How do you use silver nanoparticles?

Silver nanoparticles (AgNPs) have been one of the most attractive nanomaterials in biomedicine due to their unique physicochemical properties. In this paper, we review the state-of-the-art advances of AgNPs in the synthesis methods, medical applications and biosafety of AgNPs. The synthesis methods of AgNPs include physical, chemical and biological routes. AgNPs are mainly used for antimicrobial and anticancer therapy, and also applied in the promotion of wound repair and bone healing, or as the vaccine adjuvant, anti-diabetic agent and biosensors. This review also summarizes the biological action mechanisms of AgNPs, which mainly involve the release of silver ions (Ag + ), generation of reactive oxygen species (ROS), destruction of membrane structure. Despite these therapeutic benefits, their biological safety problems such as potential toxicity on cells, tissue, and organs should be paid enough attention. Besides, we briefly introduce a new type of Ag particles smaller than AgNPs, silver Ångstrom (Å, 1 Å = 0.1 nm) particles (AgÅPs), which exhibit better biological activity and lower toxicity compared with AgNPs. Finally, we conclude the current challenges and point out the future development direction of AgNPs.

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Considering various products containing AgNPs, such as dressings, creams, solvents, and scaffolds, it seems necessary to assess the potential toxicity of AgNPs in cells, tissues, and organs. Generally speaking, primary exposure routes include skin contact, inhalation, ingestion, and injection 36 - 38 . These routes will distribute AgNPs to different tissues and organs, such as skin, respiratory, circulatory, nervous, hepatobiliary, urinary and reproductive systems 36 - 41 . The deposited AgNPs may be potentially toxic to these tissues or organs by inducing cell necrosis, apoptosis or genetic mutations 42 - 45 . For example, AgNPs deposited in the lungs can cause pneumonia and asthma 46 . AgNPs may cross the blood testis barrier (BTB) and reduce the fertility of model animals and cause teratogenicity in offspring 42 . Some toxicological studies on model animals have shown that the potential toxicity of AgNPs is related to the inherent properties 25 , 47 . Large surface area may lead to increased silver ions (Ag + ) released from AgNPs, which may enhance the toxicity of nanoparticles. Besides the unique properties, the potential toxicity of AgNPs is closely related to dose, concentration and exposure time 24 , 48 - 50 . Exploring the pharmacodynamics of AgNPs in vivo may contribute to the development of bio-friendly and safe agents.

In recent years, the anticancer effect of AgNPs has been widely studied. AgNPs play an efficient role against a variety of cancer both in vitro and in vivo, including cervical cancer, breast cancer, lung cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, hepatocellular carcinoma, glioblastoma, colorectal adenocarcinoma, and prostate carcinoma 21 - 23 . The anticancer activity of AgNPs is affected by inherent properties, including size, shape and surface charge 24 - 26 . Generally speaking, the smaller the particle size, the higher the biological activity. To obtain an ideal anticancer agent with high biological activity, our team successfully synthesized a kind of very small silver particles which reached up to Ångstrom (Å; one-tenth of a nanometer) scale and determined the stronger anticancer activities of silver Ångstrom particles (AgÅPs) compared with AgNPs 21 . In addition, exposure time and dose are also considered as crucial factors. Longer exposure time and higher dosage will trigger stronger anticancer effects. Some possible mechanisms involving the anticancer effects of AgNPs have been proposed. AgNPs can cause apoptosis or necrosis by destroying the ultrastructure of cancer cells, inducing ROS production and DNA damage, inactivating enzymes, as well as regulating signaling pathways 27 - 29 . In addition, AgNPs can also block the invasion and metastasis of tumor cells by inhibiting angiogenesis 30 - 32 . Due to the enhanced permeability and retention (EPR) effect, tumor cells preferentially absorb NPs-sized bodies than normal tissues 33 , 34 . While the poor lymphatic drainage in the tumor enables nanoparticles to stay and penetrate 35 . This may enhance the targeted drug delivery of AgNPs. Further studies of anticancer mechanisms of AgNPs are essential to develop economical, reliable, and broad-spectrum anticancer agents.

Numerous studies focus on the synthesis of AgNPs with controlled size and shape, and a variety of specific synthetic methods have been developed, including physical, chemical, and biological methods 5 . The predominant processes of the physical methods are classified into two parts: mechanical and vapor-based processes 6 . Conventional physical methods may involve mill, pyrolysis, and spark discharging 7 . Physical synthesis can obtain AgNPs with uniform size distribution and high purity 8 . Chemical synthesis is the most commonly used method to obtain AgNPs 8 . This method involves reducing silver ions to silver atoms 9 , and the process can be divided into two steps, nucleation and growth 10 . Size- and shape-controlled AgNPs can be obtained by regulating the growth rate of nucleation. Besides reducing agents, capping agents and stabilizers also play important roles in obtaining AgNPs with good dispersion stability and uniform size distribution 11 . In addition, external energy can synergistically synthesize AgNPs, such as microwave, light, heat, and sound 12 - 15 . Although chemical synthesis methods of AgNPs are widely used, the toxicity and pollution caused by chemicals must be highlighted and more attention should be given. Compared with physical and chemical methods, the biological method proves an economical and environmental approach for AgNPs 8 . Microorganisms include bacteria, fungi, and algae, as well as plant parts, include bark, peel, callus, leaves, flower, fruit, stem, seed, and rhizome are widely used in biological synthesis 16 . The organics, like enzymes, alkaloids, phenolic compounds and terpenoids, are abundant in extracts of microorganisms and plants, which can be available to reduce silver salts 16 , 17 . Furthermore, some organic substances among these can also be used as stabilizers and capping agents 17 . Among the different methods, the additives mentioned may influence the subsequent medical applications of AgNPs.

Silver and its compounds have been used for antibacterial and therapeutic applications for thousands of years 1 , 2 . Ancient Greeks and Romans used silverwares to store water, food, and wine to avoid spoilage. Hippocrates used silver preparations to treat ulcers and promote wound healing. Silver nitrate was also used for wound care and instrument disinfection. In 1852, Sims sutured the vesicovaginal fistulas caused by delivery with fine silver wires which significantly decreased infection. At the beginning of the 19th century, silver preparations were developed for wound infection and burn care. However, in the 1940s, the medical applications of silver gave way to the clinical introduction of antibiotics 1 . With the abuse of antibiotics, bacterial resistance has become a worldwide problem especially since the 1980s, and silver began to receive attention again especially with the development of nanotechnology in the early of this century.

Plant-mediated synthesis of AgNPs, as a promising approach, has received great attention in recent years. Extracts from different parts of the plants, including bark, peel, callus, leaves, flower, fruit, stem, seed and rhizome, are involved in biosynthesis of AgNPs with various sizes and shapes 59 ( Table ). These extracts from different plant parts contain organic components such as enzymes, alcohols, flavonoids, alkaloids, quinines, oils, terpenoids and phenolic compounds 126 , 127 . There are different functional groups in these organic components 58 , like hydroxyl, carbonyl, amidogen, which may contribute to the reduction of Ag + to Ag 0 . A variety of plant extracts, including the components mentioned above and plant derivatives such as starch, cellulose, chitin, dextran and alginates, act simultaneously as reducing agents and stabilizers 128 . The plant-mediated synthesis of AgNPs is influenced by different reaction parameters such as temperature, reaction time, pH and concentration of plant extracts and precursors 129 , 130 . The AgNPs with different size and shape can be obtained by changing the synthesis parameters 129 . In summary, plant-mediated synthesis of AgNPs can be controlled by a variety of reaction conditions. In addition, different parts of plant exhibit various abilities in the synthesis of AgNPs 131 . The mechanisms of plant-mediated synthesis of AgNPs need more exploration. In conclusion, the plant-mediated synthesis of AgNPs using plant extract is a promising method due to its easy availability, nontoxicity, simplicity, cost-effectiveness and high reducing potential.

Algae, as one of the most potential coastal renewable living resources, have received more attention in the biosynthesis of nanometer materials in recent years ( Table ). Algae contain a variety of biologically active organic matters, such as carbohydrates, polysaccharides, enzymes, proteins, vitamins, pigments and secondary metabolites 17 , 121 , 122 . These abundant organic compounds make algae an ideal candidate for biosynthesis of AgNPs. These active organic matters may be used as reducing agents to form size- and shape-controlled AgNPs, including spheres, triangles, cubes, rods, wires, hexagons, pentagons and wires. The roles of many algae in biosynthesis of AgNPs are verified, including Cyanophyceae, Chlorophyceae, Phaeophyceae, Rhodophyceae 123 . These studies support algae as a promising bioresource for the synthesis of AgNPs with various shapes and sizes. Biomolecules in algae extracts, such as amino acids, proteins and sulfated polysaccharides, can also act as stabilizers or capping agents in the biosynthesis of AgNPs with variable properties 124 . The specific factors involved in the algae-mediated synthesis of AgNPs are necessary to be identified and determined, including the ratio of silver precursor to algae extract, mixture pH, incubation time and temperature 125 . In conclusion, the biosynthesis of AgNPs using algae extract provides a facile, sustainable and eco-friendly method. Various algae can be considered as candidates in the biosynthesis of AgNPs due to their unique properties of rapid growth, high metal accumulation ability and abundant organic content.

