Keywords

1 Introduction

Biofilms are colonies of bacteria, fungi, or yeasts that form organised and heterogeneous 3D structures on biotic or abiotic surfaces protected by a matrix of extracellular polymeric substances (EPS), also known as “slime”. The EPS matrix is a dynamic compartment that has multiple functions; e.g. it resembles a defensive barrier that protects individual cells from the immune system and antibacterial agents. The matrix provides shielding of the embedded cells from antibiotics and nanoparticles by acting as a diffusion hindrance, provides protection from toxic radicals, and allows for intra- and inter-species interactions and exchange of information. These properties can ultimately lead to microbial persistence, increased tolerance, and resistance to antimicrobials, including antibiotics and nanoparticles. For further reading on the biofilm matrix and interactions with nanoparticles, please refer to the literature (Fulaz et al. 2019; Joo and Aggarwal 2018; Flemming et al. 2016).

Once the biofilm is established, a survival mechanism is activated that impedes the diffusion of antimicrobials and simultaneously induces multidrug efflux pumps to remove antimicrobials from the individual cells sticking to the biofilm (Jolivet-Gougeon and Bonnaure-Mallet 2019; Geddes-McAlister et al. 2019; Uruén et al. 2021). The tolerance to antimicrobials and, therefore, the survival of biofilm is also conditioned by the production of persister cells (Carvalho et al. 2018). Biofilms are associated with over 60% of infections in humans, and there are a number of excellent reviews on protective mechanisms against antimicrobial agents and host innate immunity (Flemming et al. 2016; Yan and Bassler 2019). There is a consensus that treatment strategies with elevated efficacy against biofilms are urgently needed.

The emergence of nanotechnology in 1959 and the advances in the design, synthesis, and manipulation of particles led to noteworthy applications of nanomaterials in biotechnology. Nano refers to any material in the size range of 0.1–100 nm (1 nm = 10−9 m), in which basic biological functions operate. As physico-chemical properties can be tailored for targeted drug delivery to any site in the body, there has been a boom in research of nanoparticles for biomedical applications over the past decade.

Nanomaterials have been successfully adopted for diagnosis and sensing, novel treatments against a wide range of diseases, and as antibacterial agents (Abdel-Karim et al. 2020). In the latter case, nanomaterials have been extensively used for antibacterial coatings for implants, prostheses, and medical devices or locally delivered to infection sites as single-agent treatments or as components in multidrug delivery systems (Rezaei et al. 2019; Kirtane et al. 2021). The main advantage compared to conventional treatments is that nanomaterials have a higher reactivity conferred by their large surface-area-to-volume ratio and that it is possible to control their physico-chemical properties, thus being able to customise their interaction with biological entities (Navya and Daima 2016). A wide variety of nanomaterials have been tested for biomedical applications including metals (e.g. silver, gold, zinc, copper, iron, titanium, selenium) and the corresponding metal oxides, polymers (e.g. nanoporous polymers, hydrogels) and polymer composites, and technologies for the encapsulation of antibacterial agents comprising liposomes and responsive smart nanomaterials, amongst others (Kirtane et al. 2021).

This chapter comprehensively summarises current knowledge and breakthroughs in metal-based nanoparticles employed as antimicrobial agents, with a special emphasis on the treatment of biofilms from concept to real-world healthcare applications.

2 Silver Nanoparticles

Extensive interrelated research has been carried out to utilise metal-based nanoparticles for biomedical applications (Sánchez-López et al. 2020). In particular, silver nanoparticles (Ag-NPs) stand out for their antibacterial properties and have found multiple applications in the health field, including antibacterial treatments, antibacterial coatings for medical devices, and textile fibres, amongst others (Richter et al. 2017; Ooi et al. 2018; Lee and Jun 2019; Dong et al. 2019; Talapko et al. 2020; Sánchez-López et al. 2020). Although there are some concerns about the safety of using Ag-NPs for biomedical applications, in low concentrations (i.e. 0.001–0.1% weight/weight, based on 0.01–1 mg/mL concentration) Ag-NPs are not considered toxic (Liu et al. 2018a). Unfortunately, there is a lack of in vivo data elucidating the mechanism of Ag-NP toxicity; the majority of knowledge is based on in vitro studies, which calls for more investigations to determine safety of Ag-NP products in animals and humans. Literature has reported oxidative stress, DNA damage, and cytokine induction to be the three main mechanisms of Ag-NP toxicity (Ferdous and Nemmar 2020; Stensberg et al. 2011). Some animal studies showed Ag-NP effects on skin (Samberg et al. 2010), circulatory (Kim et al. 2008; Sung et al. 2009), respiratory (Sung et al. 2008), central nervous (Tang et al. 2008), and hepatic systems, depending on the route of Ag-NP delivery. An accumulation of Ag+ and Ag-NPs in the liver has been observed in animals (Sung et al. 2008, 2009; Kim et al. 2008). However, there is an ongoing debate on dose-dependent antimicrobial efficacy and safety of Ag-NPs and their associated effects on intracellular functions, which are attributed to the shape, size, and mobility of Ag-NPs (Richter et al. 2017; Cheon et al. 2019; Dong et al. 2019; Anees Ahmad et al. 2020).

2.1 Mechanism of Antibacterial Action

Although it is well known that Ag-NPs possess antibacterial properties, the mechanisms by which the nanoparticles target and destroy bacteria have not yet been fully elucidated. Figure 1 summarises the mechanisms that potentially contribute to the antibacterial properties of Ag-NPs.

