Metallic Structures: Effective Agents to Fight Pathogenic Microorganisms

The current worldwide pandemic caused by coronavirus disease 2019 (COVID-19) had alerted the population to the risk that small microorganisms can create for humankind’s wellbeing and survival. All of us have been affected, directly or indirectly, by this situation, and scientists all over the world have been trying to find solutions to fight this virus by killing it or by stop/decrease its spread rate. Numerous kinds of microorganisms have been occasionally created panic in world history, and several solutions have been proposed to stop their spread. Among the most studied antimicrobial solutions, are metals (of different kinds and applied in different formats). In this regard, this review aims to present a recent and comprehensive demonstration of the state-of-the-art in the use of metals, as well as their mechanisms, to fight different pathogens, such as viruses, bacteria, and fungi.


Introduction
Over the years, multidrug-resistant pathogenic microorganisms have emerged due to the overuse and misuse of antibiotics (and other drugs) against different types of infections, and also because of pathogens' innate ability to genetically acquire drug resistance [1]. Among the biggest enemies of antimicrobial drug treatments, is biofilm formation by microorganisms, since the extracellular polymeric substances from these biostructures work as an effective metabolic and physical barrier, developing antibiotic or antifungal resistance by making drug entrance difficult and consequently reducing effectiveness [2]. This poses a serious threat to public health as it becomes a challenge to treat the infections caused by these "superbugs" [3,4].
Most recently, the COVID-19 pandemic has emphasized how unprepared humanity is to fight threats associated with epidemic outbreaks of infectious diseases. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the latest in a long list of viral diseases that have emerged as severe threats to public health [5]. There have been made many attempts to develop medicines and vaccines to fight viruses; however, their ability for quick adaptation in their current host, and the ability to switch to a new host, are matters of great concern.
An example of a possible therapeutic target against the infection induced by SARS-CoV-2 can be the main SARS-CoV-2 protease, which plays an important role in the virus lifecycle, through the application of cannabinoid receptor type 2, which is highly expressed in immune cells, inhibiting inflammatory processes [6,7]. Concerning cannabinoid use,  found that these molecules are important to inhibit viral replication of SARS-CoV-2 in two ways: by binding to the main protease and blocking translation, and

Mechanisms behind the Antimicrobial Effect
In general, metals represent a considerable portion of the periodic table, and their properties can correlate with reactivity in living cells. They are required for the correct functioning of many enzymes and are involved in nearly all biological processes, providing structural stability for proteins, cell membranes, and DNA; they are co-factors for enzymes; and redox potential to facilitate electron transport and catalysis [11,12].
Even though they are essential for many biological functions, metals can also be toxic and lethal when in excess. Among many things, they can increase intracellular reactive oxygen species (ROS) via Fenton chemistry, inducing oxidative stress; disrupting membrane function and cell growth processes; and damage DNA and negatively influencing enzyme activities and protein function [11][12][13].
The toxicity of metals is achieved through multiple mechanisms of action, such as reduction potential-which interferes with the electron transfer process, preventing cells from acquiring electrons that are essential to cellular functions, and donor atom selectivitywhere a protein binds to the wrong metal cofactor and ends up with compromised folding or function [13].
Regarding the production of ROS, relevant mechanisms have been proposed through which this could be accomplished, such as the existence of other redox-active metals (such as Fe, copper (Cu), and nickel (Ni)), which can lead to Fenton chemistry, the destruction of Fe-sulfur (S) protein clusters, resulting in the release of additional Fe into the cytoplasm, which is Fenton-active and able to produce ROS in excess; and, last but not least, the oxidation of thiols within cells, which leads to the production of S radicals, forming protein disulfides and depleting glutathione reserves, leaving protein targets susceptible to attack by metals or ROS [11].
Furthermore, some metals apply their toxicity outside the microorganism barrier, by simply binding to the membrane, while others can bind to ligands of low molecular mass that present functional groups, such as phosphate, amino acids, or peptides, and be

Method Graphical Representation Short Description Reference
Top-Down synthesis Mechanical Milling This approach includes the disintegration of particle aggregates, particle shape, and particle surface. [20][21][22][23] Chemical Etching Uses a strong acid or a corrosive liquid to cut a metal surface and create a design in the metal. [24,25] Sputtering There is a particular sputtering technique called magnetron sputtering, which consists of the delivery of a high voltage across a low-pressure gas (normally argon) to create a plasma of high energy composed of electrons and gas ions, which will strike a target containing the desired coating material. [26,27] Laser Ablation Is based on the production of micropatterns through the ablation (removal) of fractions of a substrate through the action of a focused pulsed laser beam. [20,28] This approach includes the disintegration of particle aggregates, particle shape, and particle surface. [20][21][22][23] Chemical Etching

Method Graphical Representation Short Description Reference
Top-Down synthesis Mechanical Milling This approach includes the disintegration of particle aggregates, particle shape, and particle surface. [20][21][22][23] Chemical Etching Uses a strong acid or a corrosive liquid to cut a metal surface and create a design in the metal. [24,25] Sputtering There is a particular sputtering technique called magnetron sputtering, which consists of the delivery of a high voltage across a low-pressure gas (normally argon) to create a plasma of high energy composed of electrons and gas ions, which will strike a target containing the desired coating material. [26,27] Laser Ablation Is based on the production of micropatterns through the ablation (removal) of fractions of a substrate through the action of a focused pulsed laser beam. [20,28] Uses a strong acid or a corrosive liquid to cut a metal surface and create a design in the metal. [24,25] Sputtering l. Sci. 2022, 23, x FOR PEER REVIEW 4 of 32

