Emerging Trends in Nanomaterials for Antibacterial Applications

Abstract

Around the globe, surges of bacterial diseases are causing serious health threats and related concerns. Recently, the metal ion release and photodynamic and photothermal effects of nanomaterials were demonstrated to have substantial efficiency in eliminating resistance and surges of bacteria. Nanomaterials with characteristics such as surface plasmonic resonance, photocatalysis, structural complexities, and optical features have been utilized to control metal ion release, generate reactive oxygen species, and produce heat for antibacterial applications. The superior characteristics of nanomaterials present an opportunity to explore and enhance their antibacterial activities leading to clinical applications. In this review, we comprehensively list three different antibacterial mechanisms of metal ion release, photodynamic therapy, and photothermal therapy based on nanomaterials. These three different antibacterial mechanisms are divided into their respective subgroups in accordance with recent achievements, showcasing prospective challenges and opportunities in clinical, environmental, and related fields.

Graphical Abstract

Keywords:

• nanomaterials
• metal ions
• photodynamic
• photothermal
• antibacterial mechanism
• reactive oxygen species

Introduction

Nanotechnology as a tool has rapidly taken over several fields of life including the health sector.¹ Applications of nanotechnology in the health sector are diverse and range from diagnostics to therapies. One of the therapeutic challenges at a global scale remains the treatment of bacterial infections. Indeed, the long-term use and misuse of antibiotics have led to antibiotic resistance, which threatens public health through different pathways like livestock, food, and water.^2–4 Metal ion release, photodynamic therapy (PDT), and photothermal therapy (PTT) are important methods for different biological applications. Multiple metal and carbon-based complexes (both organic and inorganic) such as copper, bismuth, silver, iron, gallium, gold, titanium, molybdenum, selenium, chitosan, polyvinylpyrrolidone, polyvinylidene fluoride, polyvinyl chloride, carboxymethyl cellulose, graphene etc, have been investigated in clinical studies for treating malaria, neurodegenerative diseases, cancer, and bacterial infections.^5–32 The variety of metals, their diverse physicochemical properties, their inherited photochemical properties, their shapes, and ligand types make them suitable for widespread use in new antimicrobial designs.^33–35

Moreover, metal atoms easily become positively charged ions through the loss of electrons, and they can then dissolve in biological fluids. Because of their electron deficiency, metal ions have a high affinity for electron-rich biomolecules, such as DNA and proteins, with which they form complexes. The formation of complexes leads to the inactivation of proteins, DNA damage, membrane impairment, and ultimately bacterial death. Furthermore, PDT and PTT use light to trigger a cascade of events leading to bacterial death. Different antibacterial phototherapy modalities exist based on how light-responsive nanomaterials act. The PDT employs photosensitizers or their precursors which can absorb visible light and produce reactive oxygen species (ROS) that are toxic to cells due to stress oxidation. Nanomaterials for PDT applications include metals, up-conversion nanoparticles, carbon-based nanomaterials, two-dimensional (2D) nanomaterials, quantum dots (QDs), etc.^36–41 PDT can be used to treat infectious diseases without the specter of drug resistance like with antibiotic treatment.⁴²,⁴³ In this way, PDT can be applied to all types of microorganisms including bacteria.⁴⁴,⁴⁵ PTT uses photothermal nanomaterials which can absorb light followed by its conversion into heat leading to hyperthermia which kills cells. With this therapy, a near-infrared (NIR) laser is preferable as a light source due to its advantages in clinical applications such as deeper tissue penetration, safety, and high absorption by photothermal agents (PTAs).⁴⁶ In this literature review, we summarize three ways of fighting bacterial infections using materials or composites at the nanometer scale: metal ion release, PDT, and PTT. Thereafter, a brief overview of trends with new approaches for antibacterial drugs is provided.

Metal Ion Release

Overview of Metal Ion Release in Antibacterial Applications

The ability of metal/metal oxide nanoparticles (NPs) to release metal ions in aqueous media has recently been extensively explored especially the aspect of their biomedical application and particularly focusing on the antibacterial properties of these metal/metal oxide NPs. The bactericidal properties of these NPs and their resulting metal ions have been extensively reported in recent years, and it was observed that metal ions are to a large extent implicated when describing the toxic mechanisms of these metal/metal oxide NPs against bacteria. These NPs can be oxidized when in biological media, releasing metal ions which can generate ROS or create oxidative stress that in turn leads to biocidal effects which can range from ribosome destabilization, DNA, protein, mitochondrial, and cell wall damage to electron transport disruption and cell death (Figure 1).^47–50 Many metal ions were reported to exert toxicity against bacteria, such as Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, Fe³⁺, Ni²⁺, and Cu²⁺, which at lower concentrations are very useful in bacterial metabolic processes but at higher concentrations become toxic to bacteria and hence have a bactericidal effect.⁵¹ The effects of these metal ions are primarily attributed to their natural affinity for certain cellular components.⁵² This is not the case with other metal ions such as Ag⁺ which exerts a very strong bactericidal effect even at very low concentrations. These metal ions are known to have a great affinity for thiol groups, which for example bind to cysteine and then indirectly trigger disruption of a cell’s enzyme functions, metabolism, or general physiology through their ability to destroy a cell’s disulfide bonds which help to maintain tertiary and quaternary protein structures.

Figure 1 Schematic illustration showing some toxic effects of metal ions from NPs.

Different modes of bacterial execution mechanisms have been identified for different kinds of metal/metal oxide NPs. Some examples include reorganization of the histone-like nucleoid structuring protein of bacteria, which was observed when Escherichia coli (E. coli) was treated with an Ag⁺ solution,⁵³ and bacterial death by the release of metal ions and generation of ROS via destruction of cell membranes and DNA damage, observed as antibacterial effects of Fe₃O₄@copper (II) metal-organic framework core-shell magnetic microspheres and Ru₂O nanocomplexes.⁵⁴,⁵⁵ Furthermore, it is important to also note that these metal ions possibly trigger these toxic effects on cells via a coordinated Haber-Weiss reaction, Fenton reaction, or Fenton-like reaction in which all three reactions yield hydroxyl radicals that adversely affect bacterial cells.^56–58 The nanomaterials in these cases serve as powerful catalysts by enhancing ROS production through any of the above-mentioned pathways.

Several key factors have been identified that influence the generation of ROS and hence the evident toxicity exhibited by some nanomaterials. These factors include solubility, shape, size, oxidation status, surface area, surface coating, surface species, and the degree of agglomeration and aggregation of a nanomaterial.^59–61 These chemical and physical factors can to a great extent influence the mode of action of these NPs in terms of how they are able to enhance catalytic properties by converting less-toxic oxidants into more-reactive free radicals, which in turn have destructive effects on bacteria, just as Ag⁺, Cu²⁺, and Zn²⁺ exert bactericidal properties by inducing the generation of ROS.⁶² Furthermore, when in aqueous solution, multicomponent metal ions act synergistically to produce increased antibacterial effects than single metal ions in solution. This increased synergistic effect occurs as a result of Fenton reactions and ROS production via electron transfer in enzymes.⁶³

Recently, the concept of metal ion release has been incorporated into antimicrobial-based biomedical applications. For example, ionic solutions/colloidal suspensions of metallic NPs of Ag, Cu, and Li were incorporated into some dental materials, and the resulting metal ions had an antibacterial effect on Staphylococcus aureus (S. aures).⁶⁴ Other application include pit and fissure sealants designed with antibacterial properties,⁶⁵ wound healing,^66–68 implant technology,⁶⁹,⁷⁰ improved disinfection materials,⁷¹ eco-friendly design of NPs with antibacterial properties,⁷² and antibacterial-Ag/plasma polymer nanocomposites for cotton fabrics,⁷³ among others. The antibacterial capabilities of Ag ions can be preserved while the harmful effect on biological environment is reduced by synthesizing Ag with hydroxyapatite from phosphorus-deficient precursors using carbonate substitution, Ag containing calcium phosphate, and Ag NPs carriers of hydroxyapatite powder.^74–76 In general, the release of metal ions in aqueous solutions has been explored for quite exciting biomedical applications especially in antibacterial-based biodesigns.