Fig. 1
figure 1

Mechanisms of action that collectively contribute to antibacterial effects of silver nanoparticles (Ag-NPs). (1) Release of silver ions (Ag+) from Ag-NPs disrupts the bacterial cell wall and membrane; (2) Ag+ block ATP production and inhibit respiration; (3) Reactive oxygen species (ROS, generated as a downstream result of Ag+/Ag-NPs inhibiting bacterial respiration and metabolic pathways) damage the bacterial membrane; (4) Ag+, Ag-NPs, and ROS can impede DNA replication; (5) Ag+ can damage ribosomes; (6) Denaturation of the bacterial cell wall and membrane by Ag-NPs; (7) Creation of membrane pores by Ag-NPs inducing cytoplasmic leakage

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There is an ongoing discussion on how physico-chemical properties affect the antibacterial activity of Ag-NPs and how they enter cells (Slavin et al. 2017; Shaikh et al. 2019; Lin et al. 2020).

  1. 1.

    It has been suggested that the antibacterial properties of Ag-NPs are predominantly dependent on the release of silver ions (Ag+) from Ag-NPs surfaces, which produce pores and react with the peptidoglycan component, and then penetrate through the bacterial cell wall (Fig. 1.1; Jung et al. 2008; Singh et al. 2021). Once inside the bacterial cell, Ag+ can block adenosine triphosphate (ATP) production and inhibit respiration by deactivating respiratory enzymes on the bacterial cell membrane (Fig. 1.2), resulting in the generation of reactive oxygen species (ROS) that can further damage the bacterial membrane (Fig. 1.3; Morones-Ramirez et al. 2013). Moreover, Ag+ (as well as Ag-NPs and ROS) can impede DNA replication (Fig. 1.4) by preventing the hybridisation of single-stranded DNA to double-stranded DNA, damage ribosomes by inhibiting protein synthesis (Fig. 1.5), and disrupt intracellular metabolic pathways, creating more ROS (Fig. 1.3; Yakabe et al. 1980; Bao et al. 2015; Sadoon et al. 2020). An excellent review on the bactericidal mechanisms of silver is available elsewhere (Möhler et al. 2018).

  2. 2.

    Some studies linked the bactericidal activity of Ag+ ions and Ag-NPs to the morphological and structural changes produced in the bacterial cell (Markowska et al. 2013). Ag+ ions and Ag-NPs have the capacity to alter the permeability of bacterial membranes/cell wall through attachment to the surface (interfering with membrane proteins, such as sulfur proteins), changing the membrane/cell wall structure and modifying the cell potential (Liao et al. 2019, Lok et al. 2007). This can contribute to the denaturation or disruption of the membrane/cell wall (Fig. 1.6), the creation of membrane pores, and cytoplasmic leakage (Fig. 1.7), leading to bacterial cell death (Yin et al. 2020).

  3. 3.

    Physico-chemical properties such as particle size, shape, and zeta potential (i.e. the potential difference at the interface between the surface of the particle and the bulk solution) affect the nanoparticles’ ability to overcome biological barriers to reach the site of action. The mechanisms of action of the antibacterial properties of Ag-NPs can be classified as:

    • Influence of size: Due to their small size and large surface area, Ag-NPs are capable of penetrating into bacterial cells (Lin et al. 2020). The surface area increases with smaller particle size, which is associated with greater interactions between NPs and the surrounding area (Markowska et al. 2013). When 10–100 nm sized Ag-NPs were tested against Pseudomonas aeruginosa biofilms, the results showed that smaller Ag-NPs (10 and 20 nm) were more effective in reducing the bacterial viability and showed a lower minimum biofilm eradication concentration than 100 nm particles (Habash et al. 2014; Thieme et al. 2019). Similar findings have been reported in Vibrio natriegens, where 10 nm sized Ag-NPs completely inhibited bacterial and biofilm growth, and caused more cell damage and oxidative stress response by inducing more ROS than larger particles (Dong et al. 2019).

    • Influence of shape: It has been reported that 9 nm-sized spherical and 21 nm-sized quasi-spherical Ag-NPs showed selective size- and shape-dependent antibacterial activity against various pathogenic fungi and Gram-positive and Gram-negative bacteria. However, cubic Ag-NPs were not effective (Osonga et al. 2020). Another study showed significant activity of triangular and spherical Ag-NPs against P. aeruginosa and Escherichia coli. Spherical Ag-NPs at 50 nm were most effective, followed by triangular Ag-NPs at 150 nm, whereas spherical Ag-NPs at 90 nm were least effective (Raza et al. 2016). There is an understanding that the shape of Ag-NPs determines the surface-area-to-volume ratio. The larger the specific surface, the faster the Ag-NPs dissolve. Recent data suggest that Ag-NPs dissolve in aqueous media if oxygen is present, leading to the release of Ag+ ions, and that the rate of dissolution is highly dependent on the surface area and the shape of the Ag-NPs (Cheon et al. 2019). Considering that antibacterial assays are typically performed in aqueous media in vitro and in vivo (culture medium, body fluids, blood), it is key to further investigate the relationship between the shape of Ag-NPs, Ag+ release, and its influence on the antibacterial activity.

    • Influence of zeta potential: A positive zeta potential was reported to promote interactions of Ag-NPs with negatively charged bacterial membranes causing membrane disruption, which led to bacterial flocculation and a reduction in viability (Seil and Webster 2012). In contrast, Ag-NPs with a negative zeta potential could also exhibit antibacterial effects, though due to reduced electrostatic interactions with bacterial membranes, higher Ag-NPs concentrations or a small particle size is required for an extended antibacterial effect (Seil and Webster 2012). As an example, spherical Ag-NPs with a zeta potential of −30.8 mV effectively interacted with E. coli and S. aureus, disrupted bacterial respiration and cell permeability, and induced reactive nitrogen and oxygen species, thus generating oxidative stress on DNA, which in turn caused cell death. This was, however, mainly relying on the small Ag-NP size of 7 nm (Riaz et al. 2021). These examples show that the zeta potential has an effect on antibiofilm activity, but there are multiple factors that need to be taken into account to fully understand the antibacterial properties of Ag-NPs. If NPs have a similar size, the ones with a positive zeta potential will show greater antibacterial activity than the ones with a negative zeta potential (Seil and Webster 2012).