Fungi-mediated synthesis of AgNPs is an effective and straightforward approach 111 , 112 . According to the location of nanoparticles, fungi-mediated synthesis can obtain intracellular and extracellular AgNPs using mycelia and fungal cell-free filtrate, respectively 113 , 114 ( Table ). Compared with intracellular synthesis, the extracellular synthesis of AgNPs using fungi is preferred due to the advantages of convenient collection and downstream processing. Plenty of fungi, due to their unique abilities of metal bioconcentration, high tolerance in the metal-rich environment, rapid mycelial growth, various extracellular enzymes secretion, and economic viability, are selected for biosynthesis of AgNPs 115 , such as Fusarium oxysporum 116 , Trichoderma harzianum 57 , Penicillium polonicum 117 , Phomopsis liquidambaris 118 . However, some fungi, such as F. oxysporum 111 , are recognized to be potentially pathogenic, which may result in health risk in subsequent applications. While the AgNPs synthesized by extracellular method using the fungal extract can be purified by washing or precipitating unnecessary fungal components. Various organic components of fungi play an important role in the synthesis of AgNPs, such as nitrate-dependent reductase, xylanases 119 , naphthoquinones and anthraquinones, and quinine derivates of the latter two, are involved in the reduction of silver precursor. In addition, some proteins secreted by fungi can be used as capping agents to form shape-controlled AgNPs 120 . Various incubation conditions might influence the characteristics of AgNPs, such as the types of carbon and nitrogen sources, temperature and light source 56 . In conclusion, fungi mediated synthesis of AgNPs is a convenient, effective, low-cost and energy-saving biological method. However, reducing potential pathogens on the surface of AgNPs should be considered to obtain safe products.

Since Tanja Klaus et al. firstly reported the phenomenon of aggregation of AgNPs in Pseudomonas stutzeri AG259 in 1999 105 , series of bacteria, both Gram-negative and Gram-positive, are been screened for the synthesis of AgNPs ( Table ). The property of bacteria to survive in an extreme silver-rich environment might contribute to the accumulation of AgNPs 105 , 108 . Depending upon the location of the nanoparticles distribution, AgNPs may be synthesized intracellularly or extracellularly using biomass, supernatant, cell-free extracts, and derived components of the bacteria. Among these two modes, extracellular method is advantageous over intracellular method due to the convenience of recovery of AgNPs. The abilities and mechanisms of strains used in the biosynthesis of AgNPs are different from each other due to the organic substances. Various organic substances in bacteria can be used as reducing agents, such as exopolysaccharide, peptides, reductase, cofactors, c-type cytochromes, and silver-resistant genes. Among these, several enzymes have been involved in synthesizing AgNPs, such as nitrate reductase and lactate dehydrogenase; and peptides with special amino acid, such as methionine, cysteine, lysine, and arginine, may attach on the surface of nuclei and act as reducing agents 109 . Nitrate reductase, a kind of NADH-dependent enzymes, has gained more attention in the bacteria-mediated synthesis of AgNPs. Nitrate reductase can participate in the electron transport chain, and subsequently creates a miniature reducing environment by transferring hydrogen atoms. The enzyme gains electron from NADH, oxidizes it to NAD + , and undergoes oxidation to reduce silver ions to AgNPs 18 , 109 . Some organic substances can also act as stabilizers and capping agents for AgNPs to prevent particle aggregation 18 , 110 . The mechanisms of bacteria-mediated synthesis of AgNPs still need to be further explored. In conclusion, bacterial-mediated synthesis of AgNPs is a simple, effective, and environmentally friendly method.

In recent decades, a variety of microorganism- and plant-mediated biological syntheses of AgNPs are developed. The microorganisms can evolve metal tolerance genes and metal bioconcentration capability to survive in an extreme silver-rich environment 105 , 106 . These adaptive evolutionary mechanisms include altering and decreasing the cytotoxicity of metal and resulting formation of AgNPs. AgNPs can be regarded as “by-product” of the resistance mechanism of microorganisms against free Ag + . Plant mediated synthesis can reduce Ag + to Ag 0 using functional groups such as O-H and =C-H in organic components and their derivatives contained in the extract of plant parts 107 . Commonly used plant parts include bark, peel, callus, leaves, flower, fruit, stem, seed, and rhizome. In the process of biosynthesis, various biological components act as reducing agents, such as exopolysaccharide, peptides, nitrate reductase, reducing cofactors, c-type cytochromes, separated from microorganisms, and starch, cellulose, chitin, dextran, alginates, separated from plants. However, the organic components in the biosynthesis of AgNPs require to be further studied due to their complex interaction with AgNPs and the diversity of plants. Compared with physical or chemical methods, biological method can be carried out at normal temperature and pressure and avoid the use of toxic or hazardous additives. In this part, we will introduce several microbial and plant synthesis approaches of AgNPs, as well as the mechanisms involved in these processes.

Sonochemical method refers to the cavitation effect generated by ultrasonic irradiation, which produces a local hot spot and promotes the synthesis of AgNPs 84 . The instantaneous high pressure and microjet generated by ultrasonic irradiation can uniformly mix the solution and generate bubbles, which may suddenly collapse when the bubbles grow. The adiabatic compression of the gas phase in the bubble creates a local hot spot, which accelerates the contact of Ag + with the reducing agent and rapidly reduces it to AgNPs. Ultrasound prevents the agglomeration of nanoparticles in the aqueous solution to decrease the size of AgNPs. Besides the high temperature, other factors such as pressure, pH, high-speed microjet, and high cooling rate may also contribute to the synthesis process. In summary, the sonochemical method is a simple, economical, and environment-friendly technique for preparing colloidal silver nanoparticles.

Microwave-assisted method refers to rapid heating the silver precursor by microwave irradiation, which may promote the generation of nuclei in site 82 . Several factors may influence the microwave-assisted synthesis of AgNPs, including the concentration of precursor and the type of stabilizer, power input and irradiation time of the microwave, dielectric constant, refractive index of the medium and chirality of reducing agents 55 . Water and alcohol are ideal media for microwave heating stabilizer because of their high dielectric losses 83 . For example, polar molecules such as H 2 O attempt to orient the electric field in the microwave. When dipolar molecules attempt to reorient relative to an alternating electric field, they lose energy in the form of heat which may contribute to the reduction of Ag + . Microwave-assisted method has the advantages of high energy conversion efficiency, time-saving, cleanliness, and convenience, most importantly, it can be used to obtain large-scale production of high dispersive AgNPs.

Electrochemical method can form an electric potential in the electrolyte and reduce Ag + to Ag 0 80 . The nucleation and growth of AgNPs occur almost simultaneously under the external electric field. Electrochemical method can synthesize AgNPs with different sizes by adjusting the current density. Besides, electrode types, electrolytes, and solvents are also important in the synthesis of size-controlled AgNPs. In the synthesis process, increased precursor concentration and enhanced current intensity, as well as prolonged implementation time, will obtain more AgNPs with smaller size 81 . To obtain dispersed and stable AgNPs, stabilizers and capping agents are optional additives. The steric hindrance formed by these additives will prevent the aggregation of AgNPs. The electrochemical method has the advantages of easy reaction control, mild reaction conditions, and less environmental pollution.

The photochemical method refers to reduce the precursors to AgNPs under the illumination. The silver precursors and solution in the luminescent region produce reduced free radicals and hydrated ions, which can reduce Ag + to Ag 0 in situ directly. Light sources involving in the photochemical method include ultraviolet light, sunlight, and laser light, among which ultraviolet light is most commonly used. The source, intensity and wavelength of the light, and the irradiation time may affect the synthesis of AgNPs 79 . For example, prolonging the irradiation time and increasing the irradiation intensity during photochemical synthesis may promote the reduction of Ag + 79 . The photochemical method has the unique advantage of synthesizing highly dispersible nanoparticles in situ in the illumination region. Therefore, the photochemical method can obtain AgNPs on the surface of various media, such as polymeric films, glass, and cells which are illuminated. The photochemical method typically requires relatively simple equipment and can be carried out at room temperature without harmful or strong reducing agents. The reactions can be terminated or attenuated by stopping the illumination.

Chemical reduction is a reliable method for preparing colloidal AgNPs in organic solutions or water. AgNPs with desired shapes can be obtained by chemical reduction method, such as nanosphere, nanoprism, nanoplate, nanowire, nanocube, and nanorod. The chemical reduction method includes three components: salt precursor, reducing agent, and stabilizer. Silver precursors can be effectively reduced to AgNPs by different reducing agents with the presence of a stabilizer. There are several alternative silver precursors continuously providing monomers for nucleation, such as silver nitrate 69 , silver ammonia ( ie. Tollens reagent) 72 , silver sulfate 73 , and silver chlorate 74 . Frequently used reducing agents may affect the growth of nuclei, including NaBH4, hydrazine, N, N-dimethylformamide, TSC, ascorbic acid, ethylene glycol, polysaccharides, and formaldehyde. The types and ratio of precursors and reducers, as well as the temperature and pH of the solution, may influence the characteristics of AgNPs 75 - 78 . The nucleation and subsequent growth of the particles in the chemical reduction process can be controlled by alternating the components and adjusting the reaction parameters. For example, Jiang et al. 78 studied the role of temperature in the synthesis of AgNPs by chemical reduction method ( Figure ). At the reaction temperature range of 0 to 55 °C, the low temperature significantly slowed down the generation of nuclei and growth, therefore, it took a longer time to complete the reduction of precursors. From 17 to 55 °C, the reaction rate increased with rising temperature, as well as the size of nanoparticle ( Figure A, 2B ). There was a size jump in the reaction synthesis at around 32 °C, i.e., the size of nanoparticles increased rapidly from around 90 nm to 180 nm for the edge length of plate AgNPs and from around 25 nm to 48 nm for the diameter of spherical AgNPs ( Figure C ). The ratio of the plate to spherical nanoparticles might be fundamentally dependent on the amount of single-crystal and twin structures formed at the nucleation process. In this experiment, the amount of spherical AgNPs decreased while the plate ones increased with the temperature rising. The ratio of the plate to spherical AgNPs was 1:1 at 17 °C, while 3:1 at 55 °C ( Figure A ).

