When was silver nanoparticles invented?

Ultrafine particles of silver between 1 nm and 100 nm in size

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Electron micrograph of silver nanoparticles

Silver nanoparticles are nanoparticles of silver of between 1 nm and 100 nm in size.[1] While frequently described as being 'silver' some are composed of a large percentage of silver oxide due to their large ratio of surface to bulk silver atoms. Numerous shapes of nanoparticles can be constructed depending on the application at hand. Commonly used silver nanoparticles are spherical, but diamond, octagonal, and thin sheets are also common.[1]

Their extremely large surface area permits the coordination of a vast number of ligands. The properties of silver nanoparticles applicable to human treatments are under investigation in laboratory and animal studies, assessing potential efficacy, biosafety, and biodistribution.[2]

Synthesis methods

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Wet chemistry

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The most common methods for nanoparticle synthesis fall under the category of wet chemistry, or the nucleation of particles within a solution. This nucleation occurs when a silver ion complex, usually AgNO3 or AgClO4, is reduced to colloidal Ag in the presence of a reducing agent. When the concentration increases enough, dissolved metallic silver ions bind together to form a stable surface. The surface is energetically unfavorable when the cluster is small, because the energy gained by decreasing the concentration of dissolved particles is not as high as the energy lost from creating a new surface.[3] When the cluster reaches a certain size, known as the critical radius, it becomes energetically favorable, and thus stable enough to continue to grow. This nucleus then remains in the system and grows as more silver atoms diffuse through the solution and attach to the surface[4] When the dissolved concentration of atomic silver decreases enough, it is no longer possible for enough atoms to bind together to form a stable nucleus. At this nucleation threshold, new nanoparticles stop being formed, and the remaining dissolved silver is absorbed by diffusion into the growing nanoparticles in the solution.

As the particles grow, other molecules in the solution diffuse and attach to the surface. This process stabilizes the surface energy of the particle and blocks new silver ions from reaching the surface. The attachment of these capping/stabilizing agents slows and eventually stops the growth of the particle.[5] The most common capping ligands are trisodium citrate and polyvinylpyrrolidone (PVP), but many others are also used in varying conditions to synthesize particles with particular sizes, shapes, and surface properties.[6]

There are many different wet synthesis methods, including the use of reducing sugars, citrate reduction, reduction via sodium borohydride,[7] the silver mirror reaction,[8] the polyol process,[9] seed-mediated growth,[10] and light-mediated growth.[11] Each of these methods, or a combination of methods, will offer differing degrees of control over the size distribution as well as distributions of geometric arrangements of the nanoparticle.[12]

A new, very promising wet-chemical technique was found by Elsupikhe et al. (2015).[13] They have developed a green ultrasonically-assisted synthesis. Under ultrasound treatment, silver nanoparticles (AgNP) are synthesized with κ-carrageenan as a natural stabilizer. The reaction is performed at ambient temperature and produces silver nanoparticles with fcc crystal structure without impurities. The concentration of κ-carrageenan is used to influence particle size distribution of the AgNPs.[14]

Monosaccharide reduction

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There are many ways silver nanoparticles can be synthesized; one method is through monosaccharides. This includes glucose, fructose, maltose, maltodextrin, etc., but not sucrose. It is also a simple method to reduce silver ions back to silver nanoparticles as it usually involves a one-step process.[15] There have been methods that indicated that these reducing sugars are essential to the formation of silver nanoparticles. Many studies indicated that this method of green synthesis, specifically using Cacumen platycladi extract, enabled the reduction of silver. Additionally, the size of the nanoparticle could be controlled depending on the concentration of the extract. The studies indicate that the higher concentrations correlated to an increased number of nanoparticles.[15] Smaller nanoparticles were formed at high pH levels due to the concentration of the monosaccharides.

Another method of silver nanoparticle synthesis includes the use of reducing sugars with alkali starch and silver nitrate. The reducing sugars have free aldehyde and ketone groups, which enable them to be oxidized into gluconate.[16] The monosaccharide must have a free ketone group because in order to act as a reducing agent it first undergoes tautomerization. In addition, if the aldehydes are bound, it will be stuck in cyclic form and cannot act as a reducing agent. For example, glucose has an aldehyde functional group that is able to reduce silver cations to silver atoms and is then oxidized to gluconic acid.[17] The reaction for the sugars to be oxidized occurs in aqueous solutions. The capping agent is also not present when heated.

Citrate reduction

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An early, and very common, method for synthesizing silver nanoparticles is citrate reduction. This method was first recorded by M. C. Lea, who successfully produced a citrate-stabilized silver colloid in 1889.[18] Citrate reduction involves the reduction of a silver source particle, usually AgNO3 or AgClO4, to colloidal silver using trisodium citrate, Na3C6H5O7.[19] The synthesis is usually performed at an elevated temperature (~100 °C) to maximize the monodispersity (uniformity in both size and shape) of the particle. In this method, the citrate ion traditionally acts as both the reducing agent and the capping ligand,[19] making it a useful process for AgNP production due to its relative ease and short reaction time. However, the silver particles formed may exhibit broad size distributions and form several different particle geometries simultaneously.[18] The addition of stronger reducing agents to the reaction is often used to synthesize particles of a more uniform size and shape.[19]

Reduction via sodium borohydride

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The synthesis of silver nanoparticles by sodium borohydride (NaBH4) reduction occurs by the following reaction:[20]

Ag+ + BH4− + 3 H2O → Ag0 +B(OH)3 +3.5 H2

The reduced metal atoms will form nanoparticle nuclei. Overall, this process is similar to the above reduction method using citrate. The benefit of using sodium borohydride is increased monodispersity of the final particle population. The reason for the increased monodispersity when using NaBH4 is that it is a stronger reducing agent than citrate. The impact of reducing agent strength can be seen by inspecting a LaMer diagram which describes the nucleation and growth of nanoparticles.[21]

When silver nitrate (AgNO3) is reduced by a weak reducing agent like citrate, the reduction rate is lower which means that new nuclei are forming and old nuclei are growing concurrently. This is the reason that the citrate reaction has low monodispersity. Because NaBH4 is a much stronger reducing agent, the concentration of silver nitrate is reduced rapidly which shortens the time during which new nuclei form and grow concurrently yielding a monodispersed population of silver nanoparticles.

Particles formed by reduction must have their surfaces stabilized to prevent undesirable particle agglomeration (when multiple particles bond together), growth, or coarsening. The driving force for these phenomena is the minimization of surface energy (nanoparticles have a large surface to volume ratio). This tendency to reduce surface energy in the system can be counteracted by adding species which will adsorb to the surface of the nanoparticles and lowers the activity of the particle surface thus preventing particle agglomeration according to the DLVO theory and preventing growth by occupying attachment sites for metal atoms. Chemical species that adsorb to the surface of nanoparticles are called ligands. Some of these surface stabilizing species are: NaBH4 in large amounts,[20] poly(vinyl pyrrolidone) (PVP),[22] sodium dodecyl sulfate (SDS),[20][22] and/or dodecanethiol.[23]

Once the particles have been formed in solution they must be separated and collected. There are several general methods to remove nanoparticles from solution, including evaporating the solvent phase[23] or the addition of chemicals to the solution that lower the solubility of the nanoparticles in the solution.[24] Both methods force the precipitation of the nanoparticles.

Polyol process

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The polyol process is a particularly useful method because it yields a high degree of control over both the size and geometry of the resulting nanoparticles. In general, the polyol synthesis begins with the heating of a polyol compound such as ethylene glycol, 1,5-pentanediol, or 1,2-propylene glycol7. An Ag+ species and a capping agent are added (although the polyol itself is also often the capping agent). The Ag+ species is then reduced by the polyol to colloidal nanoparticles.[25] The polyol process is highly sensitive to reaction conditions such as temperature, chemical environment, and concentration of substrates.[26][27] Therefore, by changing these variables, various sizes and geometries can be selected for such as quasi-spheres, pyramids, spheres, and wires.[12] Further study has examined the mechanism for this process as well as resulting geometries under various reaction conditions in greater detail.[9][28]

Seed-mediated growth

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Seed-mediated growth is a synthetic method in which small, stable nuclei are grown in a separate chemical environment to a desired size and shape. Seed-mediated methods consist of two different stages: nucleation and growth. Variation of certain factors in the synthesis (e.g. ligand, nucleation time, reducing agent, etc.),[28] can control the final size and shape of nanoparticles, making seed-mediated growth a popular synthetic approach to controlling morphology of nanoparticles.

The nucleation stage of seed-mediated growth consists of the reduction of metal ions in a precursor to metal atoms. In order to control the size distribution of the seeds, the period of nucleation should be made short for monodispersity. The LaMer model illustrates this concept.[29] Seeds typically consist small nanoparticles, stabilized by a ligand. Ligands are small, usually organic molecules that bind to the surface of particles, preventing seeds from further growth. Ligands are necessary as they increase the energy barrier of coagulation, preventing agglomeration. The balance between attractive and repulsive forces within colloidal solutions can be modeled by DLVO theory.[30] Ligand binding affinity, and selectivity can be used to control shape and growth. For seed synthesis, a ligand with medium to low binding affinity should be chosen as to allow for exchange during growth phase.

The growth of nanoseeds involves placing the seeds into a growth solution. The growth solution requires a low concentration of a metal precursor, ligands that will readily exchange with preexisting seed ligands, and a weak or very low concentration of reducing agent. The reducing agent must not be strong enough to reduce metal precursor in the growth solution in the absence of seeds. Otherwise, the growth solution will form new nucleation sites instead of growing on preexisting ones (seeds).[31] Growth is the result of the competition between surface energy (which increases unfavorably with growth) and bulk energy (which decreases favorably with growth). The balance between the energetics of growth and dissolution is the reason for uniform growth only on preexisting seeds (and no new nucleation).[32] Growth occurs by the addition of metal atoms from the growth solution to the seeds, and ligand exchange between the growth ligands (which have a higher bonding affinity) and the seed ligands.[33]

Range and direction of growth can be controlled by nanoseed, concentration of metal precursor, ligand, and reaction conditions (heat, pressure, etc.).[34] Controlling stoichiometric conditions of growth solution controls ultimate size of particle. For example, a low concentration of metal seeds to metal precursor in the growth solution will produce larger particles. Capping agent has been shown to control direction of growth and thereby shape. Ligands can have varying affinities for binding across a particle. Differential binding within a particle can result in dissimilar growth across particle. This produces anisotropic particles with nonspherical shapes including prisms, cubes, and rods.[35][36]

Light-mediated growth

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Light-mediated syntheses have also been explored where light can promote formation of various silver nanoparticle morphologies.[11][37][38]

Silver mirror reaction

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The silver mirror reaction involves the conversion of silver nitrate to Ag(NH3)OH. Ag(NH3)OH is subsequently reduced into colloidal silver using an aldehyde containing molecule such as a sugar. The silver mirror reaction is as follows:

2(Ag(NH3)2)+ + RCHO + 2OH− → RCOOH + 2Ag + 4NH3.[39]

The size and shape of the nanoparticles produced are difficult to control and often have wide distributions.[12] However, this method is often used to apply thin coatings of silver particles onto surfaces and further study into producing more uniformly sized nanoparticles is being done.[12]