Although most studies investigated effects of Ag-NPs against planktonic bacteria, some studies carried out biofilm assays. Ramalingam et al. (2014) showed that Ag-NPs of 35 nm were effective against P. aeruginosa and S. aureus biofilms. Martinez-Gutierrez et al. (2013) evaluated the antibiofilm activity of Ag-NPs in P. aeruginosa biofilms, which were generated under static conditions and high fluid shear using a bioreactor. They concluded that Ag-NPs not only prevented biofilm formation, but also killed bacteria in established biofilms. Interestingly, the antibiofilm activity of Ag-NPs in this study showed less efficacy against Gram-positive bacteria (Martinez-Gutierrez et al. 2013), which is in contrast to other literature that found elevated antibiofilm effects of spherical Ag-NPs against MRSA compared to P. aeruginosa (Richter et al. 2017).

2.2 Silver Nanoparticles in Combination with Other Antimicrobial Agents

Multi-pronged treatment strategies have been investigated to maximise antibacterial efficacy, overcome biofilm tolerance to monotherapy, and circumvent potential drug resistance development.

As an example, Ag-NPs have been combined with antibiotics. Habash et al. (2014) have tested Ag-NPs in combination with aztreonam, which is an antibiotic used for the treatment of infections with Gram-negative bacteria. The sole administration of aztreonam was not effective in eradicating P. aeruginosa PAO1 biofilm, but instead stimulated its growth by 250% compared to untreated biofilms. Notably, the combination of 10 nm-sized Ag-NPs and the antibiotic achieved a 98% reduction in biofilm biomass and a 50% reduction in biofilm thickness (Habash et al. 2014). Another study confirmed substantial damage to E. coli, Salmonella typhimurium, and S aureus bacterial cell walls, resulting in 95% growth inhibition when treated with Ag-NPs and kanamycin. The mechanism of the synergistic effect was explained as a process in stages. Firstly, Ag-NPs destabilised the bacterial cell membrane, thus facilitating the penetration of the antibiotic. Secondly, increased uptake of kanamycin led to elevated antibiotic efficacy, inducing bacterial cell death (Vazquez-Muñoz et al. 2019). In contrast, when Ag-NPs were combined with ß-lactam antibiotics (e.g. ampicillin, aztreonam, and biapenem) which target the bacterial cell wall, no synergistic effect was observed. An excellent review on the synergistic effect of Ag-NPs with antibiotics is available elsewhere (Prasher et al. 2018).

Another approach is the combination of antimicrobial peptides (AMP) and Ag-NPs, which showed enhanced antimicrobial activity against several pathogens, including Klebsiella pneumoniae (Pal et al. 2019). This literature concluded that Ag-NPs were responsible for the localised delivery of AMP close to the bacterial cell membrane. This in turn facilitated the interaction between AMPs and the outer membrane of the bacterial wall, inducing its destabilisation. Moreover, Li et al. (2020) developed a multifunctional peptide (MFP)-coated Ag-NPs (MFP@Ag-NPs), which exhibited excellent antibacterial activity against Gram-positive bacteria S. aureus and methicillin-resistant S. aureus (MRSA), and Gram-negative bacteria E. coli and multidrug-resistant Acinetobacter baumannii in vitro. Furthermore, outcomes of a pneumonia model in mice infected with multidrug-resistant A. baumannii confirmed excellent antibacterial activity of MFP@Ag-NPs in vivo (Li et al. 2020).

Xu et al. (2021) synthesised a nanocomposite consisting of modified Ag-NPs with AMP using polydopamine (PDA). The incorporation of PDA served as green reducing agent and also contributed to enhance antibacterial properties. The nanocomposite inhibited the growth of E. coli, P. aeruginosa, and S. aureus by inhibiting the expression of biofilm-related genes in vitro (Xu et al. 2021).

GO-PEG-Ag (graphene oxide, polyethylene glycol, and silver) nanocomposites were synthesised by Zhao et al. (2017). The nanocomposite was tested in vitro against E. coli and S. aureus. The results revealed that the incorporation of PEG increased the compatibility and stability of the nanocomposite. Furthermore, GO-PEG-Ag nanocomposites were capable of damaging bacterial cells, inducing leakage of cytoplasm, producing ROS, and reducing bacterial metabolism, ultimately leading to bacterial cell death (Zhao et al. 2017).

2.3 Clinical Trials and Applications of Silver Nanoparticles

There are a growing number of clinical trials underway or recently completed evaluating the therapeutic potential of Ag-NPs in various conditions, including COVID-19 and biofilm co-infections, chronic sinus infections, wound care, dentistry, and implant- and medical device-related infections, amongst others (Table 1). Different routes of Ag-NP administration are being studied, such as local applications, including aerosols, injections, or wound dressings, and systemic applications through oral, intravenous, or intraperitoneal administration. Additionally, multiple clinical trials tested different types of pharmaceutical formulations, coatings, and nanocomposites for controlled and localised release of Ag-NPs, which resulted in a number of patented Ag-NP products (Sim et al. 2018). A recent review by Dhiman et al. (2019) thoroughly summarises antibacterial applications of Ag-NPs.