NP Dissolution and Metal Ion Release

NP dissolution in either a biological system or in an aqueous medium results in the release of ionic species which can elicit bacteria toxicity via specialized pathways. Horie et al assessed the physicochemical properties of 24 metal oxide NPs and their cellular effects.⁷⁷ They discovered that metal oxides which dissolved in media, such as ZnO, CuO, NiO, Sb₂O₃, CoO, MoO₃ and Gd₂O₃, released metal ions which caused detrimental cellular effects. Conversely, other metal oxide NPs which were barely able to dissolve in the media exerted very weak cellular effects. Interestingly, the authors discovered that Y₂O₃, a metal oxide NP which released metal ions in solution and also expressed increased ROS levels, showed no level of toxicity toward cells. Schiavo et al also reported similar findings with zinc oxide (ZnO) NPs toward three marine organisms: Dunaliella tertiolecta, Vibrio fischeri, and Artemia salina.⁷⁸ They however discovered that the toxicity of ZnO toward these organisms was a result of Zn²⁺ release and also the ZnO particles themselves, but the toxicity level was dependent on the exposure time. The particular physicochemical properties of NPs must be considered when assessing the exact bactericidal mechanism of a given NP. It is also noteworthy that although metal ion release arising from NP dissolution cannot solely be used to explain bactericidal properties of metal/metal oxide NPs, many NPs’ toxicity is greatly accelerated by dissolution. For example, Xia et al assessed the toxicity mechanism of ZnO and cerium oxide NPs and focused on their dissolution and oxidative stress properties; they clearly described how the key mechanism of ZnO toxicity greatly depended on its excellent dissolution property.⁷⁹

NP dissolution plays a very interesting role in the release of metal ions in biological environments. This concept is instrumental in releasing toxic metal ions to kill bacteria or other pathogens as evidenced by several reported findings (Figure 2). Furthermore, understanding the kinetics of NP dissolution can be very useful in designing safe NPs, and also in describing the etiology of some occupational health-related issues. For example, the dissolution property of ZnO was linked to pulmonary toxicity in zinc fume fever in welders.⁸⁰ Similarly, the toxicity and biopersistence of carbon nanotubes can be reduced by improving their solubility through a surface functionalization process with polyethylene glycol.⁸¹ Interestingly, it was observed that the shedding of Zn²⁺, Cu²⁺, Cd²⁺, and Ag⁺ during intracellular dissolution can trigger mitochondrial perturbation, lysosomal damage, calcium flux, cytoskeletal alterations, oxidative stress, and cell death.^82–84 In general, when released in solution, these ions can either be endocytosed by bacteria, be adsorbed onto the surface of the NPs, or undergo hydrolysis which yields insoluble metal hydroxides.^85–88 These actions would directly or indirectly result in disruption of bacterial cellular processes through halting cell wall synthesis, disintegrating bacterial membranes, disrupting biofilms, destabilizing ribosomes, degrading DNA, peroxidizing lipids, or by disrupting membrane permeability.⁸⁹

Figure 2 Illustration of possible NP toxicity mechanisms.

Wang et al reported that NP dissolution, adsorption, and hydrolysis are key factors which are likely to influence how metal ions are released by metal oxide NPs (Figure 3).⁹⁰ According to that study, the concentration of NPs in solution is initially correlated with the released metal ion concentrations; however, the free metal ion concentrations in the media at some point becomes limited via a hydrolysis process by the formation of insoluble metal hydroxides. Furthermore, the study showed that when the concentration of NPs in a medium is low, more metal ions are released, because dissolution is increased, but with a higher NP concentration, NP adsorption tends to occur and metal ions are adsorbed onto the surface of the NPs, thereby leading to a decline in available metal ions in the aqueous medium. It was observed by Wang et al that when the CuO NP concentration exceeded 180 mg/L, there were declines in both free Cu²⁺ in the supernatant and the resultant antibacterial activity. In a related study, Baek et al while studying the effects of free ions released by a group of NPs in liquid media, discovered that the dissolution rate of the NPs in the aqueous medium decreased with an increase in the NP concentration; this they attributed to aggregation of the NPs in the medium.⁹¹ Given that the modes of bactericidal effects of some NPs still remain unclear, any study to assess the bactericidal mechanisms of NP dissolution, adsorption, or aggregation properties must be carefully designed to consider other physicochemical properties which also influence the release of metal ions from NPs in biological media.

Figure 3 Schematic illustration of NPs dissolution, adsorption and hydrolysis. Reprinted from J Hazard Mater, 308,  Wang D, Lin Z, Wang T, et al. Where does the toxicity of metaloxide nanoparticles come from: the nanoparticles, the ions, or a combination of both? 328–334, Copyright (2016), with permission from Elsevier.⁹⁰

NP Size/Surface Morphology and Metal Ion Release

NP surface morphology, size, surface area, surface coatings, and the degree of aggregation and agglomeration were identified as some factors that can determine the rate of ROS generation and toxicity of a nanomaterial (Figure 4).⁹² Several studies tried to explain how the size and surface morphology affect particle dissolution. For example, some studies reported that particles of different sizes and shapes of the same material at an equivalent dissolved solute concentration dissolved at different rates.⁹³,⁹⁴ NP dissolution is a dynamic process defined by the migration of dissolving NPs through a diffusion/stagnant layer.⁹⁵ The diffusion layer is simply an interface between the NP surface and the aqueous environment. This layer is very important because in addition to the intrinsic solubility of the NPs, dissolution also depends on the concentration gradient that exists across this layer. In other words, as the concentration in the aqueous solution reaches equilibrium, the dissolution process decreases and hence metal ion release declines. The kinetics of NP dissolution in aqueous medium can be described using the Noyes-Whitney equation. This equation shows that the dissolution rate constant is dependent on the surface area of the NP. It was reported that a decreasing particle size is directly proportional to a decrease in the thickness of the diffusion layer, hence solvated molecules move faster throughout the bulk solution. In other words, with a larger surface area morphology, faster dissolution of NPs is expected as a result of a thinner diffusion layer; however, with thicker diffusion layers, dissolution will be slower. This can contextually explain why snowflake ZnO particles seem to be the most active when compared to other ZnO morphologies.⁹⁶ ZnO nanocrystals with a hexagonal plate-like morphology showed better activity than those with a rod-shaped morphology.⁹⁷ Similarly, Cha et al also reported that when compared to nanoplates and nanospheres, ZnO nanopyramids showed superior antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).⁹⁸

Figure 4 Schematic representation of several factors that can influence NPs behavior in solutions. Reprinted from Sci Total Environ, 438, Misra SK, Dybowska A, Berhanu D, et al. The complexity of nanoparticle dissolution and its importance in nanotoxicological studies. 225–232. Copyright 2012, with permission from Elsevier.⁹²

Those studies provide clues as to how different NP morphologies can influence antibacterial activity. Dissolution studies alone cannot satisfactorily be used to explain why some morphologies of NPs exert greater antibacterial effects than others or why NPs that are smaller in size have higher antibacterial activity than larger ones.⁹⁹,¹⁰⁰ This is because not all NPs show good dissolution characteristics in aqueous media, yet these NPs show bactericidal properties. In other words, for less-soluble NPs, other factors with or without metal ion release can contribute to their toxicity and ability to generate ROS (Figure 5). Other critical factors which were also identified include the presence of surface defects on the nanostructure of the NPs and the energy stability of the NP planes.¹⁰¹,¹⁰² According to those studies, surface defects on the NP nanostructure increases the surface area-to-volume ratio which influences ROS generation, whereas analysis of exposed facets (which explains why less-stable planes require less energy to form oxygen vacancies) was utilized to link the bactericidal effects of some NP to the stability of planes.

Figure 5 Schematic illustration of some NP characteristics capable of inducing ROS and subsequent bacteria death.

NP Charge and Metal Ion Release

It was also observed that the degree of electrostatic interaction between the NP and bacterial surface can also influence the bactericidal effects. It was reported that positively charged NPs can alter the function of the bacterial electron transport chain. In a study where TiO₂ and gold (Au) NPs were surface-modified, greater toxicity toward microbial cells was observed.¹⁰³,¹⁰⁴ The NPs of Ag and Zn for example are known to exert their bactericidal effects by the respective release of Ag⁺ and Zn²⁺.¹⁰⁵,¹⁰⁶ These positively charged ions are electrostatically attracted to negatively charged lipopolysaccharide (LPS) in bacterial cell walls causing disruption of the electrochemical gradient across the bacterial membrane leading to cell death. Ag⁺, for example, can interact with sulfhydryl groups in microbial cells making them dysfunctional, which in turn can lead to cell death. Furthermore, exposure of bacteria to positively charged NPs can generate ROS which induce oxidative stress in bacteria.¹⁰⁷,¹⁰⁸ In order to predict the electrostatic behavior of an NP toward an organism, it is vital to consider the NP’s point of zero charge. This point of zero charge is defined as the pH at which the charge on the surface of the NP becomes neutral. For example, at a pH value which is below the NP’s point of zero charge, a metal oxide NP has a positively charged surface, whereas at a pH value above this point of zero charge, the NP has a negatively charged surface. This is very important when predicting antibacterial effects of an NP, because positively, neutrally, and negatively charged NPs respectively exhibit strong, medium, and low bactericidal effects (Figure 6).