Method Graphical Representation Short Description Reference
Top-Down synthesis Mechanical Milling This approach includes the disintegration of particle aggregates, particle shape, and particle surface. [20][21][22][23] Chemical Etching Uses a strong acid or a corrosive liquid to cut a metal surface and create a design in the metal. [24,25] Sputtering There is a particular sputtering technique called magnetron sputtering, which consists of the delivery of a high voltage across a low-pressure gas (normally argon) to create a plasma of high energy composed of electrons and gas ions, which will strike a target containing the desired coating material. [26,27] Laser Ablation Is based on the production of micropatterns through the ablation (removal) of fractions of a substrate through the action of a focused pulsed laser beam. [20,28] There is a particular sputtering technique called magnetron sputtering, which consists of the delivery of a high voltage across a low-pressure gas (normally argon) to create a plasma of high energy composed of electrons and gas ions, which will strike a target containing the desired coating material. [26,27] Laser Ablation

Top-Down synthesis
Mechanical Milling This approach includes the disintegration of particle aggregates, particle shape, and particle surface. [20][21][22][23] Chemical Etching Uses a strong acid or a corrosive liquid to cut a metal surface and create a design in the metal. [24,25] Sputtering There is a particular sputtering technique called magnetron sputtering, which consists of the delivery of a high voltage across a low-pressure gas (normally argon) to create a plasma of high energy composed of electrons and gas ions, which will strike a target containing the desired coating material. [26,27] Laser Ablation Is based on the production of micropatterns through the ablation (removal) of fractions of a substrate through the action of a focused pulsed laser beam. [20,28] Is based on the production of micropatterns through the ablation (removal) of fractions of a substrate through the action of a focused pulsed laser beam. [20,28]  Electro Explosion Single-step process in which a delicate wire of a conductive metal is exploded by an electric discharge that is caused by a high-power DC source. This electronic discharge creates a massive temperature that vaporizes the thin wire, turning it into gas atoms, which in their turn are chilled and, finally, the NPs are synthesized. [29,30] Bottom-up synthesis

Chemical Deposition
In particular, electrochemical deposition or electrochemical precipitation involves the passage of an electric current between an anode (sacrificial) and a cathode localized in an electrolyte. The anode is oxidized into metal ions and these are then reduced to metal by the cathode with the help of stabilizers. [31,32] Spinning This method involves the use of a spinning disc reactor. The disc spins at different speeds and the spinning causes the fusion and precipitation of atoms, which are then collected. [5,33] Sol-Gel A wet-chemical process, with sol being a colloidal solution of solids suspended in a liquid phase that serves as a metal precursor and is then dispersed into the gel, a host liquid leading to the formation of a solid macromolecule submerged in the solvent. After, there is a separate phase where the gel is dried and dehydrated to recover the NPs. [20,24,[33][34][35] Single-step process in which a delicate wire of a conductive metal is exploded by an electric discharge that is caused by a high-power DC source. This electronic discharge creates a massive temperature that vaporizes the thin wire, turning it into gas atoms, which in their turn are chilled and, finally, the NPs are synthesized. [29,30]  Electro Explosion Single-step process in which a delicate wire of a conductive metal is exploded by an electric discharge that is caused by a high-power DC source. This electronic discharge creates a massive temperature that vaporizes the thin wire, turning it into gas atoms, which in their turn are chilled and, finally, the NPs are synthesized. [29,30] Bottom-up synthesis

Chemical Deposition
In particular, electrochemical deposition or electrochemical precipitation involves the passage of an electric current between an anode (sacrificial) and a cathode localized in an electrolyte. The anode is oxidized into metal ions and these are then reduced to metal by the cathode with the help of stabilizers. [31,32] Spinning This method involves the use of a spinning disc reactor. The disc spins at different speeds and the spinning causes the fusion and precipitation of atoms, which are then collected. [5,33] Sol-Gel A wet-chemical process, with sol being a colloidal solution of solids suspended in a liquid phase that serves as a metal precursor and is then dispersed into the gel, a host liquid leading to the formation of a solid macromolecule submerged in the solvent. After, there is a separate phase where the gel is dried and dehydrated to recover the NPs. [20,24,[33][34][35] In particular, electrochemical deposition or electrochemical precipitation involves the passage of an electric current between an anode (sacrificial) and a cathode localized in an electrolyte. The anode is oxidized into metal ions and these are then reduced to metal by the cathode with the help of stabilizers. Electro Explosion Single-step process in which a delicate wire of a conductive metal is exploded by an electric discharge that is caused by a high-power DC source. This electronic discharge creates a massive temperature that vaporizes the thin wire, turning it into gas atoms, which in their turn are chilled and, finally, the NPs are synthesized. [29,30] Bottom-up synthesis