  • COVID-19 and Biofilm Co-infections

Table 1 Selection of clinical trials using silver nanoparticles (source: https://clinicaltrials.gov/ct2/search)
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Whilst COVID-19 is a virus infection, multiple cases of biofilm co-infections have been reported around the world, contributing to exacerbation of disease and high mortality rates (Thorarinsdottir et al. 2020; Qu et al. 2021; Hughes et al. 2020; Lansbury et al. 2020; Rawson et al. 2020; Vaughn et al. 2021; Zhu et al. 2020). Bacterial co-infections can easily emerge during a viral lung infection due to a weakened immune system and disrupted respiratory microbiota of the host, creating a favourable environment for bacterial colonisation (Hanada et al. 2018). Most common co-infections have been associated with Haemophilus influenzae, S. aureus, K. pneumoniae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and P. aeruginosa (Lansbury et al. 2020; Zhu et al. 2020).

Specifically, bacterial co-infection in severe cases of COVID-19 can be lethal when patients require mechanical ventilation for life support and rely on endotracheal tubes to maintain an open airway. Unfortunately, the tubes can be rapidly and frequently colonised by biofilms, which are difficult to eradicate and serve as a pathogen reservoir, putting patients at risk of relapsing ventilator-associated pneumonia (Thorarinsdottir et al. 2020). In a clinical trial (NCT02284438), endotracheal tubes coated with an alloy of silver, gold, and palladium reduced high-grade biofilm formation and, therefore, the risk for ventilator-associated pneumonia, which could be a life-saving intervention for mechanically ventilated COVID-19 patients.

Another clinical trial (NCT04894409) investigated Ag-NPs for the prevention of COVID-19, which was based on in vitro findings of Ag-NPs inhibiting SARS-CoV-2 infection in cultured cells. The clinical trial tested Ag-NPs in a mouthwash and nose rinse to prevent the viral spread amongst health workers. Findings are not published yet.

  • Chronic Sinus Infections

Several clinical trials have focused on the evaluation of Ag-NPs for the treatment of chronic sinus infections. For example, Richter et al. (2017) developed a nasal rinse to treat recalcitrant chronic rhinosinusitis, which could also be used as an antibiotic-free lavage in wound care (Richter et al. 2017). After confirming substantial antibiofilm activity in vitro and in vivo with 96%, 97%, and 98% biofilm reduction of S. aureus, MRSA, and P. aeruginosa, respectively, the Ag-NPs nasal rinse was translated into human clinical trials (ACTRN12616001558415). The nasal rinse contained 0.015 mg/mL Ag-NPs and was used twice daily for 10 days. The outcomes confirmed safety of the Ag-NPs, improvement in symptoms and endoscopic scores, and similar antibiofilm efficacy of Ag-NPs compared to culture-directed oral antibiotics (Ooi et al. 2018).

Another study (NCT02403479) compared the efficacy of a commercially available nasal spray containing colloidal Ag-NPs to saline spray (Scott et al. 2017). Set up as a crossover study, chronic rhinosinusitis patients used one spray twice daily for 6 weeks and continued with the other spray twice daily for 6 weeks. Outcomes indicated no negative health effects of the Ag-NP spray, though no clinical improvements after Ag-NP treatment have been observed.

  • Wound Care

Wound dressings containing Ag-NPs, such as Ag-NP-loaded hydrogels made of biocompatible polymers (Zhong et al. 2020), have been the focus of numerous clinical trials. For example, one study (NCT02210208) investigated the wound healing capacity of a dressing containing Ag-NPs in the treatment of skin grafts in surgical burn wounds. The dressing supported adequate fixation of the skin graft and significantly improved epithelialisation and wound healing.

Two examples for successful translation from bench to bedside and market approval are the silver dressings Aquacel and Acticoat. Aquacel is a liquid absorbing hydrofibre dressing incorporating silver ions. Acticoat is a three-layered antimicrobial barrier dressing containing an absorbent core surrounded by a nanocrystalline silver-coated mesh. In a clinical study (NCT00343824) the treatment efficacy and cost-effectiveness of both wound dressings was evaluated in burn wounds. Whilst wound healing time and antibacterial efficacy were similar for both dressings, study outcomes confirmed significant benefits of Aquacel regarding ease of use and application, patient comfort, and higher cost-effectiveness than Acticoat (Verbelen et al. 2014).

In another burn wound clinical trial (NCT02108535) Acticoat was compared to silver sulfadiazine cream. Both treatments showed comparable antimicrobial efficacy and safety, whilst the use of Acticoat required fewer dressing changes/less medical material and fewer hospital visits/less professional and administrative labour and was therefore more cost-effective than silver sulfadiazine (Moreira et al. 2021).

For further reading, please refer to current systematic reviews on wound management (Nherera et al. 2017; Zhang et al. 2019) and clinical trials (Table 1).

  • Dentistry

Silver-based products have found their way into multiple dental applications, including as Ag-NPs to prevent and treat biofilm infection in the oral cavities. As an example, nano-silver fluoride was shown to prevent biofilm growth and effectively halted dentine caries without staining teeth (NCT01950546, dos Santos et al. 2014; ACTRN12618001865202; Al-Nerabieah et al. 2020). Moreover, nano-silver fluoride increased remineralisation of carious dentin (NCT03193606, Abuhashema et al. 2020). Another study (NCT03669224) used a mixture of silver and gold nanoparticles as pre-treatment for caries class II interventions and assessed the effect on marginal integrity of resin composite restoration. Findings are not published yet.

  • Implants and Medical Devices

Nanocomposite formulations consisting of biocompatible polymers and Ag-NPs (e.g. spherical Ag-NPs, nanowires, and nanolayers) have been evaluated as antimicrobial coatings for implants and medical devices, such as endotracheal tubes, catheters, tissue scaffolds, surgical meshes, and other polymeric fibres (Polívková et al. 2017). One example is an antibacterial titanium implant for the prevention of periprosthetic infection. Ag-NPs were immobilised on the implant surface via plasma immersion ion implantation. The antibiofilm activity of the implant was assessed in vivo using a model of implant-related tibia osteomyelitis in rats, which showed effective prevention of Staphylococcus epidermidis biofilms (Qin et al. 2014). Unfortunately, this innovation has not been translated to human studies. There is a lack of ongoing/upcoming clinical trials testing the antibiofilm activity of Ag-NP containing implants and medical devices.