Ion implantation

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Ion implantation has been used to create silver nanoparticles embedded in glass, polyurethane, silicone, polyethylene, and poly(methyl methacrylate). Particles are embedded in the substrate by means of bombardment at high accelerating voltages. At a fixed current density of the ion beam up to a certain value, the size of the embedded silver nanoparticles has been found to be monodisperse within the population,[40] after which only an increase in the ion concentration is observed. A further increase in the ion beam dose has been found to reduce both the nanoparticle size and density in the target substrate, whereas an ion beam operating at a high accelerating voltage with a gradually increasing current density has been found to result in a gradual increase in the nanoparticle size. There are a few competing mechanisms which may result in the decrease in nanoparticle size; destruction of NPs upon collision, sputtering of the sample surface, particle fusion upon heating and dissociation.[40]

The formation of embedded nanoparticles is complex, and all of the controlling parameters and factors have not yet been investigated. Computer simulation is still difficult as it involves processes of diffusion and clustering, however it can be broken down into a few different sub-processes such as implantation, diffusion, and growth. Upon implantation, silver ions will reach different depths within the substrate which approaches a Gaussian distribution with the mean centered at X depth. High temperature conditions during the initial stages of implantation will increase the impurity diffusion in the substrate and as a result limit the impinging ion saturation, which is required for nanoparticle nucleation.[41] Both the implant temperature and ion beam current density are crucial to control in order to obtain a monodisperse nanoparticle size and depth distribution. A low current density may be used to counter the thermal agitation from the ion beam and a buildup of surface charge. After implantation on the surface, the beam currents may be raised as the surface conductivity will increase.[41] The rate at which impurities diffuse drops quickly after the formation of the nanoparticles, which act as a mobile ion trap. This suggests that the beginning of the implantation process is critical for control of the spacing and depth of the resulting nanoparticles, as well as control of the substrate temperature and ion beam density. The presence and nature of these particles can be analyzed using numerous spectroscopy and microscopy instruments.[41] Nanoparticles synthesized in the substrate exhibit surface plasmon resonances as evidenced by characteristic absorption bands; these features undergo spectral shifts depending on the nanoparticle size and surface asperities,[40] however the optical properties also strongly depend on the substrate material of the composite.

Biological synthesis

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The biological synthesis of nanoparticles has provided a means for improved techniques compared to the traditional methods that call for the use of harmful reducing agents like sodium borohydride. Many of these methods could improve their environmental footprint by replacing these relatively strong reducing agents. The commonly used biological methods are using plant or fruit extracts, fungi, and even animal parts like insect wing extract.[42][43][44] The problems with the chemical production of silver nanoparticles is usually involves high cost and the longevity of the particles is short lived due to aggregation. The harshness of standard chemical methods has sparked the use of using biological organisms to reduce silver ions in solution into colloidal nanoparticles.[45][46]

In addition, precise control over shape and size is vital during nanoparticle synthesis since the NPs therapeutic properties are intimately dependent on such factors.[47] Hence, the primary focus of research in biogenic synthesis is in developing methods that consistently reproduce NPs with precise properties.[48][49]

Fungi and bacteria

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Bacterial and fungal synthesis of nanoparticles is practical because bacteria and fungi are easy to handle and can be modified genetically with ease. This provides a means to develop biomolecules that can synthesize AgNPs of varying shapes and sizes in high yield, which is at the forefront of current challenges in nanoparticle synthesis. Fungal strains such as Verticillium and bacterial strains such as Klebsiella pneumoniae can be used in the synthesis of silver nanoparticles.[50] When the fungus/bacteria is added to solution, protein biomass is released into the solution.[50] Electron donating residues such as tryptophan and tyrosine reduce silver ions in solution contributed by silver nitrate.[50] These methods have been found to effectively create stable monodisperse nanoparticles without the use of harmful reducing agents.

A method has been found of reducing silver ions by the introduction of the fungus Fusarium oxysporum. The nanoparticles formed in this method have a size range between 5 and 15 nm and consist of silver hydrosol. The reduction of the silver nanoparticles is thought to come from an enzymatic process and silver nanoparticles produced are extremely stable due to interactions with proteins that are excreted by the fungi.

Bacterium found in silver mines, Pseudomonas stutzeri AG259, were able to construct silver particles in the shapes of triangles and hexagons. The size of these nanoparticles had a large range in size and some of them reached sizes larger than the usual nanoscale with a size of 200 nm. The silver nanoparticles were found in the organic matrix of the bacteria.[51]

Lactic acid producing bacteria have been used to produce silver nanoparticles. The bacteria Lactobacillus spp., Pediococcus pentosaceus, Enteroccus faeciumI, and Lactococcus garvieae have been found to be able to reduce silver ions into silver nanoparticles. The production of the nanoparticles takes place in the cell from the interactions between the silver ions and the organic compounds of the cell. It was found that the bacterium Lactobacillus fermentum created the smallest silver nanoparticles with an average size of 11.2 nm. It was also found that this bacterium produced the nanoparticles with the smallest size distribution and the nanoparticles were found mostly on the outside of the cells. It was also found that there was an increase in the pH increased the rate of which the nanoparticles were produced and the amount of particles produced.[52]

Plants

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The reduction of silver ions into silver nanoparticles has also been achieved using geranium leaves. It has been found that adding geranium leaf extract to silver nitrate solutions causes their silver ions to be quickly reduced and that the nanoparticles produced are particularly stable. The silver nanoparticles produced in solution had a size range between 16 and 40 nm.[51]

In another study different plant leaf extracts were used to reduce silver ions. It was found that out of Camellia sinensis (green tea), pine, persimmon, ginko, magnolia, and platanus that the magnolia leaf extract was the best at creating silver nanoparticles. This method created particles with a disperse size range of 15 to 500 nm, but it was also found that the particle size could be controlled by varying the reaction temperature. The speed at which the ions were reduced by the magnolia leaf extract was comparable to those of using chemicals to reduce.[45][53]

The use of plants, microbes, and fungi in the production of silver nanoparticles is leading the way to more environmentally sound production of silver nanoparticles.[46]

A green method is available for synthesizing silver nanoparticles using Amaranthus gangeticus Linn leaf extract.[54]

Products and functionalization

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At small sizes silver nanoparticles typically contain twins, either Icosahedral or decagedral.[55] Synthetic protocols for silver nanoparticle production can be modified to produce silver nanoparticles with non-spherical geometries and also to functionalize nanoparticles with different materials, such as silica. Creating silver nanoparticles of different shapes and surface coatings allows for greater control over their size-specific properties.

Anisotropic structures

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Silver nanoparticles can be synthesized in a variety of non-spherical (anisotropic) shapes. Because silver, like other noble metals, exhibits a size and shape dependent optical effect known as localized surface plasmon resonance (LSPR) at the nanoscale, the ability to synthesize Ag nanoparticles in different shapes vastly increases the ability to tune their optical behavior. For example, the wavelength at which LSPR occurs for a nanoparticle of one morphology (e.g. a sphere) will be different if that sphere is changed into a different shape. This shape dependence allows a silver nanoparticle to experience optical enhancement at a range of different wavelengths, even by keeping the size relatively constant, just by changing its shape. This aspect can be exploited in synthesis to promote change in shape of nanoparticles through light interaction.[38] The applications of this shape-exploited expansion of optical behavior range from developing more sensitive biosensors to increasing the longevity of textiles.[56][57]

Triangular nanoprisms

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Triangular-shaped nanoparticles are a canonical type of anisotropic morphology studied for both gold and silver.[58]

Though many different techniques for silver nanoprism synthesis exist, several methods employ a seed-mediated approach, which involves first synthesizing small (3-5 nm diameter) silver nanoparticles that offer a template for shape-directed growth into triangular nanostructures.[7]

The silver seeds are synthesized by mixing silver nitrate and sodium citrate in aqueous solution and then rapidly adding sodium borohydride. Additional silver nitrate is added to the seed solution at low temperature, and the prisms are grown by slowly reducing the excess silver nitrate using ascorbic acid.[7]

With the seed-mediated approach to silver nanoprism synthesis, selectivity of one shape over another can in part be controlled by the capping ligand. Using essentially the same procedure above but changing citrate to poly (vinyl pyrrolidone) (PVP) yields cube and rod-shaped nanostructures instead of triangular nanoprisms.[59]

In addition to the seed mediated technique, silver nanoprisms can also be synthesized using a photo-mediated approach, in which preexisting spherical silver nanoparticles are transformed into triangular nanoprisms simply by exposing the reaction mixture to high intensities of light.[60][61][38]

Nanocubes

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Silver nanocubes can be synthesized using ethylene glycol as a reducing agent and PVP as a capping agent, in a polyol synthesis reaction (vide supra). A typical synthesis using these reagents involves adding fresh silver nitrate and PVP to a solution of ethylene glycol heated at 140 °C.[62]

This procedure can actually be modified to produce another anisotropic silver nanostructure, nanowires, by just allowing the silver nitrate solution to age before using it in the synthesis. By allowing the silver nitrate solution to age, the initial nanostructure formed during the synthesis is slightly different than that obtained with fresh silver nitrate, which influences the growth process, and therefore, the morphology of the final product.[62]

Coating with silica

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Electron micrograph of core-shell nanoparticles, which comprise dark silver cores and light silica shells

In this method, polyvinylpyrrolidone (PVP) is dissolved in water by sonication and mixed with silver colloid particles.[1] Active stirring ensures the PVP has adsorbed to the nanoparticle surface.[1] Centrifuging separates the PVP coated nanoparticles which are then transferred to a solution of ethanol to be centrifuged further and placed in a solution of ammonia, ethanol and Si(OEt4) (TES).[1] Stirring for twelve hours results in the silica shell being formed consisting of a surrounding layer of silicon oxide with an ether linkage available to add functionality.[1] Varying the amount of TES allows for different thicknesses of shells formed.[1] This technique is popular due to the ability to add a variety of functionality to the exposed silica surface.

Metrology

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A number of reference materials are available for silver nanoparticles.[63] NIST RM 8017 contains 75 nm silver nanoparticles embedded in a cake of the polymer polyvinylpyrrolidone to stabilize them against oxidation for a long shelf life. They have reference values for mean particle size using dynamic light scattering, ultra-small-angle X-ray scattering, atomic force microscopy, and transmission electron microscopy; and size distribution reference values for the latter two methods.[64][65] The BAM-N001 certified reference material contains silver nanoparticles with a specified size distribution with a number-weighted median size of 12.6 nm measured by small-angle X-ray scattering and transmission electron microscopy.[66]

Use

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Catalysis

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Using silver nanoparticles for catalysis has been gaining attention in recent years. Although the most common applications are for medicinal or antibacterial purposes, silver nanoparticles have been demonstrated to show catalytic redox properties for dyes, benzene, and carbon monoxide. Other untested compounds may use silver nanoparticles for catalysis, but the field is not fully explored.