Chemical Deposition
In particular, electrochemical deposition or electrochemical precipitation involves the passage of an electric current between an anode (sacrificial) and a cathode localized in an electrolyte. The anode is oxidized into metal ions and these are then reduced to metal by the cathode with the help of stabilizers. [31,32] Spinning This method involves the use of a spinning disc reactor. The disc spins at different speeds and the spinning causes the fusion and precipitation of atoms, which are then collected. [5,33] Sol-Gel A wet-chemical process, with sol being a colloidal solution of solids suspended in a liquid phase that serves as a metal precursor and is then dispersed into the gel, a host liquid leading to the formation of a solid macromolecule submerged in the solvent. After, there is a separate phase where the gel is dried and dehydrated to recover the NPs. [20,24,[33][34][35] This method involves the use of a spinning disc reactor. The disc spins at different speeds and the spinning causes the fusion and precipitation of atoms, which are then collected. [5,33] Sol-Gel Electro Explosion Single-step process in which a delicate wire of a conductive metal is exploded by an electric discharge that is caused by a high-power DC source. This electronic discharge creates a massive temperature that vaporizes the thin wire, turning it into gas atoms, which in their turn are chilled and, finally, the NPs are synthesized. [29,30] Bottom-up synthesis

Chemical Deposition
In particular, electrochemical deposition or electrochemical precipitation involves the passage of an electric current between an anode (sacrificial) and a cathode localized in an electrolyte. The anode is oxidized into metal ions and these are then reduced to metal by the cathode with the help of stabilizers. [31,32] Spinning This method involves the use of a spinning disc reactor. The disc spins at different speeds and the spinning causes the fusion and precipitation of atoms, which are then collected. [5,33] Sol-Gel A wet-chemical process, with sol being a colloidal solution of solids suspended in a liquid phase that serves as a metal precursor and is then dispersed into the gel, a host liquid leading to the formation of a solid macromolecule submerged in the solvent. After, there is a separate phase where the gel is dried and dehydrated to recover the NPs. [20,24,[33][34][35] A wet-chemical process, with sol being a colloidal solution of solids suspended in a liquid phase that serves as a metal precursor and is then dispersed into the gel, a host liquid leading to the formation of a solid macromolecule submerged in the solvent. After, there is a separate phase where the gel is dried and dehydrated to recover the NPs. [20,24,[33][34][35] [18,19,23,[36][37][38] Metalsynthesis is bio-mediated by microbes or through biosynthesis. Biological synthesis of nanomaterials is the best alternative being cost-effective, environmentally friendly, advantageous and does not comprise any input of toxic chemicals. Between the candidates to biosynthesize M-NPs are bacteria, fungi and yeast; algae; plants, flowers and fruits; and viruses. [18,19,23,[36][37][38] These metallic nanostructures have several applications in numerous fields, such as in diagnostics, drug delivery, antimicrobial activity, and the treatment of several diseases [39]. M-NPs are widely recognized to have antimicrobial properties and are already widespread as antimicrobial agents with several products for antimicrobial purposes having been approved by regulatory agencies and commercialized. Some of these NPs have been used in combination with certain antibiotics to help overcome resistant bacteria, enhancing the antibiotic effect [40]. In addition, nanomaterials can be moulded to incorporate conventional antiviral properties with modifications that are exclusive to nanosystems, such as small and controllable sizes that can represent a solution for the treatment of viral infections [41,42].
The main components of the bacterial cell wall are peptidoglycans linked to teichoic acid, glycolipids, and phospholipids, which provide a negative charge to the cell wall. The interaction between the positively charged metal ions and the cell wall is enabled by this negative charge, which leads to a loss of membrane integrity. This damaged membrane can cause a leakage of cytoplasmatic content, such as minerals and genetic material, with a consequent rupturing of the cell, leading to cell death. Furthermore, if M-NPs enter the bacterial cell, they poison the microbial cells by generating ROS (which mediate cellular damage) or through metal-catalyzed oxidation reaction that could lead to disfunction of proteins or DNA and interfere with the assimilation of nutrients by the cell [43].
Nguyen and co-workers studied the antimicrobial mechanisms of magnesium oxide MgO-NPs, and SEM images of Gram-negative bacteria, such as Escherichia coli (E. coli), showed membrane damage when they were seeded with high doses of MgO-NPs. These Gram-negative bacteria present a thin layer of peptidoglycan between the phospholipid layers so the MgO-NPs can easily pass through the cell wall and bind to the membrane, causing shape distortion and cell death. On the contrary, Gram-positive bacteria present a thick surface layer made of peptidoglycan that retains the MgO-NPs. This characteristic can inhibit cell growth and prevent cell adhesion to surfaces [44]. Shankar and Rhim also reported a difference between Gram-positive and Gram-negative bacteria, regarding antimicrobial activity when Ag-NPs were used. Similarly, Ag-NPs also experience more difficulty in penetrating the peptidoglycan layer (20-80 nm) in Gram-positive bacteria, in contrast to what happens to the outer membrane of Gram-negative bacteria (7-8 nm) [45].
In particular, with Ag-NPs, it has been proposed that the release of positively charged silver ions (Ag + ) may interact with the negatively charged proteins or nucleic acids, damaging the structure and deforming the bacterial cell walls and membranes. This phenomenon will disrupt metabolic processes, leading to eventual cell death. It has also been suggested that ROS derived from the NP surface causes membrane damage by increasing membrane permeability, which will lead to cell death [45][46][47]. Chatterjee and collaborators (2015) also reported that, after treatment with AG-NPs, the cellular DNA of E. coli and Staphylococcus aureus (S. aureus) showed condensation, and E. coli cells were more susceptible to the treatment compared to S. aureus, probably due to the difference in structure, which has been previously described in the literature [46]. Iron NPs could be applied per se as antimicrobial agents or can act as hyperthermia agents to treat bacterial infections (this kind of microorganism shows a higher temperature susceptibility than healthy human host cells) [48]. When placed under an alternating magnetic field with a high frequency and amplitude, these particles will absorb electromagnetic radiation and subsequently convert the magnetic energy into localized heat, leading to bacteria death [49]. The study by Kim et al. (2013) can be cited as a good example of an effective application of Fe-magnetic NPs combined with hyperthermia to provide antimicrobial efficacy in a mouse infection model caused by S. aureus [50].
Similar to what happens with bacteria, antifungal mechanisms provided by M-NPs can be associated with a disturbance of fungal cell membrane integrity, and with the generation of ROS, which enhances the membrane disintegration process [51]. With metal toxicity comes a depletion of glutathione and a loss of antioxidant response to oxidative stress [52,53]. Table 2 provides different examples of experiments regarding the antimicrobial action of M-NPs against several pathogens.        Antiviral mechanisms of M-NPs can occur either inside or outside host cells, as can be seen in Figure 1. These mechanisms include a competition between NPs and the virus for host cell-binding sites, to prevent viral attachment, and blocking virus-host binding or penetration. This kind of mechanism could be observed in recent research work performed by Jeremiah and co-workers, where Ag-NPs effectively inhibited extracellular SARS-CoV-2 by protecting target cells from infection. Another mechanism consists of inactivating virus particles before cellular entry, interacting with the viral genome, or binding to the viral particles, which can also be considered as potential mechanisms of action [84]. Furthermore, the intracellular compartment of an infected cell is abundant in the virally encoded and host cellular factors required for viral replication and production of virions. Hence, another antiviral mechanism of action is the interaction of M-NPs with these replication factors [9,86]. Overall, different NPs present different antiviral mechanisms of action, for example Au-NPs (similarly to Ag-NPs) include the blocking of glycoprotein envelope (gp120) attachment to inhibit viral entry or replication (as can be seen in Figure 1); Cu-NPs can destroy the viral genome and disrupt the capsid, Zn-NPs interfere with viral DNA polymerase activity, resulting in viral replication inhibition, and iron (Fe) NPs (Fe-NPs) bind to the virus to inhibit its binding to cells [9,87]. On the left, a normal mechanism by which a virus infects a host cell (in this case, a eukaryotic cell) can be seen: firstly, viral attachment to the cell membrane occurs, followed by penetration into the cytoplasm; then the virus proceeds to use the cellular mechanisms to replicate its genetical material, generating virions to continue the infectious cycle. On the right, is shown a mechanism of inhibition by which Au-NPs can intervene, either by attaching themselves to the virus, blocking its attachment to the cell and consequent entry in the cell and ultimately interfering with the mechanisms of viral replication inside the cell (adapted from [88]).
Overall, there are many studies in the literature stating that the complete mechanism of action underlining the antimicrobial activity and efficacy of M-NPs is not fully clear or understood; however, among the parameters that influence the biocidal effects of metals are the size, shape, and metal ions involved [87]. With bacteriathis influence diverges between Gram-negative and Gram-positive cells, where M-NPs have more difficulty in passing through the thick layer of peptidoglycan in the Gram-positive cell wall, but, once inside the cell, the principles are the same. The antifungal mechanisms of M-NPs are similar to the antibacterial ones, which are the rupture of the cell membrane by oxidative stress and leakage of DNA and proteins. Regarding the antiviral mechanisms of M-NPs, they compete with the virus for cell-binding sites, therefore, inhibiting virus attachment to the cell, or preventing viral replication inside the cell.