3 Gold Nanoparticles

In contrast to Ag-NPs, the antimicrobial activity of gold nanoparticles (Au-NPs) is less pronounced due to metallic gold being inert and nontoxic in nature (Boda et al. 2015). However, Au-NPs are also considered the most biocompatible metal nanoparticles with relatively low toxicity because of their high chemical and physical stability (Aminabad et al. 2019; Hu et al. 2020b). Therefore, Au-NPs are found in various medical fields such as imaging, diagnostics, or as drug delivery carrier and therapeutic agent in cancer and infectious diseases (Hu et al. 2020b).

The antimicrobial activity of Au-NPs has been determined for both the planktonic and the biofilm form in a wide range of Gram-positive and Gram-negative bacteria (Qayyum and Khan 2016; Abdalla et al. 2020). Whilst the mechanism for antibiofilm activity is not yet determined, it has been suggested that Au-NPs induce ROS production (Sathyanarayanan et al. 2013), create electrostatic interactions between NPs and bacterial cell membrane or cell membrane proteins (Sathyanarayanan et al. 2013; Giri et al. 2015), inhibit the exopolysaccharide synthesis (Manju et al. 2016; Ali et al. 2020), and inhibit quorum-sensing-controlled virulence factors (Qais et al. 2021).

Overall, Au-NPs were studied as monotherapy, in combination with other antimicrobial agents, and in combination with photothermal therapy. Whilst in vitro studies of new nanoparticle structures are still ongoing, first in vivo results are available for specific Au-NPs, including therapeutic approaches such as band aids (Wang et al. 2017), dental aligners (Zhang et al. 2020), and ureteral stents (Gao et al. 2020). These outcomes hold promise to progress Au-NP therapy to first human clinical trials in the future.

3.1 Gold Nanoparticle Effects on Biofilms

Similar to Ag-NPs, the shape and size of Au-NPs plays an important role in the antimicrobial activity and is influenced by the synthetic procedure. Ultrasmall nanoparticles with sizes below or around 10 nm have been chemically synthesised and showed in vitro antibiofilm activity against S. aureus, P. aeruginosa, and Shigella (Boda et al. 2015; Giri et al. 2015; Wu et al. 2021). Wu et al. (2021) described the antibiofilm effect of ultrasmall Au-nanoclusters against Shigella based on the induction of ROS. Furthermore, in a mouse model of Shigella-induced colitis the treatment with Au-nanoclusters resulted in reduced bacterial load in the faeces and an overall recovering of mice health. Furthermore, the intestinal mucosa and epithelial cell damage caused by Shigella gradually improved after treatment with Au-nanoclusters (Wu et al. 2021).

Chatterjee et al. (2021) investigated the antibiofilm activity of spherical and rod-shaped Au-NPs against Vibrio cholerae. Whilst all tested Au-NPs showed no toxicity against human colonic epithelial cells, only spherical Au-NPs destroyed biofilms. After oral treatment of V. cholerae-infected mice with spherical Au-NPs, disruption of V. cholerae attachment onto the epithelial surface and biofilm inhibition was observed. The antibiofilm activity of spherical Au-NPs is based on the disruption and crossing of the V. cholerae membrane, inhibition of ATP synthesis, and DNA damage, consequently resulting in bacterial death (Chatterjee et al. 2021).

Chemical synthesis allows us to control the specific shape and size of Au-NPs. However, as chemical procedures are linked to a range of problems, including energy loss, environmental effects, and safety challenges, the focus has shifted towards green synthesis of Au-NPs in recent years (Sánchez-López et al. 2020). Table 2 highlights examples of biogenic procedures using extracts of plants, bacteria, and fungi.

Table 2 Examples of Au-NPs developed with green methods and their in vitro antibiofilm activity
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Some Au-NPs that were synthesised with different green methods were investigated for their in vivo activity. Au-NPs synthesised with Woodfordia fruticosa flowers have shown antibiofilm activity in vitro against the fungus strains Candida albicans and Cryptococcus neoformans, whilst a topical application of the Au-NPs in a rat model promoted wound healing and prevented scar formation (Raghuwanshi et al. 2017). Furthermore, treatment with Au-NPs from the hyper-thermophilic bacterial strain Caldicellulosiruptor changbaiensis was investigated on catheters causing periprosthetic infection in mice. These small and moderate sized Au-NPs coupled with H2O2 exhibited high peroxidase activity, therefore increasing the ROS production causing the destruction of the lipid membrane of bacteria and biofilms. After 7 days, no sign of infection or S. aureus colonisation remained, the catheters were unstained, and low side effects on the mice were observed (Bing et al. 2018).

3.2 Gold Nanoparticles in Combination with Other Antimicrobial Agents

Dual treatment with other antimicrobial agents has been investigated to reduce Au-NP dosages and increase antibiofilm activity. Antimicrobials can either be directly coated onto the surface of Au-NPs or be attached through an intermediate coating. As an example, when cinnamaldehyde was directly coated onto the Au-NPs surface, the nanostructure showed in vitro antibiofilm activity against E. coli, P. aeruginosa, S. aureus, and MRSA. In addition, the bacterial virulence was reduced and viability of S. aureus-infected C. elegans was prolonged, indicating strong antimicrobial activity in vivo (Ramasamy et al. 2017). Similarly, chlorhexidine-coated Au-NPs inhibited the formation and eradicated preformed biofilms of Klebsiella pneumoniae (Ahmed et al. 2016). In another example, glucosamine-based Au-NPs (Au-GluN) synthesised by Yang et al. (2019) showed antibiofilm activity against MRSA. After exhibiting no haemolytic activity in vitro, cytotoxicity was assessed by intraperitonetal injection in vivo. No side effects were observed, no serious immune response over a 7-day period, and no histopathological changes in the heart, liver, spleen, lung, and kidney, indicating low pathological toxicity of Au-GluN in mice. After intraperitonetal MRSA injection, treatment with Au-GluN increased the survival of the mice. Furthermore, Au-GluN treatment of MRSA-infected wounds in mice resulted in smaller wound size, lower inflammation, and re-epithelialisation, suggesting Au-GluN to be a promising treatment for MRSA wound infections (Yang et al. 2019).