NOTE: This paragraph is a general description of nanoparticle properties for catalysis; it is not exclusive to silver nanoparticles. The size of a nanoparticle greatly determines the properties that it exhibits due to various quantum effects. Additionally, the chemical environment of the nanoparticle plays a large role on the catalytic properties. With this in mind, it is important to note that heterogeneous catalysis takes place by adsorption of the reactant species to the catalytic substrate. When polymers, complex ligands, or surfactants are used to prevent coalescence of the nanoparticles, the catalytic ability is frequently hindered due to reduced adsorption ability.[67] However, these compounds can also be used in such a way that the chemical environment enhances the catalytic ability.

Supported on silica spheres – reduction of dyes

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Silver nanoparticles have been synthesized on a support of inert silica spheres.[67] The support plays virtually no role in the catalytic ability and serves as a method of preventing coalescence of the silver nanoparticles in colloidal solution. Thus, the silver nanoparticles were stabilized and it was possible to demonstrate the ability of them to serve as an electron relay for the reduction of dyes by sodium borohydride.[67] Without the silver nanoparticle catalyst, virtually no reaction occurs between sodium borohydride and the various dyes: methylene blue, eosin, and rose bengal.

Mesoporous aerogel – selective oxidation of benzene

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Silver nanoparticles supported on aerogel are advantageous due to the higher number of active sites.[68] The highest selectivity for oxidation of benzene to phenol was observed at low weight percent of silver in the aerogel matrix (1% Ag). This better selectivity is believed to be a result of the higher monodispersity within the aerogel matrix of the 1% Ag sample. Each weight percent solution formed different sized particles with a different width of size range.[68]

Silver alloy – synergistic oxidation of carbon monoxide

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Au-Ag alloy nanoparticles have been shown to have a synergistic effect on the oxidation of carbon monoxide (CO).[69] On its own, each pure-metal nanoparticle shows very poor catalytic activity for CO oxidation; together, the catalytic properties are greatly enhanced. It is proposed that the gold acts as a strong binding agent for the oxygen atom and the silver serves as a strong oxidizing catalyst, although the exact mechanism is still not completely understood. When synthesized in an Au/Ag ratio from 3:1 to 10:1, the alloyed nanoparticles showed complete conversion when 1% CO was fed in air at ambient temperature.[69] The size of the alloyed particles did not play a big role in the catalytic ability. It is well known that gold nanoparticles only show catalytic properties for CO when they are ~3 nm in size, but alloyed particles up to 30 nm demonstrated excellent catalytic activity – catalytic activity better than that of gold nanoparticles on active support such as TiO2, Fe2O3, etc.[69]

Plasmonic effects have been studied quite extensively. Until recently, there have not been studies investigating the oxidative catalytic enhancement of a nanostructure via excitation of its surface plasmon resonance. The defining feature for enhancing the oxidative catalytic ability has been identified as the ability to convert a beam of light into the form of energetic electrons that can be transferred to adsorbed molecules.[70] The implication of such a feature is that photochemical reactions can be driven by low-intensity continuous light coupled with thermal energy.

The coupling of low-intensity continuous light and thermal energy has been performed with silver nanocubes. The important feature of silver nanostructures that are enabling for photocatalysis is their nature to create resonant surface plasmons from light in the visible range.[70]

The addition of light enhancement enabled the particles to perform to the same degree as particles that were heated up to 40 K greater.[70] This is a profound finding when noting that a reduction in temperature of 25 K can increase the catalyst lifetime by nearly tenfold, when comparing the photothermal and thermal process.[70]

Biological research

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Researchers have explored the use of silver nanoparticles as carriers for delivering various payloads such as small drug molecules or large biomolecules to specific targets. Once the AgNP has had sufficient time to reach its target, release of the payload could potentially be triggered by an internal or external stimulus. The targeting and accumulation of nanoparticles may provide high payload concentrations at specific target sites and could minimize side effects.[71]

Chemotherapy

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The introduction of nanotechnology into medicine is expected to advance diagnostic cancer imaging and the standards for therapeutic drug design.[72] Nanotechnology may uncover insight about the structure, function and organizational level of the biosystem at the nanoscale.[73]

Silver nanoparticles can undergo coating techniques that offer a uniform functionalized surface to which substrates can be added. When the nanoparticle is coated, for example, in silica the surface exists as silicic acid. Substrates can thus be added through stable ether and ester linkages that are not degraded immediately by natural metabolic enzymes.[74][75] Recent chemotherapeutic applications have designed anti cancer drugs with a photo cleavable linker,[76] such as an ortho-nitrobenzyl bridge, attaching it to the substrate on the nanoparticle surface.[74] The low toxicity nanoparticle complex can remain viable under metabolic attack for the time necessary to be distributed throughout the bodies systems.[74] If a cancerous tumor is being targeted for treatment, ultraviolet light can be introduced over the tumor region.[74] The electromagnetic energy of the light causes the photo responsive linker to break between the drug and the nanoparticle substrate.[74] The drug is now cleaved and released in an unaltered active form to act on the cancerous tumor cells.[74] Advantages anticipated for this method is that the drug is transported without highly toxic compounds, the drug is released without harmful radiation or relying on a specific chemical reaction to occur and the drug can be selectively released at a target tissue.[74][75]

A second approach is to attach a chemotherapeutic drug directly to the functionalized surface of the silver nanoparticle combined with a nucelophilic species to undergo a displacement reaction. For example, once the nanoparticle drug complex enters or is in the vicinity of the target tissue or cells, a glutathione monoester can be administered to the site.[77][78] The nucleophilic ester oxygen will attach to the functionalized surface of the nanoparticle through a new ester linkage while the drug is released to its surroundings.[77][78] The drug is now active and can exert its biological function on the cells immediate to its surroundings limiting non-desirable interactions with other tissues.[77][78]

Multiple drug resistance

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A major cause for the ineffectiveness of current chemotherapy treatments is multiple drug resistance which can arise from several mechanisms.[79]

Nanoparticles can provide a means to overcome MDR.[80] In general, when using a targeting agent to deliver nanocarriers to cancer cells, it is imperative that the agent binds with high selectivity to molecules that are uniquely expressed on the cell surface. Hence NPs can be designed with proteins that specifically detect drug resistant cells with overexpressed transporter proteins on their surface.[81] A pitfall of the commonly used nano-drug delivery systems is that free drugs that are released from the nanocarriers into the cytosol get exposed to the MDR transporters once again, and are exported. To solve this, 8 nm nanocrystalline silver particles were modified by the addition of trans-activating transcriptional activator (TAT), derived from the HIV-1 virus, which acts as a cell-penetrating peptide (CPP).[82] Generally, AgNP effectiveness is limited due to the lack of efficient cellular uptake; however, CPP-modification has become one of the most efficient methods for improving intracellular delivery of nanoparticles. Once ingested, the export of the AgNP is prevented based on a size exclusion. The concept is simple: the nanoparticles are too large to be effluxed by the MDR transporters, because the efflux function is strictly subjected to the size of its substrates, which is generally limited to a range of 300-2000 Da. Thereby the nanoparticulates remain insusceptible to the efflux, providing a means to accumulate in high concentrations.[citation needed]

Antimicrobial

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Introduction of silver into bacterial cells induces a high degree of structural and morphological changes, which can lead to cell death. As the silver nanoparticles come in contact with the bacteria, they adhere to the cell wall and cell membrane.[83] Once bound, some of the silver passes through to the inside, and interacts with phosphate-containing compounds like DNA and RNA, while another portion adheres to the sulfur-containing proteins on the membrane.[83] The silver-sulfur interactions at the membrane cause the cell wall to undergo structural changes, like the formation of pits and pores.[84] Through these pores, cellular components are released into the extracellular fluid, simply due to the osmotic difference. Within the cell, the integration of silver creates a low molecular weight region where the DNA then condenses.[84] Having DNA in a condensed state inhibits the cell's replication proteins contact with the DNA. Thus the introduction of silver nanoparticles inhibits replication and is sufficient to cause the death of the cell. Further increasing their effect, when silver comes in contact with fluids, it tends to ionize which increases the nanoparticles' bactericidal activity.[84] This has been correlated to the suppression of enzymes and inhibited expression of proteins that relate to the cell's ability to produce ATP.[85]

Although it varies for every type of cell proposed, as their cell membrane composition varies greatly, It has been seen that in general, silver nanoparticles with an average size of 10 nm or less show electronic effects that greatly increase their bactericidal activity.[86] This could also be partly due to the fact that as particle size decreases, reactivity increases due to the surface area to volume ratio increasing.[citation needed]

Silver nanoparticles have been shown to have synergistic antibacterial activity with commonly used antibiotics such as; penicillin G, ampicillin, erythromycin, clindamycin, and vancomycin against E. coli and S. aureus.[87] Furthermore, synergistic antibacterial activity has been reported between silver nanoparticles and hydrogen peroxide causing this combination to exert significantly enhanced bactericidal effect against both Gram negative and Gram positive bacteria.[88] This antibacterial synergy between silver nanoparticles and hydrogen peroxide can be possibly attributed to a Fenton-like reaction that generates highly reactive oxygen species such as hydroxyl radicals.[88][89][90]

Silver nanoparticles can prevent bacteria from growing on or adhering to the surface. This can be especially useful in surgical settings where all surfaces in contact with the patient must be sterile. Silver nanoparticles can be incorporated on many types of surfaces including metals, plastic, and glass.[91] In medical equipment, it has been shown that silver nano particles lower the bacterial count on devices used compared to old techniques. However, the problem arises when the procedure is over and a new one must be done. In the process of washing the instruments a large portion of the silver nano particles become less effective due to the loss of silver ions. They are more commonly used in skin grafts for burn victims as the silver nano particles embedded with the graft provide better antimicrobial activity and result in significantly less scarring of the victim.These new applications are direct decedents of older practices that used silver nitrate to treat conditions such as skin ulcers. Now, silver nanoparticles are used in bandages and patches to help heal certain burns and wounds.[92] An alternative approach is to use AgNP to sterilise biological dressings (for example, tilapia fish skin) for burn and wound management.[93]

They also show promising application as water treatment method to form clean potable water.[94] This doesn't sound like much, but water contains numerous diseases and some parts of the world do not have the luxury of clean water, or any at all. It wasn't new to use silver for removing microbes, but this experiment used the carbonate in water to make microbes even more vulnerable to silver.[95] First the scientists of the experiment use the nanopaticles to remove certain pesticides from the water, ones that prove fatal to people if ingested. Several other tests have shown that the silver nanoparticles were capable of removing certain ions in water as well, like iron, lead, and arsenic. But that is not the only reason why the silver nanoparticles are so appealing, they do not require any external force (no electricity of hydrolics) for the reaction to occur.[96] Conversely, post-consumer silver nanoparticles in waste water may adversely impact biological agents used in waste water treatment.[97]

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There are instances in which silver nanoparticles and colloidal silver are used in consumer goods. Samsung for example claimed that the use of silver nanoparticles in washing machines would help to sterilize clothes and water during the washing and rinsing functions, and allow clothes to be cleaned without the need for hot water.[98] The nanoparticles in these appliances are synthesized using electrolysis. Through electrolysis, silver is extracted from metal plates and then turned into silver nanoparticles by a reduction agent.[99] This method avoids the drying, cleaning, and re-dispersion processes, which are generally required with alternative colloidal synthesis methods.[99] Importantly, the electrolysis strategy also decreases the production cost of Ag nanoparticles, making these washing machines more affordable to manufacture.[100] Samsung has described the system:

