compounds (Minoshima et al., 2016). As expected, the infectivity was decreased to a higher degree than genome integrity, which is likely to be reduced only when the viral particles are severely damaged. The layer of AgNPs-ppHMDSO coating maintained similar antiviral effects to the layer of AgNPs. Its favorable cytotoxicity profile (Figure 2) and antibacterial activity (Figure 3) make the AgNPs-ppHMDSO combination very promising for medical applications. A similar conclusion emanated from a study of antibacterial activity of CuNPs on polyether-ether-ketone substrates overcoated with a polytetrafluorethylene using Escherichia coli ATCC 25922, complemented by a cytotoxicity study on human osteosarcoma cells MG63 (Kratochvíl et al., 2018b). They showed that by choosing appropriate conditions (mainly the amount of NPs and the thickness of the polymeric film), it is possible to create a material with simultaneously antibacterial and biocompatible properties.

Compared to the Ag-based layers (AgNPs, AgNPs-ppHMDSO, Ag) tested in our study, the smooth Cu layer was generally highly antimicrobial effective, but at the same time highly cytotoxic. Cu layer without any protective coating used in our study was clearly highly cytotoxic in direct (“on carrier“) and indirect (“around carrier“) contact (Figure 2), which is consistent with other papers concerning the cytotoxicity of Cu and its alloys on different cell types (Craig and Hanks, 1990; Wigle, 1992; Suska et al., 2005; Čolić et al., 2010). The Cu layer displays a high level of toxicity, not only to eukaryotic, but also bacterial cells or virus particles. The copper coating appears to be, according to our experiments, the most effective antibacterial agent that inhibits bacterial cells even in dry conditions (Figure 3). Moreover, despite fluctuations in the data, a clear trend toward a robust antiviral effect of the Cu layer in both dry and wet conditions is visible (Figure 4). This is consistent with several studies on the antiviral impact of copper, such as Noyce et al. (2007), who reported rapid inactivation of influenza A virus on the copper surface or the review reporting the effect of Cu and its alloys on SARS-CoV-2 virus (Govind et al., 2021). The Cu coating in our study was selected as a cheaper alternative to Ag layers, but its profound cytotoxicity (Figure 2) prevents applications in contact with the human body.

The obtained results in summary indicate that the AgNPs-ppHMDSO coatings represent an ideal surface for various types of implants, scaffolds and other intracorporal applications. On the other hand, the Cu layer could be practical for coating medical instruments in which cytotoxicity can be tolerated, as it is an efficient antibacterial and antiviral agent.

This study directly compares the biological properties of four metallic coatings (AgNP coating, AgNPs overcoated with a protective ppHMDSO film, Ag and Cu metallic coatings) and identifies the most suitable ones for medical applications. The obtained results show that regardless of the ambient environment, Cu is the most efficient antimicrobial agent, but highly cytotoxic at the same time. In summary, these properties make it suitable for use outside of the human body, efficient even in dry conditions. For all Ag-based coatings, the antimicrobial properties are greatly enhanced in the presence of liquid (wet conditions and environment simulating the human body). Moreover, the AgNPs appear to be more effective against bacterial cells and viral particles than the smooth Ag layer. The antibacterial and antiviral activity of AgNPs covered by the ppHMDSO film is generally comparable to or slightly higher than pure AgNPs. Together with the fact that the ppHMDSO coating minimizes the cytotoxicity of the AgNPs, these layers are promising for applications in close contact with or even inside the human body. Overall, the layer of AgNPs-ppHMDSO is non-toxic to lung epithelial tissue cells in its vicinity and effective against P. aeruginosa and rhinovirus. Therefore, this coating represents an excellent choice for a simple modification of 3D printed instruments applicable, for example, as various scaffolds inserted into the human body.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AM, EV, and KO were responsible for conceptualization and design of the study. EV and KO were responsible for the project administration. AM, PF, KO, and TK were responsible for the investigation and methodology. KO performed the statistical analysis. AM wrote the first draft of the manuscript. EV, KO, and JH wrote sections of the manuscript. EV, KO, JH, TL, and VS were responsible for funding and supervision. All authors contributed to the review and editing of the manuscript and approved the submitted version.

This study was financially supported by the FWF/GACR grant FWF I 5293-B/GF21-39019L.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.