Figure 6 Schematic illustration of point of zero charge/NPs surface charge affects NPs bactericidal effects.

The pH value of the environment in which the NP exists was shown to influence the bactericidal outcome of the NP. Wang et al reported that ZnO NPs exhibited excellent dissolution properties at lower pH values. In other words, Zn²⁺ was rapidly released in aqueous media whose pH was acidic than in neutral and alkaline solutions.¹⁰⁹ Similarly, Bian et al earlier described how a lower pH favors high dissolution and hence metal ion release.¹¹⁰ However, MgO and CaO were found to exert very strong bactericidal characteristics at very high pH conditions. Dong et al showed that at pH 10, a Mg(OH)₂ NP suspension triggered strong bactericidal effects on E. coli;¹¹¹ the same was also observed with a Ca(OH)₂ suspension.¹¹² The high alkalinity of MgO and CaO which results in their strong bactericidal properties could be linked to their instability in water, which leads to the formation of the hydroxides, Mg(OH)₂ and Ca(OH)₂, in the presence of water, hence, reactive oxygen species are easily generated and are toxic to bacteria.

Metal Ions Release and Infection Sites

One of the recent bioapplications of metal ion release is in the design of pit and fissure sealants which yielded high fluoride ion release and a strong antimicrobial performance. Fei et al reported a very good antibacterial effect against Streptococcus mutans (S. mutans) biofilms from a pit and fissure sealant which was designed by combining calcium fluoride NPs and dimethylaminohexadecyl (DMAHDM).⁶⁵ This combination yielded more ions, increased biofilm pH, and improved antibacterial action against S. mutans biofilms compared to existing commercial units, hence making it a promising dental material for dental caries prevention. Similarly, Zhou et al also reported a multifunctional composite containing DMAHDM and amorphous calcium phosphate NPs as a dental material against recurrent dental caries.¹¹³ This composite inhibited the growth of S. mutans biofilms, and yielded less enamel demineralization and good enamel hardness. Furthermore, Rawashdeh et al incorporated an ionic solution/colloidal suspension of metallic NPs of Ag, Cu, and Li with some dental materials and tested the resulting antibacterial effect on S. aureus. They reported a dental material-dependent antibacterial outcome. The metal ion-incorporated dental materials showed high antibacterial effects which were attributed to the release of Ag⁺, Cu²⁺, and Li⁺ metal ions from the dental materials.

Kumar et al also reported fabrication of polyvinyl alcohol- and CS-loaded AgNP hydrogels with good mechanical strength, antibacterial properties, and biocompatibility for wound dressings.⁶⁶ The material showed good inhibition toward S. aureus and E. coli. Li et al showed that silver NP-loaded collagen chitosan (CS) dressing (AgNP-CCD) can yield excellent antibacterial effects and play a good role in the healing of a second-degree burns.⁶⁷ In that study, AgNP-CCD showed an antibacterial rate of ˃99% against E. coli, Sta. aureus, and Pseudomonas aeruginosa when the concentration of silver NPs exceeded 0.3 mg/cm². Similarly, Paterson et al reported the use of copper-containing mesoporous glass NPs with antibacterial and proangiogenic effects for chronic wounds.⁶⁸ The efficacy of this material was attributed to the release of Cu²⁺ ions which showed both proangiogenic and antibacterial properties.

The concept of metal ion release was incorporated into the design of implants with improved bactericidal properties. Hengel et al explored the synergistic antibacterial capabilities of Ag and Cu NPs by functionalizing TiO₂ surfaces with those NPs.⁶⁹ They observed a 10-fold reduction in the concentration of silver ions while sustaining the same level of antibacterial activity on MRSA. Their work sheds light on how the synergistic antibacterial properties of some metal ions can be harnessed to design more-biocompatible implants with efficient antibacterial effects. It was also observed that reinforcing alloys with silicon carbide (SiC) which is hemocompatible and biocompatible and has good antibacterial qualities, can be very useful in improving the performance of some medical equipment. Javadhesari et al showed that TiCu/SiC nanocomposites showed good bactericidal effects against E. coli and S. aureus.⁷⁰ This observed bactericidal effect was due to the release of Cu²⁺ which was directly proportional to the amount of SiC. At the end of this part, we have collected recent achievements by the uses of metal ion release in antibacterial applications as shown in Table 1.

Table 1 Summary of NPs and Their Reported Mechanisms of Toxicity

Nanomaterial	Mechanism of Toxicity	Reference
Ag2O	Metal ion release, direct contact with the bacterial cell envelope, ROS generation	[^Citation52]
CuO	Metal ion release, direct contact with the bacterial cell envelope, ROS generation	[^Citation52,^Citation114]
Cu2O	Metal ion release, direct contact with the bacterial cell envelope, ROS generation	[^Citation52,^Citation114]
Al2O3	Direct contact with the bacterial cell envelope, ROS generation	[^Citation52]
NiO	Metal ion release, ROS generation	[^Citation77]
MoO3	Metal ion release, ROS generation	[^Citation77]
WO3	Metal ion release, ROS generation	[^Citation77]
Y2O3	Protein adsorption ability, ROS generation	[^Citation77]
Co3O4	Effect from nanoparticles	[^Citation90]
Cr2O3	Effect from nanoparticles	[^Citation90]
MgO	Direct contact with the bacterial cell envelope, ROS generation, high alkalinity	[^Citation111]
CaO	Direct contact with the bacterial cell envelope, ROS generation, high alkalinity	[^Citation112]
ZnO	Metal ion release, direct contact with the bacterial cell envelope, ROS generation	[^Citation115^–^Citation117]
Fe2O3	Metal ion release, ROS generation	[^Citation118,^Citation119]

Abbreviations: Ag₂O, silver oxide; ZnO, zinc oxide; Fe₂O₃, iron(III) oxide; CuO, copper (II) oxide; Cu₂O, copper (I) oxide; Al₂O₃, aluminum oxide; MgO, magnesium oxide; CaO, calcium oxide; NiO, nickel oxide; MoO₃, molybdenum trioxide; WO₃, tungsten trioxide; Y₂O₃, yttrium (III) oxide; Co₃O₄, cobalt tetraoxide; Cr₂O₃, chromium (III) oxide.

Photodynamic Antibacterial Activity

Overview of Photodynamic Therapy

Light-activated materials have long been in use to treat diseases, and for the past 100 years, this has been called photodynamic therapy (PDT). PDT needs two elements to be functional: light and a material or chemical entity that can be sensitized by ultraviolet (UV) or visible regions of the electromagnetic spectrum of light irradiation. The mechanism of action is characterized by two kinds of oxygen molecules: the triplet state of oxygen (type I reaction mechanism) and the singlet state of oxygen (type II reaction mechanism). The type I reaction mechanism of triplet oxygen under light irradiation can generate ROS like hydroperoxyl (HO₂) and anionic superoxide (O₂⁻) radicals and requires higher activation energy. The type II mechanism produces hydroxyl (OH) radicals and requires lower activation energy and is more readily available. Some metallic nanostructures and metal-organic frameworks (MOFs) have been extensively utilized due to their superior photocatalytic properties, low cost, and effective antibacterial activities. NPs under visible light irradiation is believed to be effective photocatalysts and can generate ROS for effective antibacterial activity (Figure 7).^120–124

Figure 7 Schematic illustration of NP action of mechanism under light irradiation.