Microparticles
Microparticles (MPs) are spherical particles that range in size from 0.1 to 1000 μm and can be synthesized from natural and synthetic materials, such as polymers, glass, and ceramics. Similar to what happens with NPs, their small size gives them an advantage over larger particles (macroscale) and presents many desirable properties for a variety of different applications, such as drug delivery [89,90].
In general, MPs are very similar to NPs in terms of their synthesis methods and applications, except for their size, which is bigger. Similar to NPs, MPs are commonly used to promote antimicrobial effects in different industries, and can be used as a drug delivery system due to their ability to protect from degradation bioactive drugs, proteins, and small molecules; they are also capable of controlling the release rate of encapsulated drugs over an extended period, up to months, and also, they are easily processed [89,91].
Some studies have been conducted regarding the use of MPs against different pathogens. For instance, Sophee and co-workers synthesized titanium oxide (TiO2) and ZnO MPs using the sol-gel method to prevent water-borne microbial infections. From the ob- Figure 1. Representative mechanism of the antiviral properties of Au-NPs. On the left, a normal mechanism by which a virus infects a host cell (in this case, a eukaryotic cell) can be seen: firstly, viral attachment to the cell membrane occurs, followed by penetration into the cytoplasm; then the virus proceeds to use the cellular mechanisms to replicate its genetical material, generating virions to continue the infectious cycle. On the right, is shown a mechanism of inhibition by which Au-NPs can intervene, either by attaching themselves to the virus, blocking its attachment to the cell and consequent entry in the cell and ultimately interfering with the mechanisms of viral replication inside the cell (adapted from [88]).
Overall, there are many studies in the literature stating that the complete mechanism of action underlining the antimicrobial activity and efficacy of M-NPs is not fully clear or understood; however, among the parameters that influence the biocidal effects of metals are the size, shape, and metal ions involved [87]. With bacteriathis influence diverges between Gram-negative and Gram-positive cells, where M-NPs have more difficulty in passing through the thick layer of peptidoglycan in the Gram-positive cell wall, but, once inside the cell, the principles are the same. The antifungal mechanisms of M-NPs are similar to the antibacterial ones, which are the rupture of the cell membrane by oxidative stress and leakage of DNA and proteins. Regarding the antiviral mechanisms of M-NPs, they compete with the virus for cell-binding sites, therefore, inhibiting virus attachment to the cell, or preventing viral replication inside the cell.