Chemical synthesis is currently the most common method to synthesize AgNPs ( Table ). The process involves the reduction of Ag + (supply by silver salt precursor) to elemental silver (AgNPs) through electron transfer under certain conditions 8 , 69 . In general, chemical synthesis can be promoted by reducing agents such as sodium borohydride (NaBH4) and sodium citrate (TSC). The chemical method can be combined with external energy sources to prepare AgNPs, such as photochemical, electrochemical, microwave-assisted and sonochemical methods. Among these methods, the generation process of AgNPs can be divided into two parts: nucleation and growth. The monomer concentration in the solution rapid rises above the critical level of supersaturation, and triggers “burst-nucleation” and precipitation 70 , 71 . The precipitation of the monomer forms the nucleus, and the repetitive nucleation process promotes the continuous birth of new seeds. As the seed formation, monomer concentration drops below the critical level of supersaturation. After nucleation, the increased addition of monomer induces the growth of nuclei and forms NPs with a larger size. During the synthesis process, stabilizers such as PVP and CTAB are usually used to stabilize and disperse AgNPs. Even though the chemical method of AgNPs is a reliable, high-yield, time-saving and controllable route, it must be noted that chemicals used in this method may cause environmental pollution.

The basic and most commonly used physical vapor deposition processes are divided into two general categories: arc evaporation and sputtering 67 . The former refers to the utilization of a cathodic arc source in a vacuum chamber or protective gases to obtain metal vapor and deposit it on a target coating material to form a thin, adherent pure metal or alloy coating. During this process, highly ionized metal vapor generates plasma 68 . And the latter refers to using a high-energy electrical charge to bombard the target coating material and deposit metal on the substrate. In this process, ions and energetic atoms impact atoms and mechanically eject them from the target material. Recently, our team successfully synthesized a kind of very small silver particles which reached up to Ångstrom (Ång) scale for the first time with a self-developed evaporation-condensation system 21 . A pure silver wire was fed into an explosion chamber filled with protective gas Argon, following by a high voltage of 25 kV when the wire contacted the positive electrode plate. The silver wire was exploded and evaporated to yield silver vapor plasma. Then the silver vapor was rapidly cooled and coagulated to form Ag particles in the rapid cooling chamber with a water chiller at 0-4 °C. High-intensity ultrasonic and demagnetization devices were used successively to prevent re-agglomeration of Ag particles. In conclusion, the physical vapor deposition method can obtain pure and dispersible AgNPs with small particle size, but complicated devices and external energy are required.

Laser ablation method refers to a pulsed laser instantaneously heat the target bulk metal immersed in water or an organic solvent to form plasma plume, followed by nucleation and growth of metal particles during the plasma plume cooling process and eventually form nanoscale clusters 62 , 64 . During the process of laser ablation, nanoparticles can absorb photons through multiple pathways, including plasmon excitations, interband transitions, and multiphoton absorption, which are closely related to pulse time, laser wavelength, and laser fluence. These factors, as well as the type of aqueous medium, may affect the characteristics of NPs 62 . Different synthesis conditions, such as laser fluences, pulse wavelength, as well as solvent type, may affect the size of the NPs. The addition of organic stabilizers such as cetyltrimethylammonium bromide (CTAB) and PVP can enhance the dispersibility of AgNPs 11 . However, it is difficult for laser ablation method to control the size distribution of NPs 62 .

The physical synthesis of AgNPs involves mechanical processes and vapor-based processes. Energies are used to reduce particle size, including mechanical energy (ball milling method) 60 , electrical energy (electrical arc-discharge method) 61 , light energy (laser ablation method) 62 , and thermal energy (physical vapor deposition) 6 ( Table ). During the ball milling progress, high-speed collisions between rigid balls, such as ceramics, flint pebbles, and stainless steels, can produce localized high pressures, which grind the metal into very fine powders 60 . The electrical arc-discharge method can obtain NPs via arc discharge device under direct current (DC) power 63 . The device uses the powder reagent layer as the anode and the electrodes are immersed in dielectric liquids such as hydrocarbons, liquid inert gas, and deionized water. Laser ablation method refers to the ablation of a metal plate by a high-power laser, the metal target absorbs the laser beam energy and photoions, followed by nucleation and growth of metal particles during the plasma plume cooling process and eventually synthesize NPs 62 , 64 . Sputtering and evaporation are two processes in physical vapor deposition. Sputtering refers to bombarding a target coating material with a high-energy electrical charge to sputter off atom or molecule that can be deposited on the substrate. While evaporation refers to heating the coating material to the boiling point in a vacuum environment and evaporating, and the evaporated material rises in the vacuum chamber and condenses on the substrate. Although physical synthesis can produce AgNPs on a large scale, AgNPs may aggregate and form large-sized particles which will affect subsequent applications. In order to avoid the re-aggregation of AgNPs, some stabilizers are used to obtain stable colloids AgNPs. For example, polyvinyl pyrrolidone (PVP) may be used as both the electrolyte and stabilizer in the synthesis of AgNPs by laser ablation method 65 . Our team prepared Ångstrom silver particles, capped with fructose as stabilizer, can be stable for a long time 21 . In summary, the physical method can quickly produce NPs with uniform size distribution and high purity, but complex equipment and external energy are required.

The synthesis methods of nanoparticles (NPs) are mainly divided into two processes: top-down and bottom-up ( Figure ). The top-down approach refers to the formation of metal NPs from bulk materials using various physical forces to synthesis NPs, such as mechanical energy used in ball milling, crushing and grinding; electrical energy used in the electrical arc-discharge method and laser ablation method; and thermal energy used in vapor condensation method 51 . These approaches can obtain NPs between 10 and 100 nm in size. The top-down approach, mainly the physical method, may acquire pure nanoparticles without chemical additives. NPs synthesized by physical method may exhibit uniform particle size distribution and high purity. Though the physical approach does not contain chemical reagents which may harm human and environment, it brings a great challenge to prevent agglomeration due to absence of stabilizer or capping agents. Furthermore, these methods need complex equipment and external energy in the process. The bottom-up approach involves the construction of complex clusters to obtain NPs from molecular components by employing nucleation and growth processes 51 , 52 . The commonly used bottom-up approaches include chemical synthesis and biological synthesis, both can obtain NPs by reducing the precursor salt 52 . The chemical synthesis can be coupled with alternative energies, such as photochemical 53 , electrochemical 54 , microwave-assisted 55 and sonochemical methods 12 . Though the chemical method is carried out to quickly obtain various shapes of NPs, the use of harmful chemical additives may limit the medical applications of NPs. To overcome the shortcomings of the chemical method, the biological method has been regarded as an alternative option. The biological method usually relies on macromolecular substances in bacteria, fungi, and algae 16 , such as exopolysaccharide, cellulose, and enzymes, and organic components in plant extracts such as enzymes, alcohol, flavonoids, alkaloids, quinines, terpenoids, phenolic compounds 16 , 56 - 59 . Biological synthesis is an economical, environmentally friendly, simple and reliable approach, but the components on the surface of nanoparticles must be adequately considered in the application. Based on these two approaches, frequently used methods for synthesizing AgNPs, including physical, chemical and biological methods are discussed herein.

Silver nanoclusters have unique optical and electrical properties and can be used as materials for synthetic probes. While proteins have multiple chelating and functional groups, therefore, they have unique advantages as ligands in biological imaging. Cunlan Guo and Joseph Irudayaraj 307 used denatured bovine serum albumin as a stabilizer to synthesize silver clusters, which could sensitively and selectively detect the content of mercury. The probe had important application value for detecting mercury content in water, soil and food. Sun et al. 308 used glutathione as a ligand to passivate silver nanoclusters and obtained highly sensitive fluorescent probes. During the passivation of glutathione, the specific recognition of silver nanoclusters modulated from Hg 2+ to Cu 2+ . This fluorescent probe was highly sensitive and selective in detecting Cu 2+ in blood samples. The synthesis of silver nanoclusters with DNA as the backbone has excellent spectral and photophysical properties. The generation of this fluorophore is highly dependent on the DNA sequence. Oligonucleotide sequence changes may trigger the adjustment of the photoluminescence emission band, thus identifying the mutant nucleotide sequence. Guo et al. 309 designed a double-stranded DNA scaffold that hybridizes probe DNA strands and sickle cell anemia mutation target DNA to generate fluorescent silver nanoclusters. The fluorescent silver nanoclusters specifically recognized sickle cell anemia mutations. The research extended from DNA scaffold single-stranded oligonucleotide to hybrid DNA double-stranded mutation site recognition, which may have more applications in the field of biological analysis. These studies suggest that silver nanoclusters have great clinical application potential.