[A] grapefruit-sized device alongside the [washer] tub uses electrical currents to nanoshave two silver plates the size of large chewing gum sticks. Resulting in positively charged silver atoms-silver ions (Ag+)-are injected into the tub during the wash cycle.[100]

Samsung's description of the Ag nanoparticle generating process seems to contradict its advertisement of silver nanoparticles. Instead, the statement indicates that laundry cycles.[99][100] When clothes are run through the cycle, the intended mode of action is that bacteria contained in the water are sterilized as they interact with the silver present in the washing tub.[98][100] As a result, these washing machines can provide antibacterial and sterilization benefits on top of conventional washing methods. Samsung has commented on the lifetime of these silver-containing washing machines. The electrolysis of silver generates over 400 billion silver ions during each wash cycle. Given the size of the silver source (two “gum-sized” plate of Ag), Samsung estimates that these plates can last up to 3000 wash cycles.[100]

These plans by Samsung were not overlooked by regulatory agencies. Agencies investigating nanoparticle use include but are not limited to: the U.S. FDA, U.S. EPA, SIAA of Japan, and Korea's Testing and Research Institute for Chemical Industry and FITI Testing & Research Institute.[98] These various agencies plan to regulate silver nanoparticles in appliances.[98] These washing machines are some of the first cases in which the EPA has sought to regulate nanoparticles in consumer goods. Samsung stated that the silver gets washed away in the sewer and regulatory agencies worry over what that means for wastewater treatment streams.[100] Currently, the EPA classifies silver nanoparticles as pesticides due to their use as antimicrobial agents in wastewater purification.[98] The washing machines being developed by Samsung do contain a pesticide and have to be registered and tested for safety under the law, particularly the U.S. Federal Insecticide, Fungicide, and Rodenticide Act.[98] The difficulty, however behind regulating nanotechnology in this manner is that there is no distinct way to measure toxicity.[98]

In addition to the uses described above, the European Union Observatory for Nanomaterials (EUON) has highlighted that silver nanoparticles are used in colourants in cosmetics, as well as pigments.[101][102] A recently published study by the EUON has illustrated the existence of knowledge gaps regarding the safety of nanoparticles in pigments.[103]

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The U.S. National Institute for Occupational Safety and Health derived a recommended exposure limit (REL) for silver nanomaterials (with <100 nm primary particle size) of 0.9 μg/m3 as an airborne respirable 8-hour time-weighted average (TWA) concentration. This is in comparison to its REL of 10 μg/m3 as an 8-hour TWA for total silver (including metal dust, fumes, and soluble compounds).[104] It was found that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to Ag nanoparticles.[105]

Although silver nanoparticles are widely used in a variety of commercial products, there has only recently been a major effort to study their effects on human health. There have been several studies that describe the in vitro toxicity of silver nanoparticles to a variety of different organs, including the lung, liver, skin, brain, and reproductive organs.[106] The mechanism of the toxicity of silver nanoparticles to human cells appears to be derived from oxidative stress and inflammation that is caused by the generation of reactive oxygen species (ROS) stimulated by either the Ag NPs, Ag ions, or both.[107][108][109][110][111] For example, Park et al. showed that exposure of a mouse peritoneal macrophage cell line (RAW267.7) to silver nanoparticles decreased the cell viability in a concentration- and time-dependent manner.[110] They further showed that the intracellular reduced glutathionine (GSH), which is a ROS scavenger, decreased to 81.4% of the control group of silver nanoparticles at 1.6 ppm.[110]

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Since silver nanoparticles undergo dissolution releasing silver ions,[112] which is well-documented to have toxic effects,[111][112][113] there have been several studies that have been conducted to determine whether the toxicity of silver nanoparticles is derived from the release of silver ions or from the nanoparticle itself. Several studies suggest that the toxicity of silver nanoparticles is attributed to their release of silver ions in cells as both silver nanoparticles and silver ions have been reported to have similar cytotoxicity.[109][110][114][115] For example, In some cases it is reported that silver nanoparticles facilitate the release of toxic free silver ions in cells via a "Trojan-horse type mechanism," where the particle enters cells and is then ionized within the cell.[110] However, there have been reports that suggest that a combination of silver nanoparticles and ions is responsible for the toxic effect of silver nanoparticles. Navarro et al. using cysteine ligands as a tool to measure the concentration of free silver in solution, determined that although initially silver ions were 18 times more likely to inhibit the photosynthesis of an algae, Chlamydomanas reinhardtii, but after 2 hours of incubation it was revealed that the algae containing silver nanoparticles were more toxic than just silver ions alone.[116] Furthermore, there are studies that suggest that silver nanoparticles induce toxicity independent of free silver ions.[111][117][118] For example, Asharani et al. compared phenotypic defects observed in zebrafish treated with silver nanoparticles and silver ions and determined that the phenotypic defects observed with silver nanoparticle treatment was not observed with silver ion-treated embryos, suggesting that the toxicity of silver nanoparticles is independent of silver ions.[118]

Protein channels and nuclear membrane pores can often be in the size range of 9 nm to 10 nm in diameter.[111] Small silver nanoparticles constructed of this size have the ability to not only pass through the membrane to interact with internal structures but also to become lodged within the membrane.[111] Silver nanoparticle depositions in the membrane can impact regulation of solutes, exchange of proteins and cell recognition.[111] Exposure to silver nanoparticles has been associated with "inflammatory, oxidative, genotoxic, and cytotoxic consequences"; the silver particulates primarily accumulate in the liver.[119] but have also been shown to be toxic in other organs including the brain.[106] Nano-silver applied to tissue-cultured human cells leads to the formation of free radicals, raising concerns of potential health risks.[120]

• Allergic reaction: There have been several studies conducted that show a precedence for allergenicity of silver nanoparticles.[121][122]
• Argyria and staining: Ingested silver or silver compounds, including colloidal silver, can cause a condition called argyria, a discoloration of the skin and organs.In 2006, there was a case study of a 17-year-old man, who sustained burns to 30% of his body, and experienced a temporary bluish-grey hue after several days of treatment with Acticoat, a brand of wound dressing containing silver nanoparticles.[123] Argyria is the deposition of silver in deep tissues, a condition that cannot happen on a temporary basis, raising the question of whether the cause of the man's discoloration was argyria or even a result of the silver treatment.[124] Silver dressings are known to cause a "transient discoloration" that dissipates in 2–14 days, but not a permanent discoloration.[125]
• Silzone heart valve: St. Jude Medical released a mechanical heart valve with a silver coated sewing cuff (coated using ion beam-assisted deposition) in 1997.[126] The valve was designed to reduce the instances of endocarditis. The valve was approved for sale in Canada, Europe, the United States, and most other markets around the world. In a post-commercialization study, researchers showed that the valve prevented tissue ingrowth, created paravalvular leakage, valve loosening, and in the worst cases explantation. After 3 years on the market and 36,000 implants, St. Jude discontinued and voluntarily recalled the valve.

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References

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Bibliography

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• Cao H (2017). Silver Nanoparticles for Antibacterial Devices: Biocompatibility and Toxicity. CRC Press. ISBN 9781315353470.

Recent advances in nanoscience and nanotechnology radically changed the way we diagnose, treat, and prevent various diseases in all aspects of human life. Silver nanoparticles (AgNPs) are one of the most vital and fascinating nanomaterials among several metallic nanoparticles that are involved in biomedical applications. AgNPs play an important role in nanoscience and nanotechnology, particularly in nanomedicine. Although several noble metals have been used for various purposes, AgNPs have been focused on potential applications in cancer diagnosis and therapy. In this review, we discuss the synthesis of AgNPs using physical, chemical, and biological methods. We also discuss the properties of AgNPs and methods for their characterization. More importantly, we extensively discuss the multifunctional bio-applications of AgNPs; for example, as antibacterial, antifungal, antiviral, anti-inflammatory, anti-angiogenic, and anti-cancer agents, and the mechanism of the anti-cancer activity of AgNPs. In addition, we discuss therapeutic approaches and challenges for cancer therapy using AgNPs. Finally, we conclude by discussing the future perspective of AgNPs.

Cancer is a complex, multifactorial disease which has the characteristic feature of the uncontrolled growth and spread of abnormal cells caused by several factors, including a combination of genetic, external, internal, and environmental factors [ 25 ], and it is treated by various treatments including chemotherapy, hormone therapy, surgery, radiation, immune therapy, and targeted therapy [ 25 ]. Therefore, the challenge is to identify effective, cost-effective, and sensitive lead molecules that have cell-targeted specificity and increase the sensitivity. Recently, AgNPs have been shown much interest because of their therapeutic applications in cancer as anticancer agents, in diagnostics, and in probing. Taken literature into consideration, in this review we focused on recent developments in synthesis, characterization, properties, and bio-applications mainly on the antibacterial, antifungal, antiviral, anti-inflammatory, anti-cancer and anti-angiogenic properties of AgNPs in a single platform. This review also emphasizes mechanism of anticancer activity, therapeutic approaches and the challenges and limitations of nanoparticles in cancer therapy. Finally, this review ends with conclusion and the future perspective of AgNPs.

The biological activity of AgNPs depends on factors including surface chemistry, size, size distribution, shape, particle morphology, particle composition, coating/capping, agglomeration, and dissolution rate, particle reactivity in solution, efficiency of ion release, and cell type, and the type of reducing agents used for the synthesis of AgNPs are a crucial factor for the determination of cytotoxicity [ 15 ]. The physicochemical properties of nanoparticles enhance the bioavailability of therapeutic agents after both systemic and local administration [ 16 , 17 ] and other hand it can affect cellular uptake, biological distribution, penetration into biological barriers, and resultant therapeutic effects [ 18 , 19 ]. Therefore, the development of AgNPs with controlled structures that are uniform in size, morphology, and functionality are essential for various biomedical applications [ 20 , 21 , 22 , 23 , 24 ].

After synthesis, precise particle characterization is necessary, because the physicochemical properties of a particle could have a significant impact on their biological properties. In order to address the safety issue to use the full potential of any nano material in the purpose of human welfare, in nanomedicines, or in the health care industry, etc., it is necessary to characterize the prepared nanoparticles before application [ 10 , 11 ]. The characteristic feature of nanomaterials, such as size, shape, size distribution, surface area, shape, solubility, aggregation, etc. need to be evaluated before assessing toxicity or biocompatibility [ 12 ]. To evaluate the synthesized nanomaterials, many analytical techniques have been used, including ultraviolet visible spectroscopy (UV-vis spectroscopy), X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and so on [ 13 , 14 ].