Microparticles
Microparticles (MPs) are spherical particles that range in size from 0.1 to 1000 µm and can be synthesized from natural and synthetic materials, such as polymers, glass, and ceramics. Similar to what happens with NPs, their small size gives them an advantage over larger particles (macroscale) and presents many desirable properties for a variety of different applications, such as drug delivery [89,90].
In general, MPs are very similar to NPs in terms of their synthesis methods and applications, except for their size, which is bigger. Similar to NPs, MPs are commonly used to promote antimicrobial effects in different industries, and can be used as a drug delivery system due to their ability to protect from degradation bioactive drugs, proteins, and small molecules; they are also capable of controlling the release rate of encapsulated drugs over an extended period, up to months, and also, they are easily processed [89,91].
Some studies have been conducted regarding the use of MPs against different pathogens. For instance, Sophee and co-workers synthesized titanium oxide (TiO 2 ) and ZnO MPs using the sol-gel method to prevent water-borne microbial infections. From the obtained results, it was possible to enhance the antibacterial activity against Gram-negative bacteria (E. coli and K. pneumoniae) and Gram-positive bacteria (Streptococcus pyogenes) [92]. More recently, Steckiewicz and coworkers studied spherical Ag-orthophosphate-MPs, produced by chemical precipitation, against Gram-positive bacteria (S. aureus) and fungi/yeast (C. albicans and A. niger). The application of these MPs led to increased levels of ROS and first-line defense antioxidants, such as superoxide dismutase and glutathione peroxidase, which could lead to biomedical applications in implant-related infections [93].
Due to their difference in size, MPs take more time to dissolve than NPs; therefore, they release fewer metal ions in the same period of time. As such, NPs usually present a better alternative with stronger and better antimicrobial effects [10,93].