Surface-enhanced Raman scattering (SERS) has attracted the attention of noble metals with Raman signals in many application strategies, including biochemical sensing, analytical chemistry, and materials science 303 . Among these nanomaterials, AgNPs can be used as a cost-effective surface-enhanced Raman scattering substrate. Nanoparticles containing AgNPs can be used as biosensors to detect blood glucose, enzymes, molecular markers of tumor cells, pathogens, etc. For example, Jiang et al. 304 prepared silver-containing nanocomposites as acetylcholinesterase biosensors for electrochemical detection of organophosphorus pesticides. AgNPs improved the electrical conductivity and biocompatibility of nanocomposites and made them more suitable for enzyme activity and stability. Anderson et al. 305 prepared a high-sensitivity nonenzymatic biosensor for the detection of glucose using AgNPs as a conductive additive. Both the porous nanostructures of AgNPs and large surface areas of carriers enhanced the interaction sites between AgNPs and electrode/glucose, which could accelerate the electron transfer of AgNPs and therefore improve the sensitivity of the biosensor. Although the electrochemical characteristics and Raman scattering make AgNPs exhibit good application prospects in the field of biosensing, the matrix composition may affect their SERS and reduce the detection sensitivity. Therefore, it is necessary to modify AgNPs in order to improve the sensitivity of re-creating platforms. For example, Zeng et al. 306 synthesized hybrid Ag@NGO nanoparticles by a one-step reduction method. Among these platforms, the nanosized graphene oxide (NGO) worked as inert protective layers and provided an ultrathin protective layer for AgNPs. Ag@NGO exhibited the advantages of both SERS biosensing and drug delivery, ie, monitoring biomolecule signals in tumor cells through SERS biosensing and interacting with the anticancer drug doxorubicin through formation of π-π bonds. These results prove that AgNPs hold great application potential with capabilities of SERS biosensing.

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia. DM is due to either insufficient insulin secretion or insulin resistance of the cell. Commonly used hypoglycemic agents can lower blood sugar by promoting secretion of insulin or increasing cell sensitivity 300 . In recent studies, it is noticed that AgNPs synthesized by plant extracts exhibit antidiabetic potential. Arumugam et al. 301 synthesized AgNPs using leaf extract of Solanum nigrum and evaluated the anti-hyperglycemic effect in alloxan-induced diabetic rats. They found that the blood glucose level of diabetic rats decreased when treated with AgNPs for 14 days and 21 days without significant acute toxicity. And they assessed glucose tolerance of AgNPs in diabetic rats. The results showed that AgNPs exhibited a good hypoglycemic effect compared to glibenclamide, a standard antidiabetic drug. Saratale et al. 4 demonstrated that AgNPs synthesized by leaf extract of Argyreia nervosa exhibited antidiabetic activity via inhibiting α-amylase and α-glucosidase. These two carbohydrate digestive enzymes contribute to decompose carbohydrates into monosaccharides. The antidiabetic mechanism of AgNPs is still unclear. Jihan Hussein et al. 302 hypothesized that AgNPs might influence insulin signaling pathway or insulin sensitivity in diabetic rats. The results supposed that AgNPs could activate protein kinase C and PI 3 K pathway at the insulin receptor substrate level, as well as inhibit protein kinase C isozymes, thus effectively enhance insulin secretion and sensitivity. It was highlighted that AgNPs were effective in reducing insulin resistance and DNA damage.

Vaccination is one of the most effective methods to prevent infectious diseases and manage healthcare costs 296 . Traditional vaccines have good immunogenicity due to the complex nature of the formulation and the presence of adjuvants. However, purified preparations lack immunogenicity, which makes the addition of adjuvants essential. Adjuvants can simultaneously reduce the amount of antigen required, shorten the time needed for a protective threshold of antibody production and improve the intensity of the elicited responses, stimulate long-term memory responses to reduce the requirement of repeated vaccinations. Yingying Xu et al. 297 firstly reported the remarkable immunological adjuvant effect of AgNPs both in vitro and in vivo using model antigens ovalbumin and bovine serum albumin in 2013. After intraperitoneal or subcutaneous immunization of mice, AgNPs increased the production of serum antigen-specific IgG, as well as antigen-specific IgE, indicating that AgNPs stimulated Th2-biased immune responses. Further study of the mechanism of adjuvant revealed that AgNPs could recruit and activate local leukocytes and macrophages. Vahid Asgary et al. 298 evaluated AgNPs as an adjuvant for the rabies vaccine in 2014 and 2016, respectively. They found that although the load of AgNPs could significantly increase the immune responses by arising neutralizing antibody against rabies virus in mice, the lowest concentration of virus-loaded AgNPs decreased cell viability. This limited the use of AgNPs as an adjuvant in rabies virus. They then challenged the green synthesis of AgNPs using leaf extract of Eucalyptus procera and added AgNPs as an adjuvant in rabies veterinary vaccine, following by estimating vaccine efficacy in mice and dogs. They confirmed that the vaccine loaded with a suitable concentration of AgNPs was nontoxic 299 .

Plaque biofilm formation is one of the causes of dental diseases. AgNPs have been incorporated into some dental biomaterials for reducing biofilm formation due to its antibacterial activity. Polymethyl methacrylate (PMMA), also known as acrylic resins, and composite resins are applied for the fabrication of dentures, but potential harmful organisms are likely to adhere to the rough surface of dentures 155 . AgNPs incorporated in PMMA can improve the antibacterial effect of dental material. It is proved that PMMA-AgNPs showed great anti-bacterial effect by continuous releasing of Ag + even in 28 days. It is highlighted that increased loading of AgNPs in PMMA also improved the mechanical properties 155 . While Acosta-Torres et al. demonstrated PMM-AgNPs could efficiently decrease the adherence of Candida albicans and exhibit no obvious genotoxicity or cytotoxicity. Comparison study of the anti-bacterial and anti-biofilm efficacies of AgNPs capped with carboxymethyl cellulose and sodium alginate, respectively, showed that carboxymethyl cellulose-capped AgNPs exhibited stronger inhibition to Gram-negative organisms, which were primarily responsible for periodontal infection 295 .

Bone is an active tissue with regenerative and restorative capabilities. The self-repairing capability of bone is usually compromised when bacterial infection occurs in bone defects. Bone grafts are commonly implanted to replace or restore large defects that usually resulted from severe trauma, tumor resection or genetic malformation. Orthopedic infections are usually related to bone destruction and implant loose 292 . AgNPs can be used as doping materials for synthetic bone scaffolds. AgNPs-implanted crystallized hydroxyapatite (HA) or titanium scaffolds display strong antibacterial ability against both Gram-positive and Gram-negative bacterial strains 162 . AgNPs can promote fracture healing as an osteoconductive biomaterial. For example, AgNPs can naturally stimulate the osteogenic differentiation and matrix mineralization of MC3T3-1 cells 293 . In a mouse model, AgNPs has been proved to stimulate proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro, and promote the healing process of bone fracture 294 .

The wound healing is closely related to the prognosis of surgical treatment. The rapid development of nanotechnology in recent years has provided a new therapeutic strategy for healing wounds, but the specific mechanisms of AgNPs on wound healing still need more exploration. Jun Tian et al. 288 found that AgNPs could increase wound healing rate with less hypertrophic scarring in the thermal injury model. Compared with the healing time of deep partial-thickness wounds treated with silver sulfadiazine, the AgNPs treated group could heal in a shorter period and a superior cosmetic appearance was observed, including nearly normal hair growth and less hypertrophic scarring. In the healing process, lower level of TGF-β and increased level of interferon-γ were detected at the same time in AgNPs treated group, while the former was related to keloids and hypertrophic scars, and the later was involved in the inhibition of fibroblast proliferation. In addition, higher level of VEGF mRNA detected in keratinocytes at the edge of the wound suggested that AgNPs might promote wound healing by inducing angiogenesis. These results indicated that AgNPs could participate in wound healing by regulating various cytokines and achieve cosmetic effects. Other mechanisms of AgNPs in wound repair are being explored. AgNPs can remain in the cytoplasm of fibroblasts in skin biopsies, and promote the reconstruction of dermis and epidermis 289 . Some studies prove that AgNPs can induce the proliferation and migration of keratinocytes, decrease the amounts of collagen and hydroxyproline, and promote the differentiation of fibroblasts into myofibroblasts, which may help wound early adhesion, contraction and closure 290 . Besides, AgNPs can promote wound healing by regulating the production of cytokines or proteins, such as inflammatory cytokines, VEGF and MMPs 163 , 291 . The above studies of AgNPs on wound repair broaden our understanding of the activity of AgNPs in cellular events. The role of AgNPs in wound repair is positive for clinical wound care and postoperative results.

Numerous studies have confirmed that AgNPs can inhibit migration and invasion of tumor cells in concentration- and dose-dependent manners 23 , 30 , 32 , 269 . Migration and invasion are important hallmarks of cancer progression and deterioration 270 . Although it has been observed that AgNPs can inhibit tumor invasion 269 , the specific mechanism is still unclear. It is hypothesized that AgNPs may decrease the protein expression of cytokines and growth factors within cancer cells, or reduce the enzymatic activity of MMPs. VEGF is an important signaling protein involved in vasculogenesis and angiogenesis, which plays a crucial role in tumor growth and metastasis 32 . Various studies support that AgNPs can deprive cancer cells of both nutrients and oxygen via inhibiting angiogenesis. It has been demonstrated that AgNPs can inhibit VEGF-induced angiogenesis by inactivating PI3K/AKT pathway 271 . Besides, AgNPs can block VEGF-induced Akt phosphorylation, this may influence the proliferation and migration of cells 272 . Another study has proved that AgNPs can disrupt the HIF-1 signaling pathway, thus lead to inhibition of angiogenesis 161 .