Antimicrobial agents can also be attached to the surface of Au-NPs by an intermediate coating, for example in a lipid double layer of phosphatidylcholine, loading gentamicin onto Au-NPs. This complex damaged established biofilm and inhibited S. aureus, P. aeruginosa, E. coli, and Listeria monocytogenes biofilm formation. In addition, the gentamicin-loaded Au-NPs killed intracellular bacteria in infected macrophages (Mu et al. 2016). Another combination of Au-NP with imidazole and chitosan showed antibiofilm properties against S. aureus and E. coli biofilms. The cationic amine on the surface interacts with the negatively charged sites of bacterial cells. The disruption of mature biofilms occurred through synergistic effects of the Au-NPs and imidazole by interrupting bacteria–bacteria interactions at the bacterial surface. The combination showed no haemolytic activity nor significant cell toxicity and, in a rabbit wound model with S. aureus biofilm infection, treatment with modified Au-NPs led to complete animal recovery after 14 days. In addition, wound healing was accelerated and the local bacterial count was significantly decreased (Lu et al. 2018).

However, a major component limiting treatment efficacy is the biofilm matrix, which provides a protective barrier that can hinder the penetration of antimicrobial agents, shield bacteria, and facilitate tolerance and resistance to nanoparticles (Qayyum and Khan 2016). An excellent summary on nanoparticle–biofilm matrix interactions can be found elsewhere (Fulaz et al. 2019). Multiple strategies have been proposed to disrupt the biofilm matrix, e.g. by photothermal treatment combined with amoxicillin-coated Au-NPs. Benefiting from the photosensitising properties of amoxicillin, the coated Au-NPs showed synergistic effects with phototherapy, damaging P. aeruginosa and S. aureus biofilm matrixes, whilst requiring a short irradiation time and a low Au-NP concentration (Rocca et al. 2020). In another study, daptomycin was incorporated in polydopamine-coated gold nanocages and conjugated to staphylococcal specific surface proteins. This structure showed activity against S. aureus and S. epidermidis biofilms after photothermal activation (Meeker et al. 2016). Similar results were observed against P. aeruginosa biofilms with gentamicin Au-NPs conjugated to an adapted outer membrane protein (Meeker et al. 2018).

3.3 Gold Nanoparticle Delivery Systems and Applications

Au-NP delivery systems with controlled release, as well as Au-NP-embedded medical devices, such as catheters, band aids, or aligners, have been developed for medical applications.

A photothermal nanoswimmer with spherically embedded Au-NPs and enclosed vancomycin was developed by Cui et al. (2020). When exposed to near-infrared laser irradiation, the nanoswimmers quickly penetrated and diffused into S. aureus biofilm, where the encapsulated vancomycin was released. In vitro more than 90% of the biofilm mass was removed after treatment, without spreading live bacteria in the environment. Treatment of a mouse implant-related periprosthetic infection showed very few bacteria and no biofilm remaining on the catheters. In addition, no toxicity to major organs was observed and wound healing was accelerated. This new technology provides the opportunity of eliminating deep layered biofilms, without causing damage to healthy tissue (Cui et al. 2020).

Wang et al. (2017) investigated the antimicrobial activity of Au-NPs with graphitic carbon nitride in a mouse wound model and an in vivo acute lung infected model. Application of an Au-NP-band aid with H2O2 on a drug-resistant S. aureus-infected wound resulted in prevention of wound infection, reduced presence of bacteria, and significantly accelerated the wound healing process. After intranasal instillation of MRSA into mice, the Au-NPs were given intravenously and led to a reduced number of bacteria in the lung compared to vancomycin treatment. These novel nanostructures with versatile functionalities show promising advances in different fields of nanomedical application (Wang et al. 2017).

A nanoparticle consisting of a gold core with a silver shell (Ag-Au-NP) was synthesised by Gao et al. (2020). The nanoparticle was embedded in a PGA/PLGA ureteral stent and has been investigated for cytotoxic and antibacterial properties against E. coli and S. aureus in a farm pig model. The antibiofilm activity of the stent was based on the biodegradable properties of PGA/PLGA, resulting in a constant exfoliating and removal of attached bacteria. In addition, through the self-cleaning process the embedded Ag-Au-NPs were exposed, which further eliminated adherent bacteria. The stent inhibited bacterial adhesion, contributed to biofilm removal, and reduced inflammatory response in vivo, displaying excellent characteristics as novel treatment for stent-induced urinary tract infections (Gao et al. 2020).

Au-NPs can also have an impact on oral health. Zhang et al. (2020) investigated the activity of 4,6-diamino-2pyrimidinethiol-modified Au-NPs coated on aligners against Porphyromonas gingivalis biofilms, the main cause of periodontitis. In the presence of the coated aligner, the biofilm formation was prevented, and no haemolysis induction was observed. As no irritation of the mucosa was observed after a mucosal contact test on golden hamsters, the Au-NP-coated aligner exhibited favourable biocompatible characteristics and is a promising new dental device for oral bacterial related diseases (Zhang et al. 2020).