Silver nanoparticles (AgNPs) are increasingly used in various fields, including medical, food, health care, consumer, and industrial purposes, due to their unique physical and chemical properties. These include optical, electrical, and thermal, high electrical conductivity, and biological properties [ 1 , 2 , 3 ]. Due to their peculiar properties, they have been used for several applications, including as antibacterial agents, in industrial, household, and healthcare-related products, in consumer products, medical device coatings, optical sensors, and cosmetics, in the pharmaceutical industry, the food industry, in diagnostics, orthopedics, drug delivery, as anticancer agents, and have ultimately enhanced the tumor-killing effects of anticancer drugs [ 4 ]. Recently, AgNPs have been frequently used in many textiles, keyboards, wound dressings, and biomedical devices [ 2 , 5 , 6 ]. Nanosized metallic particles are unique and can considerably change physical, chemical, and biological properties due to their surface-to-volume ratio; therefore, these nanoparticles have been exploited for various purposes [ 7 , 8 ]. In order to fulfill the requirement of AgNPs, various methods have been adopted for synthesis. Generally, conventional physical and chemical methods seem to be very expensive and hazardous [ 1 , 9 ]. Interestingly, biologically-prepared AgNPs show high yield, solubility, and high stability [ 1 ]. Among several synthetic methods for AgNPs, biological methods seem to be simple, rapid, non-toxic, dependable, and green approaches that can produce well-defined size and morphology under optimized conditions for translational research. In the end, a green chemistry approach for the synthesis of AgNPs shows much promise.

The biological activity of AgNPs depends on the morphology and structure of AgNPs, controlled by size and shape of the particles [ 70 , 71 ]. As far as size and shape are concerned, smaller size and truncated-triangular nanoparticles seem to be more effective and have superior properties. Although many studies successfully synthesized AgNPs with different shape and size ranges, they still have certain limitations. To achieve control over morphology and structure, an excess of strong reducing agent such as sodium borohydride (NaBH 4 ) was used for the synthesis of monodisperse and uniform-sized silver colloids [ 72 ]. Compared to chemical methods, biological methods allow for more ease in the control of shape, size, and distribution of the produced nanoparticles by optimization of the synthesis methods, including the amount of precursors, temperature, pH, and the amount of reducing and stabilizing factors [ 9 , 73 ].

To overcome the shortcomings of chemical methods, biological methods have emerged as viable options. Recently, biologically-mediated synthesis of nanoparticles have been shown to be simple, cost effective, dependable, and environmentally friendly approaches and much attention has been given to the high yield production of AgNPs of defined size using various biological systems including bacteria, fungi, plant extracts, and small biomolecules like vitamins and amino acids as an alternative method to chemical methods—not only for AgNPs, but also for the synthesis of several other nanoparticles, such as gold and graphene [ 9 , 52 , 53 , 54 , 55 , 56 ]. Bio-sorption of metals by Gram-negative and Gram-positive bacteria provided an indication for the synthesis of nanoparticles before the flourishing of this biological method; however, the synthesized nanomaterials were as aggregates not nanoparticles [ 57 ]. Several studies reported the synthesis of AgNPs using green, cost effective, and biocompatible methods without the use of toxic chemicals in biological methods. In this green chemistry approach, several bacteria, including Pseudomonas stutzeri AG259 [ 58 ], Lactobacillus strains [ 59 ], Bacillus licheniformis [ 55 ]; Escherichia coli (E. coli) [ 9 ], Brevibacterium casei [ 60 ], fungi including Fusarium oxysporum [ 61 ], Ganoderma neo-japonicum Imazeki [ 62 ], plant extracts such as Allophylus cobbe [ 52 ], Artemisia princeps [ 63 ], and Typha angustifolia [ 64 ] were utilized. In addition to these, several biomolecules, such as biopolymers [ 65 ], starch [ 66 ], fibrinolytic enzyme [ 39 ], and amino acids [ 67 ] were used. The biological synthesis of nanoparticles depends on three factors, including (a) the solvent; (b) the reducing agent; and (c) the non-toxic material. The major advantage of biological methods is the availability of amino acids, proteins, or secondary metabolites present in the synthesis process, the elimination of the extra step required for the prevention of particle aggregation, and the use of biological molecules for the synthesis of AgNPs is eco-friendly and pollution-free. Biological methods seem to provide controlled particle size and shape, which is an important factor for various biomedical applications [ 68 ]. Using bacterial protein or plant extracts as reducing agents, we can control the shape, size, and monodispersity of the nanoparticles [ 9 ]. The other advantages of biological methods are the availability of a vast array of biological resources, a decreased time requirement, high density, stability, and the ready solubility of prepared nanoparticles in water [ 69 ].

Chemical methods use water or organic solvents to prepare the silver nanoparticles [ 37 , 38 ]. This process usually employs three main components, such as metal precursors, reducing agents, and stabilizing/capping agents. Basically, the reduction of silver salts involves two stages (1) nucleation; and (2) subsequent growth. In general, silver nanomaterials can be obtained by two methods, classified as “top-down” and “bottom-up” [ 39 ]. The “top-down” method is the mechanical grinding of bulk metals with subsequent stabilization using colloidal protecting agents [ 40 , 41 ]. The “bottom-up” methods include chemical reduction, electrochemical methods, and sono-decomposition. The major advantage of chemical methods is high yield, contrary to physical methods, which have low yield. The above-mentioned methods are extremely expensive. Additionally, the materials used for AgNPs synthesis, such as citrate, borohydride, thio-glycerol, and 2-mercaptoethanol are toxic and hazardous [ 41 ]. Apart from these disadvantages, the manufactured particles are not of expected purity, as their surfaces were found to be sedimented with chemicals. It is also very difficult to prepare AgNPs with a well-defined size, requiring a further step for the prevention of particle aggregation [ 42 ]. In addition, during the synthesis process, too many toxic and hazardous byproducts are excised out. Chemical methods make use of techniques such as cryochemical synthesis [ 43 ], laser ablation [ 44 ], lithography [ 45 ], electrochemical reduction [ 46 ], laser irradiation [ 47 ], sono-decomposition [ 48 ], thermal decomposition [ 49 ], and chemical reduction [ 50 ]. The advantage of the chemical synthesis of nanoparticles are the ease of production, low cost, and high yield; however, the use of chemical reducing agents are harmful to living organisms [ 13 ]. Recently, Abbasi et al. explained a detailed account of synthesis methods, properties, and bio-application of AgNPs [ 51 ].

Generally, the synthesis of nanoparticles has been carried out using three different approaches, including physical, chemical, and biological methods. In physical methods, nanoparticles are prepared by evaporation-condensation using a tube furnace at atmospheric pressure [ 26 , 27 , 28 , 29 ]. Conventional physical methods including spark discharging and pyrolysis were used for the synthesis of AgNPs [ 30 , 31 ]. The advantages of physical methods are speed, radiation used as reducing agents, and no hazardous chemicals involved, but the downsides are low yield and high energy consumption, solvent contamination, and lack of uniform distribution [ 32 , 33 , 34 , 35 , 36 ].

LSPR is a coherent, collective spatial oscillation of the conduction electrons in a metallic nanoparticle, which can be directly excited by near-visible light. The localized surface plasmon resonance (LSPR) condition is defined by several factors, including the electronic properties of the nanoparticle, the size and shape of the particle, temperature, the dielectric environment, and so on. Small changes in the local dielectric environment cause the dysfunction of LSPR. The frequency of the LSPR spectral peak is very sensitive to the nanostructure environment through the local refractive index. Thereby, shifts of the LSPR frequency are widely used as a method for the detection of molecular interaction close to the surface of the nanoparticle [ 161 , 162 , 163 , 164 , 165 , 166 ]. In addition, the near-field enhancement has led to a very large variety of advances in many fundamental and applied areas of science, particularly for the determination of nanoparticle shapes, dimensions, and compositions. This spectroscopy method is being used to investigate fundamental properties and processes of nanoparticles in (bio)-molecular detection devices, or (bio)-imaging tools with improved single-molecule sensitivity. LSPR spectroscopy can provide thermodynamic and real-time kinetic data for binding processes. LSPR-based tools will be helpful to analyze faster and with higher sensitivity. The application of LSPR spectroscopy is mainly used for biological and chemical sensing by transducing changes in the local refractive index via a wavelength-shift measurement, due to its sensitivity, wavelength tunability, smaller sensing volumes, and lower instrumentation cost. Single-nanoparticle LSPR spectroscopy is an important tool for understanding the relationship between local structure and spectra. In addition, single nanoparticles can provide even higher refractive-index sensitivity than nanoparticle arrays.

Generally, AFM is used to investigate the dispersion and aggregation of nanomaterials, in addition to their size, shape, sorption, and structure; three different scanning modes are available, including contact mode, non-contact mode, and intermittent sample contact mode [ 10 , 14 , 151 , 152 , 153 , 154 , 155 ]. AFM can also be used to characterize the interaction of nanomaterials with supported lipid bilayers in real time, which is not achievable with current electron microscopy (EM) techniques [ 113 ]. In addition, AFM does not require oxide-free, electrically conductive surfaces for measurement, does not cause appreciable damage to many types of native surfaces, and it can measure up to the sub-nanometer scale in aqueous fluids [ 156 , 157 ]. However, a major drawback is the overestimation of the lateral dimensions of the samples due to the size of the cantilever [ 158 , 159 ]. Therefore, we have to provide much attention to avoid erroneous measurements [ 160 ]. Furthermore, the choice of operating mode—no contact or contact—is a crucial factor in sample analysis [ 160 ].

TEM is a valuable, frequently used, and important technique for the characterization of nanomaterials, used to obtain quantitative measures of particle and/or grain size, size distribution, and morphology [ 10 , 109 , 150 ]. The magnification of TEM is mainly determined by the ratio of the distance between the objective lens and the specimen and the distance between objective lens and its image plane [ 150 ]. TEM has two advantages over SEM: it can provide better spatial resolution and the capability for additional analytical measurements [ 10 , 148 , 150 ]. The disadvantages include a required high vacuum, thin sample section [ 10 , 109 , 148 ], and the vital aspect of TEM is that sample preparation is time consuming. Therefore, sample preparation is extremely important in order to obtain the highest-quality images possible.

Recently, the field of nanoscience and nanotechnology has provided a driving force in the development of various high-resolution microscopy techniques in order to learn more about nanomaterials using a beam of highly energetic electrons to probe objects on a very fine scale [ 145 , 146 , 147 ]. Among various electron microscopy techniques, SEM is a surface imaging method, fully capable of resolving different particle sizes, size distributions, nanomaterial shapes, and the surface morphology of the synthesized particles at the micro and nanoscales [ 10 , 117 , 137 , 148 , 149 ]. Using SEM, we can probe the morphology of particles and derive a histogram from the images by either by measuring and counting the particles manually, or by using specific software [ 117 ]. The combination of SEM with energy-dispersive X-ray spectroscopy (EDX) can be used to examine silver powder morphology and also conduct chemical composition analysis. The limitation of SEM is that it is not able to resolve the internal structure, but it can provide valuable information regarding the purity and the degree of particle aggregation. The modern high-resolution SEM is able to identify the morphology of nanoparticles below the level of 10 nm.