In the development and progression of tumors, many signaling pathways are involved in controlling cell growth and proliferation, apoptosis and viability, and can participate in more complex signaling networks that contribute to tumor progression, such as tumor microenvironment (TME), angiogenesis and inflammation 263 . Some proteases and cytokines are also involved in these regulations, such as vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMPs) and fibroblast growth factor 2 (FGF-2), etc. AgNPs have been confirmed to inhibit tumor proliferation, invasion and angiogenesis by regulating the associated signaling pathways or inactivating proteins. For example, AgNPs can regulate the HIF signaling pathway 161 . In general, rapid proliferation of tumor cells and irregular vasculature cause hypoxic TME 264 - 266 . HIF-1 level is up-regulated in hypoxic TME, followed by activation of target genes that in response to hypoxia. These genes contribute to cellular activities, such as cell proliferation, angiogenesis, and eventually lead to failure of cancer treatment 161 . Therefore, HIF-1 is a potential target for cancer treatment. It has been demonstrated that hypoxia can weaken HIF-1α-mediated autophagy 247 . Tieshan Yang et al. found that AgNPs could disrupt the HIF signaling pathway by attenuating HIF-1 protein accumulation and downstream target genes expression 161 . AgNPs can also inhibit the progression of tumors by inhibiting MMPs activity. MMPs are known as protein family and classified into different evolutionary groups according to their primary sequences 267 . MMPs play a dominant role in tumor progressions, such as tumor cell proliferation, invasiveness and distant metastasis, evasion of immune surveillance, and angiogenesis 267 , 268 . Therefore, MMPs are considered as potential targets for cancer therapy 31 . In order to obtain antitumor drugs with targeting capabilities, some teams have attempted to develop inhibitors against members of MMPs.

AgNPs can induce ROS production to disrupt DNA structure, or directly contact with DNA to cause DNA mutations 209 , 241 , 248 . High levels of ROS can generate damage to DNA double helix in a concentration-dependent manner, including breaking the single or double-stranded DNA, affecting base modifications and DNA cross-links 241 , 253 , 257 . AgNPs-treated cancer cells may exhibit DNA methylation, DNA base pairing errors, DNA repair defects and increased chromosomal aberrations 209 , 248 , 258 . AgNPs may play an important role in the regulation of gene expression of cells. AgNPs inhibit the proliferation of cells and trigger DNA repair defects by down-regulating the functions of proteins involved in cell cycle progression and DNA repair 259 . For example, proliferating cell nuclear antigen (PCNA) gene plays an important role in DNA synthesis and repair as a cofactor for DNA polymerase. PCNA is down-regulated in AgNPs-exposed cells. While the up-regulation of the apoptotic precursor protein Bax suggests that AgNPs initiate apoptosis via the mitochondrial pathway 260 . AgNPs-treated cells may undergo S phase, G2/M phase and sub-G1 cell cycle arrests in a concentration-dependent manner, as well as the increased number of G0/G1 phase cells, which may be prone to apoptosis 244 , 258 , 261 . AgNPs can not only induce apoptosis through ROS-mediated DNA damage, but also destroy DNA structure directly via Ag 0 and Ag + released by AgNPs 157 . The DNA double helix structure is composed of four bases of adenine, guanine, cytosine and thymine by strictly complementary base pairing. Base pairs are bounded by hydrogen bonds to form a unit of DNA double helix. The destruction of hydrogen bonds decreases the stability of DNA structure. Tsuneo Ishida 157 analyzed the activities of AgNPs in the nucleus. Silver could form a complex containing silver within DNA. Ag + caused DNA damage by replacing the hydrogen bonds in the G≡C and A=T base pairs. The Ag atom was twofold coordinated by two N atoms to form N-Ag + -N complex in G≡C base pair, and other complex structures appearing in the base pair were O-Ag + -N (G≡C base pair), N-Ag + -O (both G≡C and A=T base pairs). DNA damage caused by these complexes might be a factor in triggering cancer cell apoptosis.

ROS are by-products of biological aerobic metabolism, including oxygen ions, peroxides and oxygenated free radicals 251 . ROS are highly active due to the presence of unpaired free electrons. ROS are controlled at a low level by normal cellular antioxidant defense mechanisms and do not affect the normal physiological activities of the cells. However, excessive ROS can produce oxidative stress that reduces the activity of biological macromolecules and damages subcellular organelles and DNA structures 252 , 253 . Oxidative stress trigger lipid peroxidation, impaired mitochondrial function, amino acid oxidation in proteins, enzyme inactivation and DNA/RNA damage 233 , which may lead to autophagy, apoptosis and necrosis of cancer cells. AgNPs distributed in tumor cells via endocytosis can result in autophagy and apoptosis through a variety of ROS-mediated stress responses. In addition, AgNPs-induced formation of ROS may affect cellular signal transduction pathways, which may participate in the activation of apoptosis 254 . For example, the mitochondrial function can be inhibited by AgNPs via disrupting mitochondrial respiratory chain, suppressing ATP production. Besides, ROS induced by AgNPs may ultimately lead to DNA damage 255 . Superoxide radicals directed to mitochondria may enhance mitochondrial outer membrane permeabilization (MOMP) and the release of Cyt c, destroy the electron transport chain, and impair mitochondrial function 256 . Some factors influence the generation of ROS induced by AgNPs. Smaller size and higher concentration of AgNPs exhibit higher induction of ROS and stronger cytotoxicity, and sharp increased ROS appear in different cancer cells treated with AgNPs 220 .

Destruction of ultrastructures such as cell membranes and intracellular organelles leads to cell apoptosis and necrosis 21 . Tumor cells exhibit intact cell structure under light microscope, such as round nuclei, intact nuclear membrane, homogeneous chromatin, normal mitochondria and rough endoplasmic reticulum 40 . The ultrastructural changes of AgNPs-exposed tumor cells are in a dose- and time-dependent manner 246 . Generally, the higher the concentration of AgNPs and the longer the exposure time, the more serious the damage of cell ultrastructure. TEM observation showed that AgNPs-exposed cells are suffering morphological change or cytoplasmic organelle damage, and undergoing different death patterns: apoptosis, necrosis and autophagy 40 . Autophagosomes associated with apoptosis and necrosis are formed in the cytoplasm of AgNPs-treated tumor cells 247 . AgNPs promote autophagosome formation through the PtdIns3K pathway, and induce autophagy in tumor cells without inhibiting lysosomal function 22 . Structural and functional disruption of the actin cytoskeleton may be the cause of morphological deterioration of tumor cells exposed to AgNPs, and may be involved in inhibiting migration and invasion of tumor cells 248 . Free Ag + released from AgNPs is involved in the destruction of cellular membranes. Ag + released by AgNPs induces oxidation of glutathione, and increases lipid peroxidation in cellular membranes, resulting in cytoplasmic constituents leaking from damaged cells 249 . Our team found time-dependent morphological changes in cancer cells treated with F-AgÅPs, such as organelle compaction, nuclear fragmentation and cell blebbing 21 . Tumor cells exposed to AgNPs lose their typical shape due to pseudopod contraction, decreased cell adhesion and reduced cell density. Scanning electron microscopy analysis of AgNPs-treated tumor cells reveal spherical appearance, foamed membrane and shorten filopodia 248 . Tumor cells exposed to AgNPs show apoptotic cell characteristics such as loss of intact membrane, decreased contact with adjacent cells, condensed and detached from the culture plate 250 .

AgNPs have broad-spectrum anticancer activity via multiple mechanisms 21 , 239 , 240 . Numerous experiments in vitro and in vivo have proved that AgNPs can decrease the proliferation and viability of cancer cells. AgNPs can cause apoptosis and necrosis by destroying the ultrastructure of cancer cells, inducing the production of ROS and DNA damage 21 , 241 . AgNPs can promote apoptosis by up- or down-regulating expression of key genes, such as p53 242 , and regulating essential signaling pathways, such as hypoxia-inducible factor (HIF) pathway 243 . Cancer cells treated with AgNPs may also show cell cycle arrest 160 , 244 . Several cancer cells exposed to AgNPs undergo sub-G1 arrest and apoptosis. Besides, AgNPs can also reduce distant metastasis by inhibiting tumor cell migration and angiogenesis 28 , 245 . Multiple anticancer mechanisms of AgNPs are described in Figure . In order to develop safe and effective anticancer agent, more mechanisms for anti-cancer effects of AgNPs remain to be explored. Here, we summarize the possible anticancer mechanisms of AgNPs both in vitro and in vivo.

Surface charges participate in the formation of AgNPs surface chemistry, which play an important role in cytotoxicity 231 - 233 . The surface charges of AgNPs determine the binding with serum albumin, as well as the adhesion and uptake of cells 25 . Negatively charged and neutrally charged AgNPs can adhere to cell membranes but internalize in small amounts, while positively charged AgNPs exhibit more efficient cell membrane penetration and internalization 25 . Besides, the positively charged AgNPs tend to stagnate on the surface of the tissue and the lumen of the blood vessels for a long time, which may be beneficial for the targeted delivery of anticancer agents 234 . AgNPs with opposite surface charges exhibit different cytotoxicity in tumor cells. The greater cytotoxicity and more ROS production are observed in tumor cells exposed to high positive charged AgNPs 234 . Nanoparticles exposed to a protein-containing medium are covered with a layer of mixed protein called protein corona 235 . The electrostatic interactions between proteins and nanoparticles contribute to the formation of protein corona 236 . Some proteins may undergo conformational changes during the formation of protein corona 235 . Protein corona has an important effect on the absorption, accumulation and subsequent behaviors of nanoparticles in cells 237 . It is proved that AgNPs with protein coronas enter cells via receptor-mediated endocytosis and subsequently induce mitochondrial dysfunction and cell apoptosis 238 . By comparing nanoparticles without protein coronas, it is realized that the formation of protein coronas around AgNPs can be a prerequisite for their cytotoxicity.