4 Other Metal-Based Nanoparticles

In contrast to silver and gold nanoparticles, other metallic NPs typically contain the metal oxide form. These NPs are less common but have also been investigated for antibiofilm properties on their own or as part of mixed metal NPs (Shkodenko et al. 2020). Zinc oxide, copper-based, iron, titanium dioxide, and selenium NPs showed in vitro antibiofilm activity against Gram-positive and Gram-negative bacteria, which is partly based on the release of free metal ions (Kaul et al. 2022; Qayyum and Khan 2016; Sánchez-López et al. 2020; Shkodenko et al. 2020). An overview of recent in vivo biofilm studies and their application in human clinical trials is presented in Table 3.

Table 3 Selection of clinical trials using metal-based nanoparticles, not including silver nanoparticles (source: https://clinicaltrials.gov/ct2/search)
Full size table

4.1 Zinc Oxide Nanoparticles

Zinc oxide nanoparticles (ZnO-NPs) exhibit beneficial properties, including high toxicity against drug-resistant microorganisms, whilst being recognised as safe material (Mahamuni-Badiger et al. 2020). ZnO-NPs alone or as nanocomposite have shown high antibacterial and antibiofilm activity in vitro and in vivo against a range of Gram-positive and Gram-negative bacteria (Yazhiniprabha et al. 2019; Saravanakumar et al. 2020). In addition, in vivo experiments resulted in effective S. aureus biofilm prevention of a wound dressing coated with ZnO-NPs and orange oil (Rădulescu et al. 2016) and of an interconnected porous granules of nanostructured hydroxyapatite incorporated with ZnO-NPs, simulating a potential use in orthopaedic and dental application (Grenho et al. 2015).

As medical device, ZnO-NPs coated socks are investigated in multiple clinical trials against foot dermatoses caused by Gram-positive bacteria, including Corynebacterium species, Kytococcus sedentarius, Dermatophilus congolensis, and Actinomyces species. In a randomised placebo-controlled clinical trial, the effect of wearing ZnO-NP-coated socks on unpleasant foot odour and fungal feet infection was investigated over 2 weeks (NCT04000386, Table 3). ZnO-NP-coated socks inhibited the development of pitted keratolysis and reduced the level of foot odour (Ongsri et al. 2021), leading to a follow-up study that started in 2021. The effect of chlorhexidine scrubs, ZnO-NP-coated socks, and the combination of both will be examined as treatment and prevention of pitted keratolysis. In these studies, the improvement (NCT04332796) and occurrence (NCT04337749) of pitted lesion after treatment and side effects will be assessed.

4.2 Copper-Based Nanoparticles

Copper-based nanoparticles (Cu-NPs) were investigated for their antibacterial properties in healthcare-associated infections (Table 3). The addition of copper zinc nano-hydrocolloid dressing to sanitary towels significantly reduced the infection rate after vaginal delivery (Arendsen et al. 2021), and the impact of copper-enriched linen on healthcare-associated infections is ongoing (NCT04873557). Another current clinical trial (NCT04775238) investigates the antimicrobial and antibiofilm activity of Cu-NPs against S. aureus and P. aeruginosa in nosocomial infections. In addition, synergistic effects of Cu-NPs with a range of antibiotics will be examined (Kaul et al. 2022; Table 1). Previously, the combination of both Cu- and Ag-NPs has been investigated in vivo. Copper oxide microspheres decorated with Ag-NPs were constructed onto an implant used for bone defect repair. The structure promoted bone regeneration and osseointegration in rabbit tibia. The mechanism of action relied on the liberation of Cu2+ and Ag+ ions, which induced cell membrane damage in S. aureus and E. coli biofilms, ROS production, and lipid peroxidation (Yan et al. 2020). In another study, a silver-copper-boron nanocomposite showed extensive elimination of S. aureus bone infection in mice after intravenous or intramuscular administration without displaying adverse immune response (Qadri et al. 2017).

Copper sulfide NPs were investigated for their antibiofilm activity in combination with near-infrared (NIR) laser irradiation on different surfaces. On glass, a monolayer of CuS-NPs showed in vitro antibiofilm activity against S. aureus and E. coli (Gargioni et al. 2020).

4.3 Iron Nanoparticles

Iron nanoparticles (Fe-NPs) are nontoxic in nature and the most common iron oxide polymorphs are Fe2O3, Fe3O4, and FeO (Fahmy et al. 2018). Fe-NPs have been shown to inhibit biofilm formation and eradicate existing biofilms in various Gram-positive and Gram-negative bacteria in vitro (Sathyanarayanan et al. 2013). Their antimicrobial activity typically relies on superparamagnetic properties by magnetically immobilising bacterial cells and making them sensitive to an external magnetic field, resulting in the inhibition of biofilm formation (Taylor et al. 2012; Ranmadugala et al. 2018), or on the synergistic combination with other agents, such as oleic acid (Velusamy et al. 2021), poly(acrylic acid), and antibiotics (Armijo et al. 2020; Yang et al. 2020). Specifically, the dual treatment with antibiotics has revealed promising results.

  • Ficai et al. (2018) observed a significant antibiofilm activity of cefepime-functionalised Fe3O4 nanoparticles dispersed in a PLGA-based matrix in vitro against E. coli and S. aureus whilst being biocompatible and safe in vivo, making it a suitable material for the treatment of implantable devices surfaces (Ficai et al. 2018).

  • Chitosan/poly(acrylic acid) particles co-loaded with superparamagnetic Fe-NPs and amoxicillin were used for Helicobacter pylori-associated biofilm infections. This system showed multiple advantages: (i) the presence of chitosan allowed the nanoparticles to adhere onto and penetrate the gastric mucus layer upon exposure to a magnetic field; (ii) the poly(acrylic acid) allowed for a continuous release of amoxicillin; and (iii) the nanocarrier protected the amoxicillin from being dissociated by gastric acid. In vitro and in vivo results showed a prolonged residual time in the stomach by applying a magnetic field, thereby increasing the eradication rate of Helicobacter pylori and its biofilm. This new approach allowed for a reduction in dosage and treatment frequency, leading to a better tolerability and efficacy of Helicobacter pylori therapy (Yang et al. 2020).