XPS is a quantitative spectroscopic surface chemical analysis technique used to estimate empirical formulae [ 109 , 140 , 141 , 142 ]. XPS is also known as electron spectroscopy for chemical analysis (ESCA), [ 141 ]. XPS plays a unique role in giving access to qualitative, quantitative/semi-quantitative, and speciation information concerning the sensor surface [ 143 ]. XPS is performed under high vacuum conditions. X-ray irradiation of the nanomaterial leads to the emission of electrons, and the measurement of the kinetic energy and the number of electrons escaping from the surface of the nanomaterials gives XPS spectra [ 109 , 140 , 141 , 142 ]. The binding energy can be calculated from kinetic energy. Specific groups of starburst macromolecules such as P=S, aromatic rings, C–O, and C=O can be identified and characterized by XPS [ 144 ].

FTIR is able to provide accuracy, reproducibility, and also a favorable signal-to-noise ratio. By using FTIR spectroscopy, it becomes possible to detect small absorbance changes on the order of 10 −3 , which helps to perform difference spectroscopy, where one could distinguish the small absorption bands of functionally active residues from the large background absorption of the entire protein [ 122 , 123 , 124 , 125 , 126 , 127 , 128 ]. FTIR spectroscopy is frequently used to find out whether biomolecules are involved in the synthesis of nanoparticles, which is more pronounced in academic and industrial research [ 10 , 68 , 129 , 130 ]. Furthermore, FTIR has also been extended to the study of nano-scaled materials, such as confirmation of functional molecules covalently grafted onto silver, carbon nanotubes, graphene and gold nanoparticles, or interactions occurring between enzyme and substrate during the catalytic process [ 68 , 131 , 132 ]. Furthermore, it is a non-invasive technique. Finally, the advantages of FTIR spectrometers over dispersive ones are rapid data collection, strong signal, large signal-to-noise ratio, and less sample heat-up [ 133 ]. Recently, further advancement has been made in an FTIR method called attenuated total reflection (ATR)-FTIR spectroscopy [ 134 , 135 , 136 ]. Using ATR-FTIR, we can determine the chemical properties on the polymer surface, and sample preparation is easy compared to conventional FTIR [ 10 , 137 , 138 , 139 , 140 , 141 ]. Therefore, FTIR is a suitable, valuable, non-invasive, cost effective, and simple technique to identify the role of biological molecules in the reduction of silver nitrate to silver.

Physicochemical characterization of prepared nanomaterials is an important factor for the analysis of biological activities using radiation scattering techniques [ 10 , 14 , 112 ]. DLS can probe the size distribution of small particles a scale ranging from submicron down to one nanometer in solution or suspension [ 10 , 14 , 113 ]. Dynamic light scattering is a method that depends on the interaction of light with particles. This method can be used for the measurement of narrow particle size distributions, especially in the range of 2–500 nm [ 78 ]. Among the techniques for the characterization of nanoparticles, the most commonly used is DLS [ 114 , 115 , 116 ]. DLS measures the light scattered from a laser that passes through a colloid, and mostly relies on Rayleigh scattering from the suspended nanoparticles [ 117 ]. Next, the modulation of the scattered light intensity as a function of time is analyzed, and the hydrodynamic size of particles can be determined [ 118 , 119 , 120 ]. To evaluate the toxic potential of any nanomaterial, its characterization in solution is essential [ 11 ]. Therefore; DLS is mainly used to determine particle size and size distributions in aqueous or physiological solutions [ 12 ]. The size obtained from DLS is usually larger than TEM, which may be due to the influence of Brownian motion. DLS is a nondestructive method used to obtain the average diameter of nanoparticles dispersed in liquids. It has the special advantage of probing a large quantity of particles simultaneously; however, it has a number of sample-specific limitations [ 101 , 121 ].

XRD is a primary technique for the identification of the crystalline nature at the atomic scale [ 10 , 14 , 88 , 105 ]. X-ray powder diffraction is a nondestructive technique with great potential for the characterization of both organic and inorganic crystalline materials [ 106 ]. This method has been used to measure phase identification, conduct quantitative analysis, and to determine structure imperfections in samples from various disciplines, such as geological, polymer, environmental, pharmaceutical, and forensic sciences. Recently, the applications have extended to the characterization of various nano-materials and their properties [ 106 ]. The working principle of X-ray diffraction is Bragg’s law [ 88 , 105 ]. Typically, XRD is based on the wide-angle elastic scattering of X-rays [ 10 , 14 , 88 , 107 , 108 , 109 ]. Although XRD has several merits, it has limited disadvantages, including difficulty in growing the crystals and the ability to get results pertaining only to single conformation/binding state [ 14 , 108 , 110 ]. Another drawback of XRD is the low intensity of diffracted X-rays compared to electron diffractions [ 110 , 111 ].

X-ray diffraction (XRD) is a popular analytical technique which has been used for the analysis of both molecular and crystal structures [ 79 , 88 ], qualitative identification of various compounds [ 89 ], quantitative resolution of chemical species [ 90 ], measuring the degree of crystallinity [ 91 ], isomorphous substitutions [ 92 ], particle sizes [ 93 ], etc. When X-ray light reflects on any crystal, it leads to the formation of many diffraction patterns, and the patterns reflect the physico-chemical characteristics of the crystal structures. In a powder specimen, diffracted beams typically come from the sample and reflect its structural physico-chemical features. Thus, XRD can analyze the structural features of a wide range of materials, such as inorganic catalysts, superconductors, biomolecules, glasses, polymers, and so on [ 94 ]. Analysis of these materials largely depends on the formation of diffraction patterns. Each material has a unique diffraction beam which can define and identify it by comparing the diffracted beams with the reference database in the Joint Committee on Powder Diffraction Standards (JCPDS) library. The diffracted patterns also explain whether the sample materials are pure or contain impurities. Therefore, XRD has long been used to define and identify both bulk and nanomaterials, forensic specimens, industrial, and geochemical sample materials [ 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 ].

UV-vis spectroscopy is a very useful and reliable technique for the primary characterization of synthesized nanoparticles which is also used to monitor the synthesis and stability of AgNPs [ 74 ]. AgNPs have unique optical properties which make them strongly interact with specific wavelengths of light [ 75 ]. In addition, UV-vis spectroscopy is fast, easy, simple, sensitive, selective for different types of NPs, needs only a short period time for measurement, and finally a calibration is not required for particle characterization of colloidal suspensions [ 76 , 77 , 78 ]. In AgNPs, the conduction band and valence band lie very close to each other in which electrons move freely. These free electrons give rise to a surface plasmon resonance (SPR) absorption band, occurring due to the collective oscillation of electrons of silver nano particles in resonance with the light wave [ 79 , 80 , 81 , 82 , 83 , 84 ]. The absorption of AgNPs depends on the particle size, dielectric medium, and chemical surroundings [ 81 , 82 , 83 , 84 , 85 ]. Observation of this peak—assigned to a surface plasmon—is well documented for various metal nanoparticles with sizes ranging from 2 to 100 nm [ 74 , 86 , 87 ]. The stability of AgNPs prepared from biological methods was observed for more than 12 months, and an SPR peak at the same wavelength using UV-vis spectroscopy was observed.

The physicochemical properties of nanoparticles are important for their behavior, bio-distribution, safety, and efficacy. Therefore, characterization of AgNPs is important in order to evaluate the functional aspects of the synthesized particles. Characterization is performed using a variety of analytical techniques, including UV-vis spectroscopy, X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Several qualified books and reviews have presented the principles and usage of various kinds of analytical techniques for the characterization of AgNPs; however, the basics of the important techniques used for the characterization of AgNPs are detailed below for ease of understanding. For example, characterization of AgNPs using various analytical techniques prepared from culture supernatant of Bacillus species was given in .

Physical and chemical properties of AgNPs—including surface chemistry, size, size distribution, shape, particle morphology, particle composition, coating/capping, agglomeration, dissolution rate, particle reactivity in solution, efficiency of ion release, cell type, and finally type of reducing agents used for synthesis—are crucial factors for determination of cytotoxicity [ 15 , 50 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 ]. For example, using biological reducing agents such as culture supernatants of various Bacillus species, AgNPs can be synthesized in various shapes, such as spherical, rod, octagonal, hexagonal, triangle, flower-like, and so on ( ). Previous studies supported the assertion that smaller size particles could cause more toxicity than larger, because they have larger surface area [ 176 ]. Shape is equally important to the determination of toxicity [ 177 ]. For example, in the biomedical field, various types of nanostructures have been used, including nanocubes, nanoplates, nanorods, spherical nanoparticles, flower-like, and so on [ 175 , 178 ]. AgNP toxicity mainly depends on the availability of chemical and or biological coatings on the nanoparticle surface [ 179 ]. AgNP surface charges could determine the toxicity effect in cells. For instance, the positive surface charge of these NPs renders them more suitable, allowing them to stay for a long time in blood stream compared to negatively-charged NPs [ 180 ], which is a major route for the administration of anticancer agents [ 181 , 182 ].

5. Biological Applications of AgNPs

Due to their unique properties, AgNPs have been used extensively in house-hold utensils, the health care industry, and in food storage, environmental, and biomedical applications. Several reviews and book chapters have been dedicated in various areas of the application of AgNPs. Herein, we are interested in emphasizing the applications of AgNPs in various biological and biomedical applications, such as antibacterial, antifungal, antiviral, anti-inflammatory, anti-cancer, and anti-angiogenic. Herein, we specifically addressed previously-published seminal papers and end with recent updates. A schematic diagram representing various applications of AgNPs is provided in .

5.1. Antibacterial Activity of AgNPs

AgNPs seem to be alternative antibacterial agents to antibiotics and have the ability to overcome the bacterial resistance against antibiotics. Therefore, it is necessary to develop AgNPs as antibacterial agents. Among the several promising nanomaterials, AgNPs seem to be potential antibacterial agents due to their large surface-to-volume ratios and crystallographic surface structure. The seminal paper reported by Sondi and Salopek-Sondi [6] demonstrated the antimicrobial activity of AgNPs against Escherichia coli, in which E. coli cells treated with AgNPs showed the accumulation of AgNPs in the cell wall and the formation of “pits” in the bacterial cell walls, eventually leading to cell death. In the same E. coli strain, smaller particles with a larger surface-to-volume ratio showed a more efficient antibacterial activity than larger particles [183]. Furthermore, the antibacterial activity of AgNPs is not only size—but also shape-dependent [70]. AgNPs were synthesized by four different types of saccharides with an average size of 25 nm, showing high antimicrobial and bactericidal activity against Gram-positive and Gram-negative bacteria, including highly multi-resistant strains such as methicillin-resistant Staphylococcus aureus. As mentioned previously, not only the size is important for determining the efficiency, but also shape, because AgNPs undergo a shape-dependent interaction with the Gram-negative organism E. coli [71]. Furthermore, a detailed study was carried out to investigate the efficiency of the antimicrobial effects of AgNPs against yeast, E. coli, and Staphylococcus aureus. The results suggest that at low concentrations of AgNPs, the complete inhibition of growth was observed in yeast and E. coli, whereas a mild effect was observed in S. aureus [184]. Biologically synthesized AgNPs from the culture supernatants of Klebsiella pneumoniae were evaluated; the efficiencies of various antibiotics, such as penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin against Staphylococcus aureus and E. coli were increased in the presence of Ag-NPs [185]. When compared to AgNPs, hydrogel–silver nanocomposites showed excellent antibacterial activity against E. coli. One-pot synthesis of chitosan–Ag–nanoparticle composite was found to have higher antimicrobial activity than its components at their respective concentrations, because one-pot synthesis favors the formation of small AgNPs attached to the polymer, which can be dispersed in media of pH ≤ 6.3 [186]. Biologically produced AgNPs using culture supernatants of Staphylococcus aureus showed significant antimicrobial activity against methicillin-resistant S. aureus, followed by methicillin-resistant Staphylococcus epidermidis and Streptococcus pyogenes, whereas only moderate antimicrobial activity was observed against Salmonella typhi and Klebsiella pneumoniae [187]. The mechanisms of AgNP-induced cell death was observed in E. coli through the leakage of reducing sugars and proteins. Furthermore, AgNPs are able to destroy the permeability of the bacterial membranes via the generation of many pits and gaps, indicating that AgNPs could damage the structure of the bacterial cell membrane [2]. Silver nanocrystalline chlorhexidine (AgCHX) complex showed strong antibacterial activity against the tested Gram-positive/negative and methicillin-resistant Staphylococcus aureus (MRSA) strains. Interestingly, the minimal inhibitory concentrations (MICs) of nanocrystalline Ag(III)CHX were much lower than those of the ligand (CHX), AgNO3, and the gold standard, silver sulfadiazine [188].