Antimicrobial Surfaces and Coatings
As mentioned above, pathogen resistance has prompted the development of antimicrobial surfaces. A variety of inorganic materials (namely metals, metalloids, metal alloys, metal ions and oxides) can be used to grant surfaces effective antimicrobial properties [1,94]. In fact, not only can they be used as alternative antimicrobial materials, but they are also advantageous as they present more stable antimicrobial action and are a "green" approach (eco-friendly) when compared to polymers and chemical surfaces [1].
To fight these multi-resistant infections, the propagation of pathogens needs to be stopped. For this matter, several researchers have developed different antimicrobial surfaces with biocidal and/or anti-adhesive properties [95,96].
Amongst the wide range of metals, Cu and Ag are the two most commonly used, with proven antimicrobial properties, such as the ability to effectively inhibit and kill different bacterial and viral strains upon direct contact, either as coatings or as the surface material itself [97,98]. This happens due to the release of metallic ions that become toxic to microorganisms at high concentrations, leading to membrane rupture and, therefore, cell death. Between both metals, Cu proves to be advantageous compared to Ag, as it exhibits the best antimicrobial results and is also cheaper to acquire. For this reason, a large number of Cu-containing surfaces have been studied and been registered to have antimicrobial activity against several microorganisms [1,94,96,98]. Several applications for Cu surfaces, as well as for other kinds of metallic surfaces with proven results on the different microorganism reductions are described below: Healthcare applications Metals have been used for healthcare applications to prevent the spread and promote dead of pathogens over human history. In this context, several researchers have been studying the effect of metallic surfaces when in contact with different pathogens. For example, Noyce and co-workers compared the antiviral effect of Cu and stainless-steel surfaces by inoculating the Influenza A virus in sterile coupons of both metals for periods between 1 and 24 h [99]. After, the number of viral particles that remained infectious after the incubation process was determined, resulting in a 75% reduction of active virus after 24 h with stainless steel. Cu managed the same reduction (75%) in just 1 h, further reducing the number of infectious viruses to under 0.1% after 6 h of initial inoculation, emphasizing Cu's great antimicrobial potential [90]. In another study, Warnes and collaborators investigated the antiviral activity of a wide range of Cu-containing alloys, including Cu-Nis and brasses, as well as Cu, Ni, and Zn in their pure metal forms (as controls) against human coronavirus 229E (HuCoV-229E) [100]. To investigate the antiviral effects of these metals, they spread HuCoV-229E over 1 cm 2 coupons of the various test surfaces before incubating at room temperature for different periods of time. Their main results indicated that plain Cu, brasses containing over 70% Cu, and Cu-Nis with over 90% Cu, displayed the best antiviral effects against HuCoV-229E, as some of the alloys completely inactivated 100% of the inoculated virus after only 20 min of exposure [100]. Coincidently, some of the alloys used against HuCoV-229E were also used against E. coli O157 by Wilks and co-workers under similar conditions (using metal coupons) and the results obtained against these Gram-negative bacteria were similar to those observed against the virus [101]. C21000 (95% Cu) and C23000 (85% Cu) alloys, which were used in both cases, showed some of the best antiviral and antibacterial results in their respective works. C21000 managed to completely eradicate HuCoV-229E in 20 min, while it took a little longer to kill 100% of E. coli O157 (90 min). As for C23000, total microbial eradication was achieved after 60 min for both HuCoV-229E and E. coli O157, displaying the best antibacterial activity in the corresponding study [99,101]. These results indicate that brasses containing over 85% Cu could be a viable option for the fabrication of antimicrobial surfaces for general use.
The antimicrobial activity of Cu is effective against several pathogenic microorganisms, namely bacteria and viruses, as evidenced in this review paper. However, Cu's inhibitory effect against pathogens also extends to fungi. Ballo and collaborators described the fungicidal activity of Cu-coated surfaces against azole-resistant C. albicans and C. glabrata [96]. In this study, Cu-sputtered polyester surfaces (Cu-PES) and uncoated PES (control) were used and inoculated with the above-mentioned fungal strains, for different periods (15, 30, or 60 min) at room temperature and under either dark or actinic light. The authors found that Cu-PES displayed fungicidal activity against both C. albicans and C. glabrata under dark and actinic light conditions within 60 min [96].
Recently, the SARS-CoV-2 pandemic hit the globe, becoming a main focus for science to determine how to prevent its spread. Concerning this, several researchers worldwide started to work hard on this problem. Among them, Hutasoit and collaborators explored the viricidal activity of cold-sprayed Cu-coated surfaces against SARS-CoV-2 in an attempt to slow the dissemination of this virus (as well as other viruses) [102]. The cold-spray coating method used in their work is highly advantageous because it allows a very rapid coating "treatment" of commonly touched steel surfaces, which is faster and cheaper than replacing every single one of these surfaces that are touched by the public. This cold-spray technique consists of propulsion of metal particles at supersonic speeds onto surfaces due to a highly pressurized carrier gas, forming a thin dense metal layer. For antiviral testing, a push plate was subjected to an annealing treatment (Cu A) while another was tested without further treatments (Cu N). After preliminary tests, they proceeded to study the viricidal effect of Cu coating by placing SARS-CoV-2 on small squares of Cu (Cu N), stainless steel and activated Cu (Cu A) at room temperature at different times (1, 10, 30, 120 and 300 min). Their main results showed a 96% viral inactivation after 2 h of exposure to the Cu N coating and a 92% inactivation on the Cu A coating after the same exposure time. After 5 h of contact, the viral inactivation efficiency increased to 99.2% and 97.9%, for the respective coatings [102].