The applications of AgNPs can be extended by tailoring the shape of nanoparticle, which may optimize the physicochemical and biological properties of AgNPs 26 , 221 . The shape-controlled AgNPs can be obtained by changing the parameters in different synthesis methods. Though AgNPs with various shapes are prepared, such as sphere, triangle, cuboid, rod, tube, disk and wire, only a few among these are chosen for anticancer therapy. The cellular uptakes of AgNPs, as well as particle-to-cell or particle-to-protein interactions, are partly dependent on the shape of nanoparticles 216 , 222 . In general, spherical AgNPs may display stronger endocytosis and more active anticancer effect than other shapes. Because it is more efficient for spherical AgNPs than non-spherical nanoparticles to pass through vascular endothelium, and their higher specific surface area is more beneficial for them to enter cancer cells 216 , 222 . In addition, the active or weak endocytosis may be related to the different membrane bending energies of various shaped AgNPs. Ying Li et al. 223 compared the internalization rates of spherical-, cubic-, disk- and rod-shaped nanoparticles to find out the shape effect on endocytosis. They realized that the spherical nanoparticles exhibited the fastest internalization rate, followed by the cubic nanoparticles, while the disk- and rod-shaped nanoparticles exhibited the slowest internalization rate. After analyzing the free energies of four shaped nanoparticles, they speculated that the membrane bending energy of nanoparticles during endocytosis might be the main factor inducing the shape effect of the nanoparticles. Among these four shaped nanoparticles, compared with the non-spherical, the spherical nanoparticles only needed to overcome a minimal membrane bending energy barrier, while the disk shaped nanoparticles faced a larger free energy barrier caused by stronger membrane deformation. In order to understand the effect of more complex shaped particles on cellular uptake, Yuanzu He et al. 224 treated LnCAP cells with particles of different keyboard character shapes and compared the cell endocytosis. Compared with shapes without sharp features, like number 0, letter O and pound key, the rod-like microparticles, such as number 1, letter I, and arrow key, were more likely to adhere, penetrate and enter the cancer cells. The results explained that the shapes of microparticles with sharper angular features and higher aspect ratio might have a higher chance to contact and be internalized by cancer cells.

Although 50 nm, 70 nm and 80 nm nanoparticles can effectively penetrate both, 100 nm nanoparticles cannot penetrate normal fenestrations, suggesting that there might be an optimal size for effective leakage of nanoparticles from the microvasculature into the tissue. Rona et al. 41 demonstrated that size of AgNPs could influence cellular uptake and toxicity. Smaller particles (10 nm, 20 nm) easily penetrate LoVo cells and then significantly increase intracellular ROS levels, while larger particles (100 nm) appeared mainly on the cell surface. Alicia et al. 220 also found that smaller AgNPs were more cytotoxic than larger AgNPs when studying the therapeutic effects of AgNPs on human hepatoma and leukemia. Our team 21 used an evaporation-condensation system to obtain silver particles approaching the Ångstrom dimension. By comparing AgNPs with larger size, we found Ångstrom-scale silver particles had greater cytotoxicity to tumor cells, but did not induce notable toxicity on normal tissues.

Nanoparticles motility in capillaries, as well as endocytosis and metabolism in tumor cells, are significantly affected by the size of AgNPs 217 , 218 . It has been found that the kinetics of uptake, intracellular accumulation and excretion, and the resulting cytotoxicity and genotoxicity, varied with the different sized AgNPs. In general, smaller AgNPs have higher endocytosis and exocytosis efficiency, therefore are supposed to produce greater cytotoxicity than larger particles 49 , 217 . To investigate the effect of nanoparticle size on distribution within tumor, Gavin Fullstone et al. 219 simulated the transport of nanoparticles in blood flow using an agent-based approach, testing the ability of 10 nm, 20 nm, 50 nm, 70 nm, 80 nm, 100 nm and 160 nm nanoparticles to traverse fenestrations with pore size of normal blood vessels and tumor-associated blood vessels.

AgNPs have been observed to exhibit good anticancer activities in breast cancer 158 , cervical cancer 208 , colon cancer 209 , ovarian cancer 210 , pancreatic ductal adenocarcinoma 211 , lung cancer 212 , hepatocellular carcinoma 213 , melanoma 214 , osteosarcoma 215 , etc. ( Table ). Several studies confirm that the anticancer activities of AgNPs with various sizes, shapes and doses/concentrations are discrepant in different cancer cells 210 - 212 , 215 . In addition, other factors, such as pH of lesions, exposure time, cell lines and tumor microenvironment, also influence the anticancer activity of AgNPs 210 , 211 , 214 . Generally speaking, AgNPs exhibit wide spectrum anticancer activity in size-, dose-/concentration- and time-dependent manners. AgNPs with smaller size can elicit enhanced endocytosis, and induce more significant cytotoxicity and genotoxicity. Compared with other shapes, spherical AgNPs exhibit better cytotoxicity due to the higher surface-to-volume ratio 216 . And higher dose of AgNPs usually leads to more apoptosis than lower one. In this section, we highlight these factors.

Cancer is currently considered an important factor in morbidity and mortality worldwide 201 . About 14 million new cancer cases are predicted by 2035, which will lead to a substantial impact on the economy and society around the world 202 . Therefore, there is an urgent need to develop effective and advanced treatment methods to reduce the adverse effects of cancer incidence. Common treatments of cancer or tumor include surgery, chemotherapy and radiotherapy. However, side effects and limitations of conventional treatments influence the outcomes. For example, standard chemotherapy may cause serious side effects, including local reactions, such as thrombophlebitis and tissue necrosis, and systemic reactions, including myelosuppression, dysfunction of liver and kidney and immunosuppression 203 . In addition, malignant tumors can develop multi-drug resistance (MDR), which may lead to chemotherapy failure 204 . Therefore, it is essential to develop novel drugs to improve the therapeutic effects. In recent years, nanoparticles have attracted more attention in cancer therapeutics due to their special physical and chemical properties, which gives rise to a new field of anticancer—cancer nanomedicine 205 , 206 . Compared to traditional anticancer agents, metallic nanoparticles (MNPs) can be used as novel therapeutic agents or drug carriers in combination with drug candidates, and undesirable side-effects can be prevented by providing a targeted approach 207 . Among these nanoparticles, AgNPs represent an ideal one in the search for anticancer or antitumor therapeutic agents 207 .

Worm infection via contact with contaminated soil is one of the most common diseases among children from middle and low-income countries 193 . Worm infections often lead to stunted growth, malnutrition and lagging academic performance 193 , 194 . According to recent studies, AgNPs may become a candidate as a novel insecticide. Saha et al. 195 confirmed that AgNPs were effective in killing filaria and larvae. AgNPs induced the cell apoptosis and destroyed parasites mainly through the generation of ROS. It was worth noting that the carbohydrate polymer not only participated in the synthesis of AgNPs, but also enhanced the filaricide activity of AgNPs. This suggested that AgNPs may be a potential preparation for filariasis control. In addition, they also tried to use AgNPs synthesized by Acacia auriculiformis to kill filaria, and also achieved impressive results 196 . Tomar et al. 197 realized the biologically synthesized AgNPs might inhibit both egg hatch and adult motility in dose-dependent manner. That was, a higher dose of AgNPs might exhibit better anthelmintic activity. Shabad et al. 198 confirmed the AgNPs synthesized by Ziziphus jujuba leaf extract showed ideal ovicidal and anthelmintic activity against Haemonchus contortus via nutrient depletion. The combination of AgNPs and organic components separated from plants can produce a synergistic effect which may enhance anthelmintic activity. Mamun et al. 199 speculated that organic substances in M. charantia fruit extracts, such as glycosides, alkaloids, reducing sugars and free acids, can help biosynthetic AgNPs to protect against parasitic infections. The phytochemicals might exert effect by adhering to the gastrointestinal tract or parasite cuticles. AgNPs exhibited larvicidal activities against larvae of Anopheles stephensi and Culex quinquefasciatus, thus contributed to the prevention of malaria and filariasis 200 . In conclusion, AgNPs may be used as an effective insecticidal agent to kill eggs, larvae and adult parasites. However, the mechanisms still need to be further explored.

The antimicrobial effect of AgNPs has been widely studied, and the mechanisms are being explored. It is observed that AgNPs can anchor and then penetrate the bacterial membrane, and subsequently trigger the destruction of cell membrane and leakage of content 187 . Besides, AgNPs can influence crucial intracellular activities, such as attacking the respiratory chain, disturbing DNA replication and inhibiting cell division 188 . The antibacterial mechanisms of AgNPs are illustrated in Figure . AgNPs also have a good microbicidal effect in drug-resistant fungi via influencing the cellular targets, which are involved in the drug resistance and pathogenicity. For example, Venkatraman et al. 189 demonstrated that AgNPs could affect drug sensitivities by acting on multiple cellular targets of Candida albicans, including fatty acids like oleic acid, which were important in the hyphal morphogenesis involved in the pathogenicity. Some studies speculate that AgNPs can saturate and adhere to the fungal hypha and eventually inactivate the fungus 180 . The antiviral mechanism of AgNPs has also been deeply explored. AgNPs can be used to prevent viral infection against several virus by blocking virus contact with cells and entry steps, or directly inactivating the virus, including herpes simplex virus (HSV), human parainfluenza virus 3, vaccinia virus, chikungunya virus and respiratory syncytial virus 182 , 190 - 192 . These studies indicate that AgNPs can be used as a novel promising virucide agent. In order to develop safe and effective antimicrobial agents, the yet-to-be-determined mechanisms of antimicrobial properties of AgNPs are needed to be further studied.