  • Another approach used magnetically propelling Fe-NPs that dig artificial channels into biofilms to promote deep penetration of antibiotics. This concept was tested in vitro and in vivo (Quan et al. (2019). The combination of magnetically propelled Fe-NPs with gentamicin resulted in significant S. aureus colony forming unit reduction without adverse effect on body weight and blood chemistry of mice (Quan et al. 2019).

Other innovative treatment approaches are antibiotic-independent. Some Fe-NPs displayed peroxidase-like activity in the presence of H2O2, as the iron oxide can catalyse H2O2 leading to extracellular matrix degeneration, cell membrane damage, and bacterial death within the biofilm (Gao et al. 2016). This process was pH related and especially interesting in the treatment of dental biofilms. The successive treatment of different Fe-NPs and H2O2 was therefore investigated in an in vivo Streptococcus mutans orally infected rat model. Fe-NPs only containing Fe3O4 (Gao et al. 2016), the nanoparticle formulation ferumoxytol (Liu et al. 2018b), and dextran-coated Fe-NPs (Naha et al. 2019) showed a disruption of the development of biofilms, a suppression of caries development, and an attenuation of severe caries lesions in the presence of H2O2. These results are highly promising, as another dextran-coated Fe-NP formulation (Feridex) and ferumoxytol already received FDA approval, making these Fe-NPs excellent candidates for the treatment of dental caries (Liu et al. 2018b; Naha et al. 2019). Ferumoxytol was investigated as topical antibiofilm treatment on dental carries in a clinical trial (NCT03678012, Table 3). Results are not available yet. To overcome the problem of a two-component application, Huang et al. (2021) developed dextran-coated Fe-NPs conjugated with glucose oxidase. This nanohybrid does not require additional H2O2, as the glucose oxidase catalyses the oxidation of glucose to H2O2. Since glucose accumulates in oral biofilms, the nanohybrid enhanced in vitro S. mutans killing without affecting other commensal bacteria. Similar results were observed in vivo; when compared to chlorhexidine, the nanohybrid had significantly increased efficacy, without affecting the gastrointestinal microbiota and host tissue (Huang et al. 2021).

4.4 Titanium Dioxide Nanoparticles

Medical devices are frequently made of a titanium base with an antimicrobial coating, such as Ag-NPs, which is mainly responsible for antibiofilm effects (Wang et al. 2016; Hu et al. 2020a). An example has been described by Guan et al. (2019), who developed a titanium plate featuring polydopamine-coated TiO2 nanorods with attached Ag-NPs. This new surface coating displayed adequate long-term antibacterial and antibiofilm activity against MRSA and E. coli through selective physical puncture of bacteria and the controlled release of Ag+. In addition, satisfying biocompatibility was observed in vitro and in vivo, making it a promising application for orthopaedics and dental implants (Guan et al. 2019).

Furthermore, the antibiofilm activity of titanium dioxide nanoparticles (TiO2-NPs) was observed independently of the presence of silver, mainly through production of ROS and resulting damage of cell membranes and cellular constituents (Ismail et al. 2019; Alexpandi et al. 2020). TiO2 nanocomposites synthesised with the extract of Diplocyclos palmatus showed antibiofilm activity against Vibrio harveyi, making this a potential anti-infection candidate in aquaculture (Alexpandi et al. 2020). In a medical setting, TiO2-NPs incorporated into a gellan gum biofilm were applied as a wound dressing material on an open excision wound model in rats. The combination showed antimicrobial activity against S. aureus and E. coli and promoted cell growth, leading to a better re-epithelialisation and no scar formation (Ismail et al. 2019).

According to clinicaltrials.gov, a phase 3 clinical trial (NCT04365270) investigated the effect of TiO2-NPs with chitosan glass ionomer on the bacterial count of children’s dentin samples. Furthermore, a clinical trial (NCT04991064) examined the effect of TiO2-NPs’ addition to denture base material on their retention (Table 3). Results for both clinical trials have not yet been published.

4.5 Selenium Nanoparticles

Selenium-NPs (Se-NPs) previously showed activity against leishmania, fungus, planktonic bacteria, and biofilms (Tan et al. 2018). Se-NPs on titanium implants inhibited MRSA and MRSE biofilm formation and reduced the viable bacteria count in surrounding tissue of an infected femur rat. Implants coated with Se-NPs have the potential to reduce the risk for antibiotic-resistant orthopaedic implant infections (Tran et al. 2019). Prateeksha et al. (2017) determined that the combination of Se-NPs with polyphenols of honey induced wound healing and prevented fatal P. aeruginosa infection in mice. The antibiofilm activity was linked to the downregulation of quorum sensing and of quorum-sensing-related virulence genes (Prateeksha et al. 2017).

5 Conclusions and Future Perspectives

Metallic nanoparticles have opened new avenues for the treatment of biofilm-related infections and present an alternative strategy to antibiotics to combat multidrug-resistant bacteria. Numerous studies have been conducted, particularly using silver and gold nanoparticles, which showed a strong antibiofilm effect against a variety of bacterial species in vitro and in vivo. Notably, synergistic combinations with other antimicrobial agents seem to be most promising. Ongoing and upcoming clinical trials will validate the potential of metallic nanoparticles as effective antimicrobial treatment in clinical applications. However, there are still considerable gaps in our knowledge about the mechanism of action (especially regarding the role of the EPS in the regulation of nanoparticle–biofilm interactions), safety, and targeted drug delivery platforms. Therefore, more research and pharmaceutical formulation development is warranted.