Other researchers have also studied surfaces with physical and/or chemical modifications. As an example, Selvamani and collaborators demonstrated the antimicrobial properties of a laser textured Cu (LT-Cu) surface and its efficiency against pathogenic bacteria, such as methicillin-resistant S. aureus (MRSA), P. aeruginosa, S. aureus, and E. coli, at different concentrations [1]. According to their findings, the LT-Cu process enhanced the bactericidal properties of the surfaces, achieving total eradication of E. coli after 120 min of direct exposure. Moreover, they studied the bacterial killing potential of simple (not modified) Cu surfaces and LT-Cu surfaces by dipping both surfaces in PBS (phosphatebuffered saline) with two multidrug-resistant bacterial strains (MRSA and P. aeruginosa) and found that LT-Cu surfaces completely eradicated P. aeruginosa and MRSA after 40 and 90 min of exposure, respectively. The pristine Cu surfaces also killed 100% of both strains but at a slower pace. These authors found that, when higher concentrations of S. aureus and E. coli were applied, only LT-Cu surfaces were able to eradicate them (at 120 and 60 min of exposure). Moreover, the LT-Cu process used by these researchers produced micro and mesoporous structures, which in turn granted anti-adhesive properties to the surfaces that might have helped to improve their antibacterial activity [1].
Taking the above-mentioned into account, and to create the "ultimate" antimicrobial surface, a combination of anti-adhesive surfaces and biocidal agents was proposed by Ellinas and co-workers, as well as Kefallinou and co-workers, due to the limitations of both types of surfaces when used separately (e.g., biofilm formation reduces the biocidal activity of surfaces without anti-adhesive properties, while in anti-adhesive surfaces with no biocidal agents the anti-adhesive properties are lost over time, as the few attached microorganisms grow and form biofilms) [4,95]. These works consist in the development and study of the antimicrobial activity of micro-nanotextured superhydrophobic poly(methyl methacrylate) (PMMA) surfaces complemented with sputtered Cu and Ag coatings, which they referred to as "hybrid" surfaces. Overall, amongst several tests and comparisons between surfaces, they concluded that Cu was the best bactericidal agent and the Cu-coated superhydrophobic PMMA surface achieved the best antibacterial activity, as it demonstrated antimicrobial action against Synechococcus spp. PCC7942 (at one of the highest concentrations ever reported) [4,95].
Food industry and other applications Metal surfaces and coatings have also been widely applied in the food industry as an attempt to fight cross-contamination (which commonly occurs between food, surfaces, and equipment). Concerning this, Akhidime and co-workers studied the leaching potential of Ag, Ti, Cu, Fe, molybdenum, and Zn into media and its effect against S. aureus, E. coli and L. monocytogenes, with silicon substrates as base surfaces (control) [97]. One of their antibacterial studies was conducted by submerging metal-coated surfaces into bacterial suspensions and registering the number of viable cells over time via plate counts. Among the main results, it was possible to observe that the biggest leaching behavior was obtained for Cu followed by Zn. Surprisingly, Ag demonstrated one of the lowest leaching behaviors. However, despite Ag's leaching potential being 350 times lower than Cu, Ag displayed one of the best antimicrobial effects. Cu's antibacterial potential was the greatest, killing 99% of the three bacterial species after just one hour of incubation. In the same period, Ag managed to kill 46%, 99%, and 34% of S. aureus, E. coli, and L. monocytogenes, respectively. Overall, Cu was the coating with the best antimicrobial activity, followed by Ag as the second-best coating against the three pathogenic bacterial species tested (E. coli, S. aureus, and L. monocytogenes) [97].
Another approach to the treatment of fungal infections using metals was presented by Bastos and co-workers with a study of the antimicrobial effect of gallium (gallium nitrate III, [Ga(NO 3 ) 3 ]) against different fungal pathogens, namely azole-resistant A. fumigatus and different species of Candida spp [103]. They performed a fungal killing assay by testing different concentrations of Ga(NO 3 ) 3 against various strains. For this, inoculated microplates were incubated at 37 • C for 2, 8, 16, 24, and 48 h in different media, and samples of each time were taken to measure cell viability. Based on their results, the researchers concluded that the antifungal activity of gallium is dependent on the Fe concentration in the medium, as gallium's MIC increased (or no antifungal effect was obtained) when Fe-containing media were used [103].
Despite all the above-mentioned information, metal surfaces and coatings alone are not the sole options for the development of antimicrobial surfaces. Wettability is a property of materials that have been extensively studied to accomplish antimicrobial surfaces, through either super hydrophobicity or super hydrophilicity, due to their ability to reduce microbial adhesion [1,103]. When pathogenic adhesion is reduced, further growth and dissemination are hindered, which passively prevents infections [4,95,103]. Moreover, roughness can also cause a surface to become superhydrophobic, due to the entrapment of air between roughened structures when a liquid comes in contact with the solid surface. This entrapped air has an anti-adhesive effect that hinders microbial adhesion, as it reduces the area available for microorganisms to attach, therefore minimizing adhesion to surfaces with moderate wettability, such as food or food packaging [104]. Table 3 presents a summary of several studies using different approaches for the development of antimicrobial surfaces.