AgNPs have been proven to effectively inhibit various pathogenic bacteria, fungi and viruses, including Staphylococcus aureus 164 , Escherichia coli 165 , Pseudomonas aeruginosa 166 , dermatophyte 167 , HIV-1, etc. 168 , 169 . The antibacterial effect of AgNPs against various strains of bacteria is different 156 . Rather than Gram-positive bacteria, AgNPs show a stronger effect on the Gram-negative ones. This may be due to the different thickness of cell wall between two kinds of bacteria 170 . Besides the bacteria strains, AgNPs may also exhibit size-, shape-, concentration-, time-, and charge-dependent antibacterial activity. In general, as particle size decreases, the antibacterial effect of AgNPs increases significantly 171 . Especially when the size is less than 10 nm, AgNPs show better antibacterial activity 172 . The antibacterial effect can be significantly enhanced by prolonging the treatment time of AgNPs 173 . The increased bacterial mortality may be ascribed to the accumulation of AgNPs and silver ions during the exposure period. Besides, the shape of AgNPs may also influence the antibacterial activity 171 , 174 . By comparing the antibacterial activity of spherical, triangular, linear and cubic AgNPs, it is observed that spherical shaped AgNPs exhibit superior antibacterial effect. This phenomenon suggests that AgNPs with larger surface to volume ratio, which relates to both higher effective contact and larger reaction surface, may show stronger antibacterial activity 174 . In addition, the antimicrobial activity of AgNPs is also affected by the surface charge 156 , 175 . Due to the presence of lipopolysaccharide, peptidoglycan and multiple groups, including carboxyl, amino and phosphate groups, bacterial membranes are primarily loaded with negative charges 170 , 176 . Positive charge can facilitate the adherence of AgNPs on bacterial membranes through electrostatic attraction 156 . Therefore, adjusting the surface charges of AgNPs may contribute to the enhanced antibacterial effect 175 . The stabilizers may influence the size, dispersion, and surface charge of AgNPs, which may involve in the antibacterial effect of AgNPs 154 , 177 . Some stabilizers, such as citrates, PVP 154 and polyvinylalcohol 177 , have been shown to influence the bacterial effect by adjusting the characteristics of AgNPs.

Antimicrobial and anticancer properties of AgNPs have been widely studied. Studies have shown that AgNPs have broad-spectrum antimicrobial properties against pathogens including bacteria, fungi and viruses 19 , 49 . Besides, AgNPs can effectively damage or kill nematodes 152 and worms 153 . A variety of factors affect the antimicrobial activities of AgNPs, including size, shape, dose and stabilizer of AgNPs 49 , 154 , 155 . Interestingly, AgNPs may have different antibacterial effects against Gram-positive and Gram-negative bacteria 156 . AgNPs exhibit broad-spectrum anticancer properties. Anticancer activity of AgNPs is also affected by a variety of factors, including size, shape, dose, and exposure time 22 , 157 , 158 . It is also realized that the surface charge of AgNPs is a potential factor. Although current specific mechanisms of antimicrobial and anticancer properties of AgNPs are still unclear, many studies have carried out hypothesis. AgNPs can inhibit the growth of bacteria or kill them by inducing membrane destruction, ROS generation, DNA damage, enzyme inactivation and protein denaturation 4 , 56 , 159 . However, the anticancer mechanisms of AgNPs are much more complicated. Until now, it has been approved that AgNPs can inhibit the growth of tumor cells by destroying the cellular ultrastructures, inducing ROS production and DNA damage 21 - 23 , 160 . In addition, AgNPs can induce tumor cell apoptosis through inactivating proteins and regulating signaling pathways, or blocking tumor cell metastasis by inhibiting angiogenesis within lesion 31 , 161 . Besides antimicrobial and anticancer properties, AgNPs can also be used in other medical applications, such as bone repair 162 and wounding repair 163 . And AgNPs can be regarded as an additive in dental materials or an adjuvant in vaccine. In this part, we will discuss the antimicrobial and anticancer properties and possible mechanisms of AgNPs, as well as other promising medical applications.

Potential Toxicity of AgNPs

The potential harm of nanomaterials to organs and systems in the body has been gradually observed 310-312, which may influence the biomedical application of nanomaterial. Therefore, it is necessary to review the dynamics of AgNPs in vivo. AgNPs can be taken and distributed to different organs through a variety of routes of administration, mainly include inhalation, ingestion, skin contact, and subcutaneous or intravenous injection (Figure ). The absorbed AgNPs are distributed in many systems 310, 311, such as the dermis, respiratory, spleen, digestive, urinary, nervous, immune and reproductive system, and mainly distributed in the spleen, liver, kidney and lung, while little deposition of AgNPs is observed in teeth and bones. The small-sized AgNPs are easy to penetrate the body and cross biological barriers like the blood-brain barrier and the blood-testis barrier, and subsequently induce potential cytotoxicity. Besides the directly exposed tissues, AgNPs can also be transported to different organs via blood circulation. Therefore, the non-specific distribution of AgNPs may produce cytotoxicities such as dermal toxicity, ocular toxicity, respiratory toxicity, hepatobiliary toxicity, neurotoxicity and reproductive toxicity, which limit the applications of AgNPs. The potential cytotoxicity of AgNPs depends on the routes of administration and the properties or characteristics of the AgNPs, such as the size, shape, and concentration. At the cellular level, Wang et al. 313 used TEM and integrating synchrotron radiation-beam transmission X-ray microscopy (SR-TXM) with 3D tomographic imaging to capture the information of the cellular uptake, accumulation, degradation, chemical transformation, and removal of AgNPs. The experiment revealed that the cytotoxicity was caused by the chemical transformation of AgNPs, ie. Ag0 transformed into Ag+, Ag-O-, and Ag-S- species, which might induce the cellular biochemical changes. However, there is still inadequate acknowledge of the potential cytotoxicity, long-term adverse health effects, and the specific mechanisms of AgNPs accumulated in the different tissues and organs. In order to develop AgNPs with better biocompatibility for medical applications, it is urgent to systematically study their potential cytotoxicity. This chapter provides a brief overview of the potential toxicity and possible mechanisms of AgNPs in different organs, including skin, eye, kidney, respiratory system, hepatobiliary system, central nervous system, immune system and reproductive system (Table ).