Metallic Nanocomposites

Nanocomposites of silver-zinc oxide (Ag/ZnO) showed notable bacteriostatic effects under visible light irradiation.¹²⁵ In Ag/ZnO nanocomposites, due to the localized surface plasmon resonance (SPR) property of silver, there is a very efficient electron-hole generation and reduction in the bandgap of ZnO which enhances the photocatalytic activity to counter bacterial growth by producing ROS.¹²⁶ The mechanism of action of Ag/ZnO nanocomposites under visible light irradiation is the creation of oxygen vacancies on ZnO NPs to produce H₂O₂, and this was enhanced by combining with silver NPs. Oxygen from the atmosphere is absorbed onto the oxygen-vacant surface of the nanocomposite that bears electrons which react to produce superoxide radicals (O₂⁻). O₂⁻ undergoes a reaction with water (H₂O) to form hydroperoxyl radicals (HO₂), and two HO₂ molecules combine again to produce H₂O₂ in a cyclically repeating process. H₂O splits into H⁺ and OH⁻ ions, and OH⁻ ions generate hydroxyl (OH) radicals (Figure 8). Electron paramagnetic resonance (EPR) studies validated the above-proposed mechanism for the efficient photocatalytic antibacterial activity of Ag/ZnO nanocomposites. Furthermore, the rupture of cell membranes of both gram-positive and gram-negative bacteria is due to the destruction of the lipid layer by peroxidation after ROS are generated as determined by lipid peroxidation assays.^127–131 Ag/ZnO nanocomposites were effective and promising antibacterial agents under visible light irradiation compared to bare ZnO.

Figure 8 Antibacterial mechanism of Ag/ZnO nanocomposites under light visible light irradiation. Reprinted from Catal Today, 339, Liu Q, Liu E, Li J, et al. Rapid ultrasonic-microwave assisted synthesis of spindle-like Ag/ZnO nanostructures and their enhanced visible-light photocatalytic and antibacterial activities.  391–402. Copyright 2020, with permission from Elsevier.¹²⁵

Abbreviations: VB, valence band; CB, conduction band; SPR, surface plasmon resonance.

Recently, Mutalik et al reported that titanium dioxide-iron disulfide (TiO₂-FeS₂) nanocomposites are an efficient photocatalyst for antibacterial applications.¹³² The mechanism of action of the photocatalytic antibacterial activity is based on the conversion of anatase titanium dioxide to rutile titanium dioxide NPs by combining iron sulfide nanocrystals to form TiO₂-FeS₂ nanocomposites, which also expands the absorption of the nanocomposite from the UV region to the visible-near infrared (NIR) region of the electromagnetic spectrum. The bandgap of FeS₂ is 0.95 eV and that of TiO₂ is 3.2 eV when combined and irradiated with simulated solar light, and due to the reduction in the bandgap of TiO₂, there was a substantial increase in the photocatalytic property which was shown to be effective in bacteriostasis against E. coli. The facile transfer of electrons from the valence band to the conduction band in TiO₂-FeS₂ nanocomposites effortlessly generates ROS to produce an efficient antibacterial effect. The TiO₂ in TiO₂-FeS₂ nanocomposites was observed to intensify the absorption of the visible region of light from the UV region of the electromagnetic spectrum to produce light-prompted ROS radicals like O₂⁻ and OH (Figure 9). The O₂⁻ and OH radicals generated from TiO₂-FeS₂ nanocomposites were effective in inhibiting E. coli, compared to their counterparts used in those studies, by causing damage to the microbial cell envelope.^133–138

Figure 9 Antibacterial mechanism of TiO₂-FeS₂ nanocomposites under visible light irradiation by narrowing bandgap. Reproduced with permission from Dove Medical Press Limited. Mutalik C, Hsiao YC, Chang YH, et al. High uv-vis-nir light-induced antibacterial activity by heterostructured TiO2-FeS2 1470nanocomposites. Int J Nanomed. 2020;15:8911..¹³²

Abbreviations: VB, valence band; CB, conduction band.

Nanocomposites of Ag-ZnO-magnetite (F₃O₄) showed better photocatalytic abilities for antibacterial applications under light-emitting diode (LED) irradiation.¹³⁹ An Ag-ZnO-F₃O₄ nanocomposite was synthesized using a green method. The reported mechanisms by which ROS are generated include the effective influence of the Fenton reaction of magnetite and SPR of silver under LED light irradiation, and these ROS substantially damage bacterial cell walls. In Figure 10, the SPR effect of silver and silver combined with ZnO induce the production of superoxide radicals and narrows the bandgap of ZnO for swift and easy electron transfer from the valence band to the conduction band of ZnO. The effortless heterojunction transfer of electrons with magnetite facilitates the easy generation of hydroxyl radicals under LED illumination. The O₂⁻ and OH radicals produced by the Ag-ZnO-F₃O₄ nanocomposite under LED light irradiation were efficient in inhibiting MRSA and Sta. epidermidis. Electrons and hole pairs are enhanced, and H₂O₂ is the end product of the Ag-ZnO-F₃O₄ nanocomposite due to the presence of silver, which in turn enhances the antibacterial efficiency. The Fenton reaction generated OH radicals to support and enhance the bacterial inhibition under LED irradiation for 3 h.^140–145

Figure 10 Antibacterial mechanism of Ag-ZnO-F₃O₄ nanocomposites under LED light irradiation. Reprinted from  Appl Surf Sci. 527, Abutaha N, Hezam A, Almekhlafi FA, et al. Rational design of Ag-ZnO-Fe3O4 nanocomposite with promising antimicrobial activity under led light illumination. 146893, Copyright 2020, with permission from Elsevier.¹³⁹

Abbreviations: VB, valence band; CB, conduction band; SPR, surface plasmon resonance.

Nanocomposites of Ag NPs and bismuth vanadium tetraoxide (BiVO₄) nanosheets cultivated on the surface of silver vanadium trioxide (AgVO₃)-(Ag@AgVO₃/BiVO₄) showed elevated photocatalytic efficiency against rhodamine B dye, E. coli, and Sta. aureus under simulated solar light irradiation.¹⁴⁶ Figure 11A–C exhibit a general proposal for a mechanism to eliminate bacteria under visible light irradiation. In Figure 11A, the SPR effect of Ag NPs is showcased as a primary cause of the antibacterial effect under visible light irradiation. In Figure 11B, after combining and arranging Ag NPs with BiVO₄ and AgVO_3, the photocatalytic efficiency improved under visible light irradiation. In Figure 11C, the Z-scheme arrangement of Ag@AgVO₃/BiVO₄ nanocomposites showed improved light-activated antibacterial activity by generating active O₂⁻ and OH radicals under visible light irradiation, that was not possible in the former arrangement of Ag@AgVO₃/BiVO₄ nanocomposites. An increase in the concentration of Ag@AgVO₃/BiVO₄ nanocomposites also showed an improvement in the photo-activated antibacterial effect. Under light irradiation, the Ag@AgVO₃/BiVO₄ nanocomposite (molar ratio=0.2) and z-scheme arrangement produced microbial cell wall destruction of E. coli and Sta. aureus and dye degradation.^147–152

Figure 11 Schematic illustration of SPR effect from AgNPs under visible light irradiation (A), the Ag position in Ag@AgVO₃/BiVO₄ nanocomposites and performance under visible light irradiation (B), and Z-scheme arrangement of Ag in Ag@AgVO₃/BiVO₄ nanocomposites and performance under visible light irradiation (C). Reprinted from J Colloid Interface Sci. 579, Ju P, Wang Y, Sun Y, et al. In-situ green topotactic synthesis of a novel z-scheme Ag@agvo3/bivo4 heterostructure with highly enhanced visible-light photocatalytic activity. 431–447, Copyright 2020, with permission from Elsevier.¹⁴⁶

Recently, ZnO-selenium (Se) nanocomposites showed effective photocatalytic activity in antibacterial applications under visible light irradiation.¹⁵³ Figure 12 shows penetration of the cell envelope of the pathogen Sta. aureus, the impairing mechanism by ZnO-Se nanocomposites under visible light irradiation due to ROS generation, and a biological pathway. Furthermore, ZnO-Se nanocomposites were found to be more efficient against Sta. aureus compared to ZnO NPs alone, as evaluated by the zone of inhibition which was found to be significantly greater in the case of ZnO-Se nanocomposites under visible light irradiation. Moreover, bacterial cell death by selenium is believed to be internally induced by proteins, and ROS generated by ZnO cause substantial rupture of microbial cell walls under visible light irradiation.^154–157

Figure 12 Schematic representation of antimicrobial mechanism of ZnO-Se nanocomposites under light irradiation. Reprinted from J Photochem Photobiol B, 203,  Ahmad A, Ullah S, Ahmad W, et al. Zinc oxide-selenium heterojunction composite: synthesis, characterization and photo-induced antibacterial activity under visible light irradiation. 111743, Copyright 2020, with permission from Elsevier.¹⁵³

Carbon-Based Nanocomposites

In Figure 13, nanocomposites of graphitic carbon nitride/chromium (g-C₃N₄/Cr)-ZnO showed exceptional photocatalytic properties in antibacterial applications.¹⁵⁸ Under simulated solar irradiation, g-C₃N₄/Cr-ZnO nanocomposite showed an improved photocatalytic mechanism by reducing the bandgap and improved electron-hole regeneration to generate ROS to eradicate bacteria by substantially damaging bacterial cell walls.^159–161 g-C₃N₄/Cr-ZnO nanocomposites at 60% resulted in more-efficient inhibition of E. coli, Sta. aureus, Bacillus subtilis, and Str. salivarius compared to its 60% g-C₃N₄/ZnO, 5% Cr-ZnO, g-C₃N_4, and ZnO counterparts under visible light irradiation. The photogenerated holes and electrons react with oxygen and water molecules present on the surface of the g-C₃N₄/Cr-ZnO nanocomposite to generate O₂⁻ and OH radicals. The ideal separation of electron-hole pairs and also less dense g-C₃N₄ sheets induce effortless electron transfer from the valence band to the conduction band for effective generation of ROS under visible light irradiation.¹⁶⁰,¹⁶² The g-C₃N₄/Cr-ZnO nanocomposite exhibited better photocatalytic performance under visible light irradiation, and the increased existence of electron-hole pairs enhanced ROS generation to eliminate bacteria and also degrade organic dyes responsible for pollution.

Figure 13 Schematic representation of antibacterial mechanism of g-C₃N₄/Cr-ZnO nanocomposites under simulated solar light irradiation. Reprinted from J Photochem Photobiol A, 401,  Qamar MA, Shahid S, Javed M, et al. Highly efficient g-C3N4/cr-ZnO nanocomposites with superior photocatalytic and antibacterial activity. 112776, Copyright 2020, with permission from Elsevier.¹⁵⁸

A recently reported copper sulfide-conjugated protonated C₃N₄ (CuS/PCN) composite showed enhanced antibacterial activity under the dual effects of visible light irradiation and a photothermal effect.¹⁶³ The antibacterial mechanism of CuS/PCN was observed against both gram-positive and gram-negative bacteria under visible light irradiation. In Figure 14, the mechanism of ROS generation under visible light irradiation is demonstrated. CuS and PCN form heterojunctions for the effortless transfer of electrons, and this combination reduces the bandgap. Electron transfer from the valence band to the conduction band facilitates charge separation in CuS/PCN composites under visible light irradiation. The adsorbed atmospheric oxygen molecules on both CuS and PCN during electron transfer are changed into O₂⁻ and OH radicals under visible light irradiation causing observable destruction of microbial cell membranes. The lower bandgap of CuS influences the higher bandgap of PCN to enhance the photocatalytic properties and production of ROS from CuS/PCN composites under visible light irradiation. Electron spin resonance (ESR) studies confirmed and validated the photodynamic ROS production from CuS/PCN composites. The photoactive CuS/PCN composite had effective bacteriostatic properties against both Sta. aureus and E. coli.^164–167

Figure 14 Schematic representation of the antibacterial mechanism of CuS/PCN composites under visible and laser light irradiation. Reprinted from J Hazard Mater. 393, Ding H, Han D, Han Y, et al. Visible light responsive CuS/protonated g-C3N4 heterostructure for rapid sterilization. 122423, Copyright 2020, with permission from Elsevier.¹⁶³

Fe₃O₄@SiO@Ag₃PO₄/ZnO-10% (Ag₃PO₄-10 wt%) (FSZA2-10%) microspheres showed enhanced photocatalytic efficiency against Sta. aureus and E. coli under visible light irradiation.¹⁶⁸ Figure 15A and B explain the mechanistic pathway for the generation of ROS and components that induce effective ROS generation under visible light irradiation. In Figure 15A, two Ag₃PO₄ and ZnO nanohybrid composites are involved in generating desired radicals which cause substantial microbial cell wall damage (Figure 15B). ZnO NPs generate electrons and are transferred at heterojunctions with their partner Ag₃PO₄ NPs that in turn generate O₂⁻ radicals under visible light irradiation and adsorb surface atmospheric oxygen. Ag₃PO₄ NPs in combination with ZnO NPs in FSZA2-10% microspheres generate electron holes to form OH radicals under visible light irradiation. In FSZA2-10% microspheres, Ag₃PO₄ and ZnO nanohybrids showed better electron-hole separation, and there was a narrowing of the valence band and conduction band for easy electron transfer and electron-hole generation at heterojunctions of Ag₃PO₄/ZnO which effectively produced ROS under visible light irradiation. Overall, FSZA2-10% microspheres were effectively photoactive leading to bacterial cell membrane damage in Sta. aureus and E. coli under visible light irradiation and showed effective bacteriostasis for up to six cycles of reuse.^169–172

Figure 15 (A) Schematic representation of antibacterial mechanism of Fe₃O₄@SiO@Ag₃PO₄/ZnO-10% (Ag₃PO₄-10 wt%) (FSZA2-10%) microspheres under visible light irradiation and (B) bacterial cell wall damage by ROS generation. Reprinted from Colloids Surf, A Physicochem Eng Asp, 603, Mao K, Zhu Y, Zhang X, et al. Effective loading of well-dopedZnO/ag3po4 nanohybrids on magnetic core via one step for promotingits photocatalytic antibacterial activity. 125187, Copyright 2020, with permission with Elsevier.¹⁶⁸

Nanocomposites of Ag/Ag₃PO₄ were combined with MOFs (MOF-5 or IRMOF-1), and a zinc-based MOF was synthesized by a hydrothermal process followed by a liquid chemical reduction method for antibacterial application under visible light irradiation.¹⁷³ In Figure 16, the mechanistic pathway is functionalized by three components of Ag, Ag₃PO₄, and IRMOF-1, and showed enhanced photocatalytic efficiency in producing bactericidal properties against E. coli and S. aureus. Nanocomposites of Ag/Ag₃PO₄-IRMOF-1 radiated with light for 2 h at a concentration 200 µg showed complete inhibition of gram-positive and gram-negative bacteria. In Ag/Ag₃PO₄-IRMOF-1 nanocomposites, Ag operates through the SPR effect leading to the formation of synergistic electrons which are directly supplied to the IRMOF-1 surface to activate ROS production to generate O₂⁻ radicals and electron-holes generated by Ag₃PO₄ under simulated light irradiation. Ag/Ag₃PO₄-IRMOF-1 showed sustained release of Ag⁺ and Zn²⁺ ions under light irradiation to eliminate E. coli and Sta. aureus by disrupting their cell walls. To sum up, Ag/Ag₃PO₄-IRMOF-1 nanocomposites were efficient at photo-actively inhibiting E. coli and S. aureus under visible light irradiation compared to their counterparts.^174–179 In Table 2, we have summarized several examples according to light-driven nanomaterials for antibacterial applications.

Table 2 Light-Driven Nanomaterials for Antibacterial Applications

Nanomaterial	Type	Light Source	Bacteria	Reference
Ag/PSCN nanocomposite	CMN	Visible light	E. coli	[^Citation180]
B-Bi2O3@BiOBr core/shell	Metallic nanocomposite	LED light	E. coli S. aureus	[^Citation181]
Ag-doped spindle-like BiFeO3 nanocomposites	Metallic nanocomposite	Visible light	E. coli M. luteus	[^Citation182]
Ag@AgCl-CA/SF composite film	CMN	Visible light	E. coli S. aureus	[^Citation183]
Ag-TiO2 nanocomposite	Metallic nanocomposite	Visible light	E. coli S. aureus	[^Citation184]
VS4/Ag2WO4 nanocomposite	Metallic nanocomposite	Visible light	E. coli B. subtilis	[^Citation185]
Gd-doped nickel spinel ferrite NPs	Metallic nanocomposite	Visible light	E. coli S. aureus	[^Citation186]
5 wt% cubic AgNPs-RGO	CMN	Visible light	E. coli S. aureus B. subtilis	[^Citation187]
ZnO/Ag nanocomposite	Metallic nanocomposite	Visible light	E. coli S. aureus	[^Citation188]
Ag3PO4-FeTiO3 heterostructure/glycol chitosan	CMN	LED light	E. coli S. aureus	[^Citation189]
I-ZnO-n	Metallic nanocomposite	Visible Light	E. coli	^Citation190]

Abbreviations: CMN, carbon-based metallic nanostructure; Ag/PSCN, silver/phosphorus- and sulfur-doped carbon nitride; B-Bi₂O₃@BiOBr, core/shell- beta-bismuth oxide-doped bismuth oxybromide; Ag-BiFeO₃ nanocomposite, silver-doped spindle-like bismuth ferrite; Ag@AgCl-CA/SF, Ag@AgCl-cellulose acetate-doped silk fibroin composite film; Ag-TiO₂ nanocomposite, silver-doped titanium dioxide; VS₄/Ag₂WO₄ nanocomposite, silver tungstate-doped vanadium tetrasulfide nanoplate nanocomposite; Cd-Gd-doped nickel spinel ferrite nanoparticle, cadmium- and gadolinium-doped nickel spinel ferrite nanoparticle; RGO, reduced graphene oxide; Ag₃PO₄-FeTiO₃ heterostructure/glycol chitosan, silver phosphate and iron titanate heterostructure-doped glycol chitosan; I-ZnO-n, Iodine modified zinc oxide-nanomaterial; LED, light-emitting diode.

Figure 16 Schematic representation of antibacterial mechanism of Ag/ Ag₃PO₄ combined MOF (MOF-5 or IRMOF-1) nanocomposites under visible light irradiation. Reproduced from Naimi Joubani M, Zanjanchi MA, Sohrabnezhad S. A novel Ag/ag3po4-irmof-1 nanocomposite for antibacterial application in the dark and under visible light irradiation. Appl Organomet Chem.2020;34:e5575. © 2020 John Wiley & Sons, Ltd.¹⁷³

Photothermal Therapy

Overview of Photothermal Therapy

Among multiple methods developed as an alternative way to fight bacterial infections in response to antibiotic resistance, PTT occupies a prominent place.^191–193 When photo-responsive agents are activated with a convenient NIR laser, the surface plasmon band leads to the conversion of electromagnetic radiation to heat. The resulting heat can reach temperatures able to destroy bacterial cells. The photothermal effect results in membrane damage followed by protein inactivation and/or leakage leading to bacterial death.¹⁹⁴,¹⁹⁵ PTT against bacteria is efficacious on both gram-positive and gram negative types and regardless of antibiotic resistance. In addition to its efficiency, it is fast, noninvasive, and can be modulated by the laser intensity and time of irradiation. More interestingly, PTT can be combined with other therapies such as PDT, chemotherapy, radiotherapy, and so on for better treatment results. Therefore, multiple photothermal NPs or nanostructures are being developed to eradicate bacterial infections. These photothermal NPs are also called PTAs and are grouped into five categories of metals, metal sulfides, oxides, carbon-based nanocomposites, small molecule-based nanomaterials, and polymeric nanomaterials.¹⁹⁶,¹⁹⁷ A set of transition metals and their oxide and sulfide forms can advantageously absorb more laser energy compared to inorganic PTAs.¹⁹⁸,¹⁹⁹ For instance, in our previous investigation of the photothermal effects on E. coli of gold nanorods (AuNRs) and gold nanobipyramids (AuNBPs), 100 µg/mL of AuNRs and 25 µg/mL of AuNBPs were used to kill E. coli with an efficiency of 100% after 808-nm laser illumination for 7 min.²⁰⁰ The principle of antibacterial PTT is illustrated in Figure 17. Among the different approaches to reducing the heat effect with good efficiency, targeted delivery of PTAs, controlled release of PTAs, and combined therapy are described below.

Figure 17 Principle of antibacterial photothermal therapy.

Approaches for the Targeted Delivery of PTAs

Molybdenum disulfide (MoS₂) nanosheets are one of the nanomaterials with efficient photothermal performance. In addition to the efficiency, this nanomaterial can be stabilized when conjugated with other biomolecules. So, Wenbo Cao and colleagues fabricated molybdenum disulfide coated with polyethylenimine (MoS₂-PEI) by means of covalent bonds in order to evaluate their photothermal antibacterial effect as illustrated in Figure 18.²⁰¹ So, S. aureus and E. coli suspensions were mixed to different concentrations of 20~80 µg/mL of MoS₂, PEI, and MoS₂-PEI. After 10 min of irradiation under an 808-nm laser (1 W/cm²), the mixture was incubated for 5 h and cultured on LB agar and then incubated at 37 °C overnight. In 10 min, a weight concentration of MoS₂-PEI at 80 μg/mL induced the temperature to increase to 67 °C. The intrinsic antibacterial effect of MoS₂ was better than that of PEI and reduced bacterial viability to 46.0% for E. coli and 45.6% for S. aureus. Meanwhile, inhibition rates of the MoS₂-PEI nanocomposite were 68.1% and 66.2%, respectively. Under NIR laser irradiation, the antibacterial effects of MoS₂ and PEI improved. MoS₂ reduced E. coli and S. aureus viability to 53.8% and 52.3%, respectively. By fluorescence microscopy, the synergistic effect of the MoS₂ -PEI nanocomposite was proven using the live/dead method. The results revealed that upon NIR irradiation, MoS₂ -PEI induced severe damage to bacterial membranes. This bactericidal effect could be attributed to the positive charge and the captive agent property of MoS₂ -PEI.

Figure 18 Schematic illustration of preparation and synergistic photothermal antibacterial activity of MoS₂-PEI nanocomposite. Reprinted from J Photochem Photobiol A, 401, Cao W, Yue L, Khan IM, et al. Polyethylenimine modified MoS2nanocomposite with high stability and enhanced photothermal antibacterial activity. 112762, Copyright 2020, with permission from Elsevier.²⁰¹

Huajuan Wang and co-authors designed gold NPs (AuNPs) conjugated with a peptide and dimethylmaleic anhydride (Pep-DA) which they named Pep-DA/Au.²⁰² Pep-DA/Au has a particular characteristic of being charge-convertible and can target bacteria. This synthesized nanocomposite has negative charges in normal physiological conditions of pH 7.4. But in a bacterial infection environment at pH 6.0, the DA is hydrolyzed by the acidity. In this situation, the amino groups on the peptide are displayed leading to a positive charge. So, the new Pep-DA/Au with positive charges can interact with negative charges of bacterial cell surfaces allowing the nanocomposite to attach to the bacteria. Under NIR laser illumination, the generated heat from the AuNPs will kill the bacteria and precisely minimize damage to surrounding tissues. The principle is shown schematically in Figure 19. In the SEM images of MRSA treated with a control and Pep-DA/Au under irradiation and the bacteria cultured on agar plates after treatment, the survival rate of bacteria confirmed the efficiency of the anti-MRSA photothermal effect of the investigated nanocomposite. More interesting, in a mouse model, in vivo wound healing was evaluated and has provided consistent results compared to in vitro results.

Figure 19 Photothermal bacterial selective killing therapy: (1) The charge conversion of Pep-DA/Au. (2) Bacterial targeting by nanocomposite. (3) Photothermal selective killing of bacteria.

Polymeric antimicrobials such as guanidine NPs can efficiently adhere to bacteria. This adhesion occurs through electrostatic bonds between the positive charges of polymeric antimicrobials and negative charges of bacterial cell walls.²⁰³,²⁰⁴ However, the drawback of cationic NPs is their interactions with protein corona once in the peripheral blood.²⁰⁵,²⁰⁶ To overcome this disadvantage, Congyu Wang and co-workers used a strategy based on charge reversal to design caged guanidine NPs (CGNs). These CGNs were synthesized through two main steps: first, preparation of amphiphilic diblock copolymers denoted P(GEMADA-co-DMA)-b-PBMA utilizing N,N-dimethylacrylamide (DMA), and 2-aminoethyl methacrylate hydrochloride (AEMA) and second, conjugation of the hydrophilic block (DMA) with dimethylmaleic anhydride (DA) for the final P(GEMADA-co-DMA)-b-PBMA as described in Figure 20.²⁰⁷ By means of confocal laser scanning microscopic imaging, CGNs were seen to effectively accumulate in bacterial biofilms. Under NIR irradiation, CGNs revealed about a ~40.9% photothermal conversion efficiency. They were also an efficient treatment in mouse models with catheters infected with Sta. aureus biofilm where they exhibited a 99.6% bacteria inhibition ratio. The mechanisms of nanocomposite preparation and bacterial killing are given in Figure 20.

Figure 20 Synthesis of Caged Guanidine NPs and in vivo biofilm eradication via NIR irradiation. Reprinted with permission from Wang C, Zhao W, Cao B, et al. Biofilm-responsive polymeric nanoparticles with self-adaptive deep penetration for in vivo photothermal treatment of implant infection. Chem Mater.2020;32:7725–7738. Copyright (2020) American Chemical Society.²⁰⁷

To combat food-borne diseases, Shimayali and colleagues developed a nanocomposite for rapid bacterial detection and NIR laser-reinforced antibacterial activity. Graphene oxide (GO), polyethylene glycol (PEG), gold NPs (AuNPs), and specific bacterial antibodies (Abs) were used to design a composite called Ab-PEG-GO-AuNPs. GO with multiple functional groups on its surface, such as carboxyl, epoxy, and hydroxyl, facilitates diverse functionalization of the surface.²⁰⁸,²⁰⁹ Due to the presence of the Abs and AuNPs, the designed nanoprobe was able to combine bacterium-specific targeting and photothermal ablation properties as summarized in Figure 21.²¹⁰ E. coli and S. typhimurium as food-borne bacteria were used to test the specificity through colorimetric detection. The results confirmed the performance of this investigated nanoprobe in terms of specificity and color change. Using bicinchoninic acid and fluorescence methods, the amount of Ab functionalized with PEG-GO-AuNPs was greater than that on PEG-AuNPs. That is why the limit of visual detection was ten-fold higher for GO-PEG-GO-AuNPs compared to PEG-AuNPs. The results showed that the GO coating of the AuNPs enhanced the photothermal performance with a rise in temperature of up to 76 °C under 808-nm laser irradiation for 30 s. As to the photothermal ablation of bacteria, results showed effective bacterial killing up to 95% within 15 min.

Figure 21 Schematic representation of specific bacterial recognition through Ab-PEG-GO-AuNPs via colorimetric detection and its photothermal ablation upon NIR irradiation. Reprinted from Sens Actuators B Chem, 329, Kaushal S, Pinnaka AK, Soni S, et al. Antibody assisted grapheneoxide coated gold nanoparticles for rapid bacterial detection and near infrared light enhanced antibacterial activity. 2021;329:129141, Copyright 2021, with permission from Elsevier.²¹⁰

Approach of the Controlled Release of PTAs

One of challenges with antibacterial treatment is the efficient delivery of drugs into sites of infection. Biomimicry, in situ polymerization and porous nanomaterial techniques are some of methods to resolve this issue.²¹¹ The uses of nanomaterials as nanocarriers are rapidly cleared by the immune system, thereby reducing the treatment efficiency. The biomimicking technique uses specific cell membranes such as red blood cells (RBCs),²¹² macrophages,²¹³ platelets,²¹⁴ and stem cells²¹⁵ in drug biominimization to bypass the immune system. RBC membranes (RBCMs) possess immunomodulatory markers, and their use as nanomaterial camouflage improves the circulation time of NPs in the body. With this logic Luoxiao Ran and co-authors designed a biomimic nanoantibiotic using gentamicin (GM), gold-silver (AuAg) alloy NPs, polydopamine (PDA), and RBCMs. This nanoantibiotic was named RBCM-NW-G, and a summary of it preparation and treatment under 808-nm NIR laser irradiation is given in Figure 22.²¹⁶ The biomimic nanocomposite revealed photothermal performance, effective accumulation in infected areas, and longer blood circulation times with good biocompatibility. With the synergistic effect of photothermal treatment, silver ions, and gentamicin, the prepared nanoantibiotic showed antibacterial potential highlighted in in vitro and mouse models against E. coli and S. aureus.

Figure 22 Diagram of RBCM-NW-G preparation and the treatment principle of bacterial infections in vivo. Reprinted from Bioact Mater, 6, Ran L, Lu B, Qiu H, et al. Erythrocyte membrane-camouflaged nanoworms with on-demand antibiotic release for eradicating biofilms using near-infrared irradiation. 2956–2968, Copyright 2021, with permission from KeAi Publishing.²¹⁶

Xueqin Yang and co-workers produced a photothermal nanoantibiotic (PTNA) by means of oxidative polymerization of pyrrole and anionic vesicles formed from sodium bis-(2-ethylhexyl) sulfosuccinate (AOT).²¹⁷ They examined the antibacterial and antibiofilm activities on multidrug-resistant (MDR) S. typhimurium. The entire process is shown in schematic form in Figure 23. Bacterial death was confirmed in two ways; bacterial growth on agar plates was inhibited and scanning electron microscopy clearly showed bacterial membrane damage. PTNA revealed an intrinsic antibacterial effect, with a reduction of S. typhimurium to 2log¹⁰ (CFU/mL). However, after laser irradiation (at 1 W/cm² for 10 min), PTNA reduced the bacterial viability by 6log¹⁰ (CFU/mL) confirming reinforcement of the antibacterial effect. Both effects were compared to a control and the nanocomposite without irradiation. The in vivo antibacterial potential of PTNA was also assessed in that work by determining the survival rate of mice that orally received 100 μL of 10⁹ CFU of S. typhimurium. Twenty-four hours later, the mice received treatment with phosphate-buffered saline (PBS), light irradiation, PTNA, and PTNA with light irradiation. Mice under treatment with light or PBS did not survive beyond day 6 or 5. Those treated with PTNA were alive beyond 6 days but did not reach day 15. However, 50% of mice treated with PTNA followed by irradiation under a 1064-nm laser (1 W/cm²) for 10 min remained alive by day 15.

Figure 23 PPy-based photothermal nano-antibiotic (PTNA) for the treatment of multidrug-resistant bacterial infection. Reprinted with permission from Yang X, Xia P, Zhang Y, et al. Photothermal nano-antibiotic for effective treatment of multidrug-resistant bacterial infection. ACS Appl Bio Mater. 2020;3:5395–540. Copyright (2020). American Chemical Society.²¹⁷

Patel and co-authors developed a nanocomposite consisting of AuNRs coated with mesoporous silica.²¹⁸ This complex played a role of nanocarrier onto which the antituberculotic medicine, bedaquiline (BDQ), was loaded. The entire composite was wrapped in thermo-sensitive liposomes (TSLs) under the name GNR@MSNP@BDQ@TSL@NZX with NZX responsible for the bond between the nanocomposite and mycobacteria. This engineered nanomaterial possessed three interesting features: targeted delivery of a nanoantibiotic to mycobacteria due to the affinity between NZX and the bacterial surface; a synergistic effect from BDQ and PTT; and the remotely triggered release of a synthesized bacterial drug under an NIR laser. The designed nanoantibiotic was shown to be 20-times more efficient than an equivalent amount of the free drug against M. smegmatis. It induced 99.9% inhibition of intracellular mycobacteria in lung A549 cells. A schematic illustration is shown in Figure 24.

Figure 24 (A) Schematic illustration of stepwise synthesis of the final nano‐assembly. (B) Application of final nano‐assembly (GNR@MSNP@BDQ@TSL@NZX) for targeting and killing M. smegmatis using 808-nm NIR laser. Reprinted with permission from Patel U, Rathnayake K, Jani H, et al. Near-infrared responsive targeted drug delivery system that offer chemo-phototherma therapy against bacterial infection. Nano Select. 2021. © 2021 The Authors. Nano Select published by Wiley-VCH GmbH.²¹⁸

Approach to Combinational Therapy

Due to the heat effect of PTT, several strategies were developed to reduce the heat and also to include targeted synergistic therapies, deliver and control the release of PTAs, and regulate irradiation.²¹⁹ Black phosphorus (BP) is one of the attractive photosensitizers (PSs) for phototherapeutic applications.^220–222 To develop a nanostructure with a synergistic effect, Aksoy and collaborators synthesized BP and AuNPs nanocomposites to fight pathogenic bacteria especially E. faecalis in planktonic form and as a biofilm. The principle is summarized in Figure 25. They found that the photothermal antibacterial activity of the nanocomposite was better than those of BP and a nanocomposite without irradiation. By a densitometric method at a weight concentration of 128 μg/mL and under 808-nm NIR laser irradiation, the BP/Au nanocomposite inhibited the growth of E. faecalis. Moreover, the BP/AuNPs composite also inhibited E. faecalis biofilm formation with and without light irradiation to 58% and 33%, respectively. In view of this investigation, the antibacterial activity of nanocomposites was due to membrane damage, and photothermal and photodynamic effects. The membrane damage was due to BP/AuNPs disrupting bacterial membranes due to the sharp edges of the BP nanosheets through simple contact.²²³

Figure 25 Design of BP/Au nanocomposite and its photothermal killing of bacteria. Reprinted with permission from Aksoy I, Kucukkececi H, Sevgi F, et al. Photothermal antibacterial and antibiofilm activity of black phosphorus/gold nanocomposites against pathogenic bacteria. ACS Appl Mater Interfaces. 2020;12:26822–26831. Copyright (2020), American Chemical Society.²²³

Amino-conjugated GO (AGO) nanosheets were fabricated by Bo-Yao Lu and co-authors to simultaneously exploit three properties: the natural power of AGO to cut bacteria, positive charges for bacterium targeting, and effective photothermal performance.^224–226 With this synthesized nanoantibiotic, the antibacterial effect was investigated on S. mutans, the main cause of dental caries. The positive charge of AGO due to the presence of amino groups favored electrostatic attraction with bacterial cell surfaces possessing negative charges. With 30 min of incubation, 100 µg/mL of AGO killed 27% of the bacteria. Under NIR laser irradiation for 5 min, the synergistic effect from the three properties of AGO caused 98% bacterial death. An illustration of this engineered nanosheet is summarized in Figure 26.²²⁷

Figure 26 Schematic illustration of natural and photothermal antibacterial effect of AGO based on its positive charge, natural cutting effect, and photothermal property.

Yingnan Liu and colleagues constructed a nanocomposite with antimonene nanosheets (AMNSs) incorporated in chitosan (CS) and named them CS/AMNSs hydrogels. CS can attach to bacteria via electrostatic forces, van der Waals forces, and hydrophobic interactions with cell membranes, and it also possesses an intrinsic antibacterial effect.²²⁸ Furthermore, the photothermal performance of AMNSs was confirmed under an NIR laser.²²⁹ In this design, the hydrogel acts as a carrier of the nanomaterials. CS-based hydrogels have various advantages such as low cost, biocompatibility, biodegradability, and an intrinsic antibacterial effect.²³⁰ The principle of this design is shown in Figure 27.²³¹ The photothermal conversion efficiency of AMNSs was evaluated to be 45.2%. The synergistic antibacterial effect including capture, intrinsic antibacterial effect, and photothermal performance killed 97% of E. coli and 100% of S. aureus. In Table 3, we have collected several important studies based on the applications of nanomaterials for photothermal killing of bacteria.

Table 3 Recap of Photothermal Bacterial Killing with Different Photothermal Agents (PTAs)

Nanomaterial	Mechanism	Light Source	Bacteria	Reference
2D C/Cu	PTT, Cu^2+ release	808 nm	E coli S. aureus MRSA	[^Citation71]
Au@Aβ NCs	PTT, binding affinity to bacteria	808 nm	S. aureus MRSA E. coli	[^Citation232]
HAuNS@PVP	PTT	808 nm	E. coli	[^Citation233]
VAT hydrogel	PTT, spatiotemporal antibiotics release	808 nm	S. aureus	[^Citation234]
Y2O3:Yb/Er@SiO2/SiO2@Cu2S	PTT	980 nm	S. aureus E. coli	[^Citation235]
NbTe2/TA	PTT	808 nm	E. coli	[^Citation236]
Tet-SiPc@AuNR@SiO2	PTT	808 nm	E. coli Antibiotic-resistant E. coli	[^Citation237]
AgNPs/N-CD@ZnO	PTT, Ag^+ and Zn^2+ release	808 nm	E. coli S. aureus	[^Citation238]
Au–ZnO–BP (AZB)	PTT	808 nm	S. aureus MRSA	[^Citation239]
Bi2S3/Ti3C2Tx-5 MXene	PTT, ROS generation	808 nm	E. coli S. aureus	[^Citation240]
Gel-PDA@Cur	PTT, curcumin release	808 nm	E. coli S. aureus	[^Citation241]

Abbreviations: Au@Aβ NCs, gold and amyloid peptide Aβ25-35 nanocomposite; HAuNS@PVP, hollow gold nanosphere conjugated with polyvinylpyrrolidone; VAT hydrogel, vancomycin-agarose-ferric tannate; Y₂O₃: Yb/Er@SiO₂/SiO₂@Cu₂S, nanocomposite from yttrium oxide (Y₂O₃), yttrium/erbium conjugated with silicon dioxide (Yb/Er @SiO₂), and silicon dioxide conjugated with copper sulfide (SiO₂@Cu₂S); NbTe2/TA, tannic acid (TA)-modified NbTe2 nanosheet; Tet-SiPc@AuNR@SiO₂, mesoporous silica-coated gold nanorod loaded with tetrazolyl phthalocyanine; 2DC/Cu, two-dimensional carbon nanosheet (2D C) decorated with Cu nanoparticles (NPs); CNSs@FeS₂, carbon nanospheres (CNSs) decorated with ultrasmall FeS₂ NPs; AgNPs/N-CD@ZnO, Ag NPs and ZnO NPs decorated by N-doped carbon dots (N-CD@ZnO); AZB, black phosphorus nanosheets conjugated with gold NPs and assembled with ZnO NPs; Bi₂S₃/Ti₃C₂T_x-5, bismuth sulfide (Bi₂S₃) conjugated with titanium carbide (Ti₃C₂T_x-5) MXene; Gel-PDA@Cur, dibenzaldehyde-grafted poly (ethylene glycol) (PEGDA), lauric acid-terminated chitosan (Chi-LA), and curcumin (Cur)-loaded mesoporous polydopamine nanoparticles (PDA@Cur); PTT, photothermal therapy; MRSA, methicillin-resistant Staphylococcus aureus.

Figure 27 Schematic illustration of (A) the preparation of CS/AM NSs hydrogel and (B) its use in treating bacterial wound infection. Reproduced with permission from Liu Y, Xiao Y, Cao Y, et al. Construction of chitosan-based hydrogel incorporated with antimonene nanosheets for rapid capture and elimination of bacteria. Adv Funct Mater. 2020;30:2003196. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Reproduced with permission from ref²³¹

Challenges and Opportunities

Recent advances in photothermal, photodynamic, and ion release antibacterial therapies using nanomaterials were brought together in the present review article. The nanostructures reported in these studies could soon be potential antibacterial candidates in food protection, water purification, and medicine. Metal, metal-polymer nanocomposites, bimetallic nanoparticles and polymer nanoparticles are promising antibacterial agents that can be harnessed for this purpose. The photothermal effects shown by the nanomaterials were presented by describing the mechanism of action and thermal efficiencies to show microbial inhibition in vitro and in vivo. Through their photothermal effect, the nanostructures affect the biological activities of bacteria to foster bacterial inhibition in addition to their enhanced thermal efficiencies under NIR light irradiation. The photodynamically active nanostructures recently investigated were demonstrated with their antibacterial mechanisms, ROS production abilities, and microbial inhibition under simulated solar light or similar light sources. Metal ion release is similar to photodynamic activity under dark conditions and completely depends on the dissolution and dissociation of metal nanostructures to produce ROS and show potential bacteriostatic and bactericidal effects. One of the crucial unanswered questions is clinical acceptance of these metallic nanostructures and their bacterial inhibition capacity (they are required to exhibit 4-log reduction or about 99.99% efficiency). Another crucial question is the cytotoxicity and biocompatibility of these metallic nanostructures which also remain unanswered. PTT can also be coupled with other therapies such as metal ion release, PDT, chemotherapy, radiation, and so on to improve treatment outcomes. Bacterial pestilences are likely to be the most frequent diseases globally which will need to be contained, and evaluating the safety and efficacy of available nanomaterials will play a crucial role before their administration in living beings and may have significant impact surrounding environment.

Acknowledgments

We acknowledge support from the Ministry of Science and Technology, Taiwan (Contracts MOST 109-2113-M-038-005-MY2), the Higher Education Sprout Project from the Ministry of Education (MOE) in Taiwan and Taipei Medical University. We acknowledge the academic and science graphic illustration service provided by TMU Office of Research and Development and Ms. Pei-Hsuan Tseng for her graphic illustration support at TMU Office of Research and Development.

Disclosure

The authors report no conflicts of interest in this work.