Biofilms are not only leads to antimicrobial resistance, but are involved in the development of ocular-related infectious diseases, such as microbial keratitis [189]. Kalishwaralal and co-workers demonstrated the potential anti-biofilm activity against Pseudomonas aeruginosa and Staphylococcus epidermidis. Similarly, guava leaf extract reduced AgNPs (Gr-Ag-NPs) showed significant antibacterial activity and stability against E. coli compared to chemically synthesized AgNPs; the reason for this higher activity could be the adsorption of biomolecules on the surface of the Gr-Ag-NPs [190]. AgNPs synthesized by Cryphonectria sp. showed antibacterial activity against various human pathogenic bacteria, including S. aureus, E. coli, Salmonella typhi, and Candida albicans. Interestingly, these particular AgNPs exhibited higher antibacterial activity against both S. aureus and E. coli than against S. typhi and C. albicans. shows the effectiveness of dose-dependent antibacterial activity of biologically synthesized AgNPs in E. coli.

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Besinis et al. [191] compared the toxic efficiency of different nanomaterials, such as AgNPs, silver, and titanium dioxide against routine disinfectant chlorhexidine in Streptococcus mutans. Among various nanomaterials, AgNPs had the strongest antibacterial activity of the NPs tested. Agnihotri et al. [192] demonstrated that the mechanisms of AgNPs on bactericidal action using AgNPs immobilized on an amine-functionalized silica surface. They found that contact killing is the predominant bactericidal mechanism, and surface-immobilized nanoparticles show greater efficacy than colloidal AgNPs, as well as a higher concentration of silver ions in solution. The nanocomposite containing silver/polyrhodanine nanocomposite-decorated silica nanoparticles shows potential and enhanced antibacterial activity against E. coli and S. aureus, which is due to the particular combination of AgNPs and the polyrhodanine [155]. Interestingly, Khurana et al. [193] investigated the antimicrobial properties of silver based on its physical and surface properties against S. aureus, B. megaterium, P. vulgaris, and S. sonnei. The enhancement of antibacterial action was observed with particles having a hydrodynamic size of 59 nm compared to 83 nm. Gurunathan et al. [68] reported that the antibacterial and anti-biofilm activity of antibiotics, AgNPs, or combinations of AgNPs against important pathogenic bacteria Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, and Streptococcus pneumoniae. The results suggest that, the combination of both antibiotics and AgNPs showed significant antimicrobial and anti-biofilm effects at the lowest concentration of antibiotics and AgNPs compared to AgNPs or antibiotics alone. Nanocomposite spheres composed of AgNPs decorated on the polymer colloids exhibited excellent antibacterial activity [194]. Recently, nanocomposites containing graphene and AgNPs showed much interest in antibacterial activity. Graphene oxide (GO)-Ag nanocomposite showed enhanced antibacterial activity against E. coli and S. aureus using the conventional plate count method and disk diffusion method [195]. The GO-Ag nanocomposite exhibited an excellent antibacterial activity against methicillin-resistant S. aureus, Acinetobacter baumannii, Enterococcus faecalis, and Escherichia coli. In addition, GO-Ag nanocomposite is a promising antibacterial agent against common nosocomial bacteria, particularly antibiotic-resistant MRSA [196]. AgNPs derived from fungal extracts as reducing agents (F-AgNPs) showed enhanced antibacterial activity both in Pseudomonas aeruginosa and Staphylococcus aureus when compared to AgNPs derived from the culture supernatant of bacteria (B-AgNPs) ( ). The minimum inhibitory concentration of F-AgNPs is lesser than B-AgNPs. Nano-silver interacts with peptides and bacteria and serves as nanomedicine in various bacteria, fungi, and virus-mediated diseases [197].

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5.2. Antifungal Activity of AgNPs

Fungal infections are more frequent in patients who are immunosuppressed, and overcoming fungi-mediated diseases is a tedious process, because currently there is a limited number of available antifungal drugs [198]. Therefore, there is an inevitable and urgent need to develop antifungal agents, which should be biocompatible, non-toxic, and environmentally friendly. At this juncture, AgNPs play an important role as anti-fungal agents against various diseases caused by fungi. Nano-Ag showed potent anti-fungal activity against clinical isolates and ATCC strains of Trichophyton mentagrophytes and Candida species with concentrations of 1–7 μg/mL. Esteban-Tejeda et al. [199] developed an inert matrix containing AgNPs with an average size of 20 nm into a soda-lime glass which shows enhanced biocidal activity. Monodisperse Nano-Ag sepiolite fibers showed significant antifungal activity against Issatchenkia orientalis. AgNPs exhibited good antifungal activity against Aspergillus niger and a MIC of 25 μg/mL against Candida albicans [200]. Biologically-synthesized AgNPs showed enhanced antifungal activity with fluconazole against Phoma glomerata, Phoma herbarum, Fusarium semitectum, Trichoderma sp., and Candida albicans [201]. AgNPs stabilized by sodium dodecyl sulfate showed enhanced antifungal activity against Candida albicans compared to conventional antifungal agents [20]. The size-dependent antifungal activities of different AgNPs were performed against mature Candida albicans and Candida glabrata biofilms. Biologically synthesized AgNPs exhibited antifungal activity against several phytopathogenic fungi, including Alternaria alternata, Sclerotinia sclerotiorum, Macrophomina phaseolina, Rhizoctonia solani, Botrytis cinerea, and Curvularia lunata at the concentration of 15 mg [202,203]. Similarly, The AgNPs synthesized by Bacillus species exhibited strong antifungal activity against the plant pathogenic fungus Fusarium oxysporum at the concentration of 8 μg/mL [204]. Carbon nanoscrolls (CNSs) composed of graphene oxides and AgNPs exhibited enhanced and prolonged antifungal activity against Candida albicans and Candida tropical compared to GO–AgNP nanocomposites containing graphene oxide and AgNPs [205]. The antifungal efficacy of AgNPs was evaluated in combination with nystatin (NYT) or chlorhexidine (CHX) against Candida albicans and Candida glabrata biofilms. The results from this investigation suggest that AgNPs combined with either nystatin (NYT) or chlorhexidine digluconate (CHG) showed better synergistic anti-biofilm activity; however, this activity depends on the species and drug concentrations [206].

The biologically synthesized AgNPs exhibited strong antifungal activity against Bipolaris sorokiniana by the inhibition of conidial germination [207]. Interestingly, AgNPs not only inhibit human and plant pathogenic fungi, but also indoor fungal species such as Penicillium brevicompactum, Aspergillus fumigatus, Cladosporium cladosporoides, Chaetomium globosum, Stachybotrys chartarum, and Mortierella alpine cultured on agar media [208].

5.3. Antiviral Activity of AgNPs

Viral mediated diseases are common and becoming more prominent in the world; therefore, developing anti-viral agents is essential. The mechanisms of the antiviral activity of AgNPs are an important aspect in antiviral therapy. AgNPs have unique interactions with bacteria and viruses based on certain size ranges and shapes [70,209,210]. The antiviral activity nano-Ag incorporated into polysulfone ultrafiltration membranes (nAg-PSf) was evaluated against MS2 bacteriophage, which shows that significant antiviral activity was a result of increased membrane hydrophilicity [21]. Lara et al. [211] showed the first mechanistic study demonstrating anti-HIV activity at an early stage of viral replication. Poly vinyl pyrrolidone (PVP)-coated AgNPs prevented the transmission of cell-associated HIV-1 and cell-free HIV-1 isolates [211]. AgNPs have demonstrated efficient inhibitory activities against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) [212]. A study was attempted to investigate the antiviral action of the AgNPs; the data showed that both macrophage (M)-tropic and T-lymphocyte (T)-tropic strains of HIV-1 were highly sensitive to the AgNP-coated polyurethane condom (PUC) [213]. Although several studies have shown that AgNPs could inhibit the viability of viruses, the exact mechanism of antiviral activity is still obscure. However, the studies from Trefry and Wooley found that AgNPs caused a four- to five-log reduction in viral titer at concentrations that were not toxic to cells [214]. Interestingly, in the presence of AgNPs, virus was capable of adsorbing to cells, and this viral entry is responsible for the antiviral effects of AgNPs. Hemagglutination assay indicated that AgNPs could significantly inhibit the growth of the influenza virus in Madin-Darby canine kidney cells. The study from intranasal AgNP administration in mice significantly enhanced survival, lower lung viral titer levels, minor pathologic lesions in lung tissue, and remarkable survival advantage after infection with the H3N2 influenza virus, suggesting that AgNPs had a significant role in mice survival [215]. Biologically-synthesized AgNPs inhibited the viability in herpes simplex virus (HSV) types 1 and 2 and human parainfluenza virus type 3, based on size and zeta potential of AgNPs [216]. The treatment of Vero cells with non-cytotoxic concentrations of AgNPs significantly inhibited the replication of Peste des petits ruminants virus (PPRV). The mechanisms of viral replication are due to the interaction of AgNPs with the virion surface and the virion core [217]. Tannic acid mediated the synthesis of various sizes of AgNPs capable of reducing HSV-2 infectivity both in vitro and in vivo through direct interaction, blocked virus attachment, penetration, and further spread [218]. The antiviral property of Ag+ alone and a combination of 50 ppb Ag+ and 20 ppm CO32− (carbonate ions) was performed on bacteriophage MS2 phage. The results from this study showed that 50 ppb Ag+ alone was unable to affect the phage, and the combination of 50 ppb Ag+ and 20 ppm CO3 was found to have an effective antiviral property within a contact time of 15 min [219]. Treatment with AgNPs for 24 h in Bean Yellow Mosaic Virus (BYMV) decreased virus concentration, percentage of infection, and disease severity [220].