Table 6

ObjectsExposureToxicityReferencesAnimal modelIn vitro/vivoCell lines/TissuesSize; ShapeDosagesRouteTimeEffectToxicity mannersPigIn vitro and vivoHEKs and porcine skin20, 50 and 80 nm0.34, 1.7 μg/mLIncubation; skin contactAcute: 18 and 24 h;
chronic: 14 dFocal inflammationDose-dependent350MiceIn vivoLiverLess than 30 nm10 ppmSkin contact2, 7 and 14 dCentral venous dilation; hyperemia, cell swelling, Kupffer and inflammatory cells increaseTime-dependent332MiceIn vivoSpleen, liver, lung and kidney12-20 nm7.5, 30 and 120 mg/kgIntravenous administration7 and 14 dInduction of inflammatory reactions in lung and liver cellsGender-, concentration- and time-dependent346MiceIn vivoLung10-20 nm; spherical10, 100, 1000 and 10,000 ppmIntratracheally administration1, 3, 7 and 15 dAcute lung inflammation and bronchitis; hyperplasia of alveolar epithelial cellsDose-dependent330MiceIn vivoLiver, spleen, kidneys, heart, lungs, testes, stomach, intestine and seminal vesicles3±1.57 nm; spherical11.4-13.3 mg/kgIntravenous injection1, 28 dInflammatory response; alteration of hematological factors; change of gene expression; ROS generationDose-dependent351MiceIn vivoLiver, kidneys and lung10, 75 and 110 nm; spherical25 μg/miceIntravenous administration1, 3 and 7 dPeripheral inflammation in liver, kidneys and lungsTime-, concentration- and size-dependent343MiceIn vivoLung20 and 110 nm0.05, 0.15, 0.45 and 1.35 mg/kgIntratracheal instillation1, 7 and 21 dAlter SP-D level; deficit immune defense functionSize- and stabilization-dependent44MiceIn vivoBrain, lung, liver, kidney and testis22, 42 and 71 nm0.25 mg/kg, 0.50 mg/kg, 1.00 mg/kgOral administration14 and 28 dInduce organ toxicity and inflammatory responsesDose-dependent352MiceIn vivoheart, lung, kidney, liver and blood1.4-250nm11.4-13.3mg/kg body weightIntravenous administration28 dInduce gene expression; ROS generation; apoptosisDose-dependent351MiceIn vitro and in vivoA549, BxPC-3; PC-3; Hep G2, CNE; AsPC-1; U-87 MG; SW480; EC109; MDA-MB-231; VSMC; HMEC; LO2; 293FT; tumor, brain, heart, kidney, lung, spleen, and liver19.2±3.8 Ång, spherical or ellipsoidal0-32ng/µl, 1.875 mg/kgIntravenous administrationAcute: 24 h; chronic: 28 dNoneDose- and time-dependent21Micein vivoLung20 and 110 nm, spherical0.1, 0.5 and 1.0 mg/kgInhalationAcute: 40 h; chronic: 21 dPulmonary fibrosisSize- and coating-dependent353Micein vivoKidney, liver and spleen2.45-19.53 nm0.37, 0.65, 13 and 21 mg/kgOral administration27 dTissue destruction; cell necrosis and apoptosisdose-dependent354RatIn vivoBrain>100 nm5 and 50 mg/kgOral administration79 dCell death, disturbed neurotransmitter and cytokine production, ROS generation--355RatIn vivoSperm and testicular tissue60-80 nm30, 125 and 300 mg/kgIntraperitoneal injection28 dDecrease normal sperm morphology, sperm vitality and sperm countDose-dependent348RatIn vivoKidneys, liver and blood20-65 nm2,000 mg/kgIntraperitoneal administration3 dLiver and kidney damageTime- and dose-dependent356RatIn vivoLung, spleen, liver, kidney, thymus and heart6.3-629 nm0.5 mg/kgIntravenous administration24 hLiver and kidney damage; chromosome breakage; genotoxicityDose-dependent357RatIn vivoEpididymal sperm20-30 nm50, 100 and 200 mg/kgOral administration90 dSperm anomalies; decrease sperm viabilityDose-dependent37RatIn vivoBrain3-10 nm, spherical1 and 10 mg/kgIntragastric administration14 dNeuron shrinkage; cytoplasmic or foot swelling of astrocytesDose-dependent38RatsIn vivoKidney, liver and blood20-60 nm, spherical2,000 mg/kg bw, twice injectionsIntraperitoneal injection5 dLiver and kidney damage; blood parameters disruptDose- and time-dependent356RatIn vivoSpleen, liver, and lymph nodes and blood20 nm and 100 nm6 mg/kgIntravenous administration28 dSuppression of the natural killer cell activity; stimulate LPS mitogen; increase cytokine productionDose-dependent358RatIn vivoLiver and kidney56 nm30, 125 and 500 mg/kgOral administration90 dLiver damage; bile-duct hyperplasiaDose- and gender-dependent341RatIn vivoKidney52.7-70.9 nm10 ml/kgOral administration90 dDeposite in kidneysDose-dependent340Female ICR mice; male guinea pigsIn vivoOral, skin and eye10-20 nm, spherical5,000 mg/kg (oral);
50 and 5,000 ppm (eye);
50 and 100,000 ppm (skin)Oral administration;
eye drops;
skin contact1, 2 and 3 dayConjunctivae irritation—326Male ICR miceIn vivoBlood, liver, spleen, kidney, lungs and brain10, 40 and 100 nm, spherical10 mg/kgIntravenous injection24 hBleeding or necrosis of multiple internal organsSize- and tissue-dependent331BN and SD ratsIn vivoLung20, 110 nm, spherical0.1 mg/kg or 90 breaths/minuteIntratracheal administration1, 7 and 21 dLung eosinophilia and bronchial hyperresponsiveness; distruction of blood/alveolar epithelial permeability barrierDose- and size-dependent; rat strains related46Mice and guinea pigsin vivoLung, lymph node, heart, liver and kidney10-20 nm, spherical5,000 mg/kg, 5000 ppmOral administration, eye and skin contact14 dNo mortality and toxic signs326Freshwater fishIn vivoEmbryo25.9-36.7 nm, sphericalAcute: 0.3, 0.6, 1.2, 2.4 and 4.8 mg/L; subchronic: 0.05, 0.1, 0.25 and 0.5 mg/LIncubation14 dLiver damage; deplete glutathione; deactivate lactate dehydrogenase and antioxidant enzymesTime- and dose-dependent39Japanese medakain vivoEmbryo20-37 nm, spherical0, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/LOral administration48 hdeathdose-dependent327ZebrafishIn vivoEmbryo20 and 110 nm, spherical0.08, 0.4, 2, 1,0 and 50 mg/LHatch5 dMultiple developmental abnormalitiesSize- and surface coating-dependent359ZebrafifishIn vitro and in vivoBrain, heart, yolk and blood of embryo5-20 nm5, 10, 25, 50 and 100 µg/mLHatch24, 48 and 72 hMultiple developmental abnormalitiesConcentration-dependent43Drosophila melanogasterin vivoParents, egg and offspring2-20 nm10, 20, 30, 40, 50 and 100 mg/LOral administration5,10, 15, 30, 45, 60 and 95 minAbdominal pigmentationDose-dependent360Drosophila melanogasterIn vivoGermline stem cell; testis20 nm2, 3.5 and 5 mg/LOral administration24h, 5 dDelay the development of the F1 offsprings; ROS generation; premature GSC differentiationDose-dependent42Caenorhabditis elegansIn vivoThe worms' body96.4±35.6 nm, spherical0-1mg/LCulture6 and 24 hDNA damage, ROS generation, inhibition of growthDose- and time-dependent361Caenorhabditis elegansIn vitroThe worms' body< 100 nm, spherical0.025, 0.05 and 0.075 µg/mLCulture24 hROS generation; DNA damageSize-dependent152—In vitrorat brain microvascular endothelial cells, pericytes, and astrocytes7±2 nm1 and 10 μg/mLIncubation24 hTrx system, Nr4a1 and Dusp1 regulaion , inflammation and apoptosisDose-dependent338—In vitroMouse ESCs20.2 ±4.1 nm, spherical5.0 µg/ml—24 hHeat shock protein and the metallothionein families regulation, induce oxidative stress and apoptosisDose-dependent362—In vitroMouse microglia N9 cell line, N27 neuronal cells49.7±10.5 nm, spherical50 μg/mLIncubation24 hNitric oxide and TNFα productionDose-dependent363—In vitroMouse lymphoma cell line, human lymphoblastoid cells20, 50 and 100 nm, spherical0-400 μg/mLIncubation4, 8, and 24 hDNA mutantsSize-, concentration- and coating-dependent47—In vitroRat primary cerebral astrocytes24.18±4.14 nm, spherical0.01, 0.1, 1 and 10 mg/mLIncubation24 hNeuroinflammation and apoptosis; increase caspase activitiesDose-dependent337—In vitroPrimary astrocyte cell, rat glioma C6 cell line6.9-8.7 nm, spherical0.1, 1, 10, 50, 75 and 100 µg/mLIncubation24 hNecrosis and apoptosisDose-dependent364—In vitroMurine brain ALT astrocytes, murine microglial BV-2 cells and mouse neuroblastoma Neuro-2a (N2a) cells3-5 nm0.5, 1, 5, 10 and 12.5µg/mLIncubation24 hCytokine secretion, Aβ amyloid deposition, inflflammatory responseDose-dependent339—In vitroUMR-1066 nm, cubic10, 25, 50, 100, 150 and 225 μMIncubation24hDecrease lysosomal and mitochondrial activityDose-dependent365—In vitroU937 cell4,20 and 70 nm, round1.56, 3.12, 6.25, 12.5, 25 and 50 µg/mLIncubation24 hOxidative stress; cytokines releaseSize-dependent50—In vitroHepG2 cell line20 nm2.5 to 50 µg/cm3Incubation24 hEndogenous antioxidant defence regulationDose-dependent366—In vitroJurkat T, NCI-H460, HeLa cells, HepG2, MCF-7, Beas-2B5-10 nm0.2, 0.5 and 1 mg/LIncubation4, 12 and 24 hDNA damage; p38 MAPK activation; cell arrest; apoptosisTime- and concentration- dependent27—In vivoHuman lymphocytes and sperms8-10 nmDensity gradient: 1:9, 1:3, 1:1—30 and 60 minsCell viability decreaseConcentration- and time-dependent367—In vitroPk1561.2±33.9 nm, nonuniform50 mg/LIncubation24 and 48 hGenotoxicity in Pk15 cellsDose-dependent342—In vitroZebrafish ovarian follicle cells30-55 nm30 μg/mLIncubation2 hApoptosis of ovarian follicle cells; germinal vesicle breakdownConcentration-dependent349Open in a separate window

Skin Toxicity

Even as early as in 1614, Angelo Sala reported the first case of a definitive diagnosis of argyria, a kind of disease induced by the deposition of silver in tissues 314. Since the mid-19th century, it has been recognized that silver or silver compounds may induce some tissues to turn gray or blue-grey, especially involving the skin. The skin, as the largest organ and the first-line barrier of the human body, can isolate the external pathogens from the internal environment. Topically applied AgNPs may induce cytotoxicity in the site and penetrate the skin and subsequently access the systemic circulation followed by adverse effects on other organs. For example, applying AgNPs gel and covering dressings will allow particles to penetrate and accumulate in the skin and produce potential cytotoxicity 315. Before AgNPs, there are several reports on the skin toxicity of elemental silver, known as Argyria 316. Argyria is a disease characterized by permanent gray-blue pigmentation of mucous membranes, eyes and skin, occurring in individuals exposed to high concentrations of silver for a long period. G D DiVincenzo et al. 317 previously reported that the skin of workers exposed to silver aerosols showed a distinctive gray bluish hue change, and deposited silver was also detected in worker's hair, urine and feces. Jennifer et al. 318 reported a Argyria case. The patient showed uniform accumulation of silver on the skin after long-term consumption of silver solution. Current studies show that AgNPs can enter the hair follicles to induce local deposition and deposit into the subcutaneous structure by penetration pathways. The follicular penetration pathway is most commonly used to explain the penetration of particles into the skin 319-322. Yu Kyung Tak et al. observed that AgNPs of different shapes would remain at different layers of skin. Rod-shaped, spherical, and triangular AgNPs penetrated the dermis, epidermis and stratum corneum layers, respectively. They observed the behavior of AgNPs in subcutaneous capillaries