Synergic Combination with Drugs
Metallic particles could work with standard drugs to increase their antimicrobial activity. Moreover, the majority of practical trials in the literature regarding the synergistic effect between metals and drugs use Ag-NPs combined with antibiotics. Figure 2 presents an example of the combined action of antibiotics with Ag-NPs. M-NPs can disrupt the bacterial membrane of both Gram-negative (left) and Gram-positive (right) bacteria, enabling the entrance of antibiotics into the cell and leading to DNA damage. Moreover, the combined action of antibiotics with M-NPs induces oxidative stress and creates ROS; consequently, it also damages the bacterial DNA (as noted above) [106][107][108][109][110][111].

Synergic Combination with Drugs
Metallic particles could work with standard drugs to increase the tivity. Moreover, the majority of practical trials in the literature regar effect between metals and drugs use Ag-NPs combined with antibiotic an example of the combined action of antibiotics with Ag-NPs. M-N bacterial membrane of both Gram-negative (left) and Gram-positive ( bling the entrance of antibiotics into the cell and leading to DNA dam combined action of antibiotics with M-NPs induces oxidative stress an sequently, it also damages the bacterial DNA (as noted above) [106-1 Figure 2. Representative illustration of the interaction between antibiotics a combined action inside both Gram-negative (left) and Gram-positive (righ there is a thicker path for Ag-NPs and antibiotics to travel through with the G membrane, once, inside the cell, the mechanisms are the same as for Gram-p mulation of particles inside the cell; production of ROS; and ultimately cell d bacterial DNA (adapted from [111]). This effect, increased by the dual antimicrobial vehicles produce different drugs, has already been demonstrated by several authors, as 4. Some authors have studied the synergy of M-NPs in combination w Although there is a thicker path for Ag-NPs and antibiotics to travel through with the Gram-negative bacteria membrane, once, inside the cell, the mechanisms are the same as for Gram-positive bacteria: accumulation of particles inside the cell; production of ROS; and ultimately cell death by damaging the bacterial DNA (adapted from [111]). This effect, increased by the dual antimicrobial vehicles produced using M-NPs and different drugs, has already been demonstrated by several authors, as presented in Table 4. Some authors have studied the synergy of M-NPs in combination with antibiotics, such as amoxicillin and polymyxin B, and found higher bactericidal activity by binding Ag-NPs with these antibiotics [112,113]. For example, Abo-Shama and co-workers detected the antimicrobial activity of Ag-NPs and ZnO-NPs alone and combined with a range of antibiotics [114]. From nearly all the different antibiotic classes against Gram-positive and Gram-negative bacteria (S. aureus, E. coli, Salmonella spp. and C. albicans), their findings showed that the combination of Ag-NPs with some of the tested antibiotics presented a better synergistic effect than ZnO-NPs with the same antibiotics [114]. In addition, Vazquez-Muñoz and collaborators evaluated the antimicrobial activity of Ag-NPs combined with several antibiotics, such as chloramphenicol, kanamycin, ampicillin, aztreonam, and biapenem in both Gram-positive and Gram-negative bacteria [106]. They determined that Ag-NPs change the cell membrane integrity and increase cell permeability, enabling antibiotics intracellular access, which will enhance the efficiency of the antibiotics on their intracellular targets [106].
In another study, Pereira and co-workers produced chitosan microspheres and evaluated their efficiency in the adsorption of Ag ions for the formation of NPs [115]. Then, they evaluated the bactericidal activity and potential as a system for the release of ibuprofen. They found that the concentration of ibuprofen retained in chitosan microspheres was higher than in Ag-NPs microspheres, which indicates that microspheres with Ag-NPs released more drug (77%), exhibiting bactericidal characteristics and a greater drug release capacity than the material without Ag-NPs [115].   [124]
The bonding reaction between the antifungal and AgNPs might be chelation, which altered the membrane permeability and morphology.
When alone, the MIC values were 2.5 and 5 mg/mL for Ag-NPs and AmB, respectively, whereas the MFC values were 10 and >10 mg/mL for Ag-NPs and AmB, respectively. However, when they were combined, the MIC and MFC values decreased to 0.156 and 2.5 mg/mL, respectively. [118] tebuconazole, propineb, fludioxonil Bipolaria maydis Antifungals and NPs were used individually and not complexed.
The synergism between antifungal compounds and Ag-NPs was measured by the percentage of inhibition towards B. maydis ranging from 46-58% for the Ag-NPs alone 48.28, 47.27 and 52.13% for the fungicides Tebuconazole, Propineb, Fludioxonil, respectively, and with values of 73.08, 64.56 and 62.13% for the combination of Ag-NPs with tebuconazole, propineb, fludioxonil, respectively, therefore proving a synergistic interaction between the NPs and the fungicides. [125] zineb Neoscytalidium dimidiatum.
Chitosan was used to functionalized with Ag-NPs and improve the NPs stability, then this compound was combined with zineb and meant to improve fungicidal effect.
To prove the synergistic effects between Ag-NPs and zineb, the inhibition zones were measured and were high when in combination. [126]
Ag-NPs and OTV were synthesized together, being the OTV on the surface of the Ag-NPs. It is believed that Ag-NPs can facilitate OTV's entry into the cell to exert its antiviral action by reducing ROS and p53 levels.
For the infection growth in MDCK cells, the virus alone diminished cell viability to below 40%, virus+OTV presented viability below 60% and the virus+Ag-NPs close to 70%. Virus+Ag-NPs+OTV reached almost 100% of cell viability. Similar results were obtained for the mitochondrial membrane potential, proving the synergism between Ag-NPs and the antiviral OTV. [127] Ag & Au

FluPep
Influenza type-A virus.
Ag-NPs and Au-NPs were used as probes to deliver FluPep onto the cell. The system was based on a shell of NPs around the peptideFluPep.
Ag-NPs and Au-NPs in combination with the peptide provide better antiviral activity than FluPep alone. FluPep alone had IC 50 values ranging from 1-5 nM, whilst in combination with Au-NPs and Ag-NPs, the value of IC 50 decreased to 0.015 nM, proving a positive synergistic activity between the antiviral peptide and the NPs. [128] Although the majority of the research performed until now has focused on the synergistic interaction between Ag-NPs and antibiotics, other (fewer) experiments have been performed on other types of M-NPs conjugated with different drugs, such as antifungal or antiviral medicines, proving the versatility of M-NPs for a wide range of biomedical applications.

Conclusions and Future Perspectives
Metals are very interesting and promising vehicles to promote antimicrobial effects. Through this review, it is possible to access a compilation of several studies, showing that different metals can be used in different types of "structures" to achieve/increase biocidal behavior. For example, metal particles have been shown as efficient approaches to promote antimicrobial effects. Although they can be applied as micro-or nanostructures, the nano size is usually desirable to be applied in biomedical fields. Some works have reported that NPs, particularly Ag-NPs, can present good synergistic relations with antibiotics, resulting in an enhanced antibacterial effect on resistant strains of bacteria. Some authors have even proposed that this relation and the type of response could be transposed to studies with viruses, although there is a need for additional research in this field. Although several advantages regarding the use of M-NPs have been highlighted, there is still a need to improve many aspects, such as their production methodologies, which should be mostly achieved through biological synthesis methods, trying to use few chemicals, as well as generating lesser amounts of waste.
Regarding metallic surfaces and coatings, according to the examples presented here, Cu has proven to be the metal with the best biocidal properties against several microorganisms. In addition, anti-wetting properties were revealed to be important to improve the antimicrobial activity of metal surfaces or metal-coated surfaces. Overall, a variety of approaches for the development of these self-disinfecting surfaces were able to accomplish just that-create surfaces that kill pathogenic microorganisms, such as bacteria, viruses, and fungi upon direct contact. This is true even against the most recent threat to humanity, the SARS-CoV-2 (COVID-19) pandemic.
As for future projects, the study of dual functionality (involving the combination of biocidal agents with anti-adhesive surfaces) might be the key to the development of superefficient multifunctional and multimicrobial killing agents.