5.4. Anti-Inflammatory Activity of AgNPs

Inflammation is an early immunological response against foreign particles by tissue, which is supported by the enhanced production of pro-inflammatory cytokines, the activation of the immune system, and the release of prostaglandins and chemotactic substances such as complement factors, interleukin-1 (IL-1), TNF-α, and TGF-β [221,222,223,224]. In order to overcome inflammatory action, we need to find effective anti-inflammatory agents. Among several anti-inflammatory agents, AgNPs have recently played an important role in anti-inflammatory field. AgNPs have been known to be antimicrobial, but the anti-inflammatory responses of AgNPs are still limited. Bhol and Schechter [225] reported the anti-inflammatory activity in rat. Rats treated intra-colonically with 4 mg/kg or orally with 40 mg/kg of nanocrystalline silver (NPI 32101) showed significantly reduced colonic inflammation. Mice treated with AgNPs showed rapid healing and improved cosmetic appearance, occurring in a dose-dependent manner. Furthermore, AgNPs showed significant antimicrobial properties, reduction in wound inflammation, and modulation of fibrogenic cytokines [226]. Continuing the previous study, Wong et al. [222] investigated to gain further evidence for the anti-inflammatory properties of AgNPs, in which they used both in vivo and in vitro models and found that AgNPs are able to down-regulate the quantities of inflammatory markers, suggesting that AgNPs could suppress inflammatory events in the early phases of wound healing [222]. A porcine contact dermatitis model showed that treatment with nanosilver significantly increases apoptosis in the inflammatory cells and decreased the levels of pro-inflammatory cytokines [227]. Biologically-synthesized AgNPs can inhibit the production of cytokines induced by UV-B irradiation in HaCaT cells, and also reduces the edema and cytokine levels in the paw tissues [228].

5.5. Anti-Angiogenic Activity of AgNPs

Pathological angiogenesis is a symbol of cancer and various ischemic and inflammatory diseases [229]. There are several research groups interested in discovering novel pro- and anti-angiogenic molecules to overcome angiogenic-related diseases. Although there are several synthetic molecules having anti-angiogenic properties, the discovery of a series of natural pro- and anti-angiogenic factors suggests that this may provide a more physiological approach to treat both classes of angiogenesis-dependent diseases in the near future [230]. Recently, several studies provided supporting evidence using both in vitro and in vivo models showing that AgNPs have both anti-angiogenic and anti-cancer properties. Herein, we wish to summarize the important contribution in cancer and other angiogenic related diseases.

Kalishwaralal et al. [231] demonstrated the anti-angiogenic property of biologically synthesized AgNPs using bovine retinal endothelial cells (BRECs) as a model system, in which they found the inhibition of proliferation and migration in BRECs after 24 h of treatment with AgNPs at 500 nM concentration. The mechanisms of inhibition of vascular endothelial growth factor (VEGF) induced angiogenic process by the activation of caspase-3 and DNA fragmentation, and AgNPs inhibited the VEGF-induced PI3K/Akt pathway in BRECs [232]. Followed by this study, Gurunathan et al. [23] provided evidence for the anti-angiogenic property of AgNPs by using pigment epithelium derived factor (PEDF) as a bench mark, which is known as a potent anti-angiogenic agent. Using BRECs as an in vitro model system, they found that AgNPs inhibited VEGF-induced angiogenic assays. Furthermore, they demonstrated that AgNPs could block the formation of new blood microvessels by the inactivation of PI3K/Akt. The same group also demonstrated the anticancer property of AgNPs using various cytotoxicity assays in Dalton’s lymphoma ascites (DLA) cells, and a tumor mouse model showed significantly increased survival time in the presence of AgNPs [24]. AgNPs reduced with diaminopyridinyl (DAP)-derivatized heparin (HP) polysaccharides (DAPHP) inhibited basic fibroblast growth factor (FGF-2)-induced angiogenesis compared to glucose conjugation [232]. Kim et al. [233] developed an anti-angiogenic Flt1 peptide conjugated to tetra-N-butyl ammonium modified hyaluronate (HA-TBA), and it was used to encapsulate genistein. Using human umbilical vein endothelial cells (HUVECs) as in vitro model system, they found that genistein/Flt1 peptide–HA micelle inhibited the proliferation of HUVECs, and the same reagents could drastically reduce corneal neovascularization in silver nitrate-cauterized corneas of Sprague Dawley (SD) rats. Ag2S quantum dots (QDs) used as nanoprobes to monitor lymphatic drainage and vascular networks. Ag2S-based nanoprobes showed long circulation time and high stability. In addition, they were able to track angiogenesis mediated by a tiny tumor (2–3 mm in diameter) in vivo [5]. Recently, Achillea biebersteinii flowers extract-mediated synthesis of AgNPs containing a concentration of 200 μg/mL showed a 50% reduction in newly-formed vessels [234]. shows the inhibitory effect of AgNPs on VEGF induced angiogenic activity in bovine retinal endothelial cells (BRECs) and human breast cancer cells MDA-MB 231.

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5.6. Anticancer Activity of AgNPs

In our lifetime, 1 in 3 people has the possibility to develop cancer [235]. Although many chemotherapeutic agents are currently being used on different types of cancers, the side effects are enormous, and particularly, administrations of chemotherapeutic agents by intravenous infusion are a tedious process [235]. Therefore, it is indispensable to develop technologies to avoid systemic side effects. At this juncture, many researchers are interested in developing nanomaterials as an alternative tool to create formulations that can target tumor cells specifically. Several research laboratories have used various cell lines to address the possibility of finding a new molecule to battle cancer. Here we summarized the work from various laboratories reporting anticancer activity using both in vitro and in vivo model systems. Gopinath et al. [236] investigated the molecular mechanism of AgNPs and found that programmed cell death was concentration-dependent under conditions. Further, they observed a synergistic effect on apoptosis using uracil phosphoribosyltransferase (UPRT)-expressing cells and non-UPRT-expressing cells in the presence of fluorouracil (5-FU). In these experimental conditions, they observed that AgNPs not only induce apoptosis but also sensitize cancer cells. The anticancer property of starch-coated AgNPs was studied in normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251). AgNPs induced alterations in cell morphology, decreased cell viability and metabolic activity, and increased oxidative stress leading to mitochondrial damage and increased production of reactive oxygen species (ROS), ending with DNA damage. Among these two cell types, U251 cells showed more sensitivity than IMR-90 [237]. The same group also demonstrated that the cellular uptake of AgNPs occurred mainly through endocytosis. AgNP-treated cells exhibited various abnormalities, including upregulation of metallothionein, downregulation of major actin binding protein, filamin, and mitotic arrest [237]. The morphology analysis of cancer cells suggests that biologically synthesized AgNPs could induce cell death very significantly. Jun et al. [238] elegantly prepared multifunctional silver-embedded magnetic nanoparticles, in which the first type consist of silver-embedded magnetic NPs with a magnetic core of average size 18 nm and another type consist of a thick silica shell with silver having an average size of 16 nm; the resulting silica-encapsulated magnetic NPs (M-SERS dots) produce strong surface-enhanced Raman scattering (SERS) signals and have magnetic properties, and these two significant properties were used for targeting breast-cancer cells (SKBR3) and floating leukemia cells (SP2/O).

The antineoplastic activities of protein-conjugated silver sulfide nano-crystals are size dependent in human hepatocellular carcinoma Bel-7402 and C6 glioma cells [239]. Instead of giving direct treatment of AgNPs into the cells, some researchers developed chitosan as a carrier molecule for the delivery of silver to the cancer cells. For example, Sanpui et al. [240] demonstrated that chitosan-based nanocarrier (NC) delivery of AgNPs induces apoptosis at very low concentrations. They then examined cytotoxic efficiency using a battery of biochemical assays. They found an increased level of intracellular ROSin HT 29 cells. Lower concentrations of nanocarrier with AgNPs showed better inhibitory results than AgNPs alone. Boca et al. [241] reported that chitosan-coated silver nanotriangles (Chit-AgNTs) show an increased cell mortality rate. In addition, human embryonic cells (HEK) were able to take up Chit-AgNTs efficiently, and the cytotoxic effect of various sizes of AgNPs was significant in acute myeloid leukemia (AML) cells [242]. Recently, the anticancer property of bacterial (B-AgNPs) and fungal extract-produced AgNPs (F-AgNPs) was demonstrated in human breast cancer MDA-MB-231 cells. Both biologically produced AgNPs exhibited significant cytotoxicity [62,243]. Among these two AgNPs, fungal extract-derived AgNPs had a stronger effect than B-AgNPs, which is due to the type of reducing agents used for the synthesis of AgNPs. Similarly, AgNPs derived from Escherichia fergusoni showed dose-dependent cytotoxicity against MCF-7 cells [62]. Plant extract-mediated synthesis of AgNPs showed more pronounced toxic effect in human lung carcinoma cells (A549) than non-cancer cells like human lung cells, indicating that AgNPs could target cell-specific toxicity, which could be the lower level of pH in the cancer cells [63]. Targeted delivery is an essential process for the treatment of cancer. To address this issue, Locatelli et al. [244] developed multifunctional nanocomposites containing polymeric nanoparticles (PNPs) containing alisertib (Ali) and AgNPs. PNPs conjugated with a chlorotoxin (Ali@PNPs–Cltx) showed a nonlinear dose–effect relationship, whereas the toxicity of Ag/Ali@PNPs–Cltx remained stable. Biologically synthesized AgNPs showed significant toxicity in MCF7 and T47D cancer cells by higher endocytic activity than MCF10-A normal breast cell line [245]. Banti and Hadjikakou explained the detailed account of anti-proliferative and anti-tumor activity of silver(I) compounds [246]. Biologically synthesized AgNPs capable of altering cell morphology of cancer cells ( ), which is an early indicator for apoptosis. Apoptosis can be determined by structural alterations in cells by transmitted light microscopy.

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5.7. Diagnostic, Biosensor, and Gene Therapy Applications of AgNPs

The advancement in medical technologies is increasing. There is much interest in using nanoparticles to improve or replace today’s therapies. Nanoparticles have advantages over today’s therapies, because they can be engineered to have certain properties or to behave in a certain way. Recent developments in nanotechnology are the use of nanoparticles in the development of new and effective medical diagnostics and treatments. The ability of AgNPs in cellular imaging in vivo could be very useful for studying inflammation, tumors, immune response, and the effects of stem cell therapy, in which contrast agents were conjugated or encapsulated to nanoparticles through surface modification and bioconjugation of the nanoparticles. Silver plays an important role in imaging systems due its stronger and sharper plasmon resonance. AgNPs, due to their smaller size, are mainly used in diagnostics, therapy, as well as combined therapy and diagnostic approaches by increasing the acoustic reflectivity, ultimately leading to an increase in brightness and the creation of a clearer image [247,248]. Nanosilver has been intensively used in several applications, including diagnosis and treatment of cancer and as drug carriers [249,250,251]. Nanosilver was used in combination with vanadium oxide in battery cell components to improve the battery performance in next-generation active implantable medical devices [250]. Recently, silver has been used to develop silver-based biosensors for the clinical detection of serum p53 in head and neck squamous cell carcinoma [252]. In addition, it has been explored for the location of cancer cells, and can absorb light and selectively destroy targeted cancer cells through photothermal therapy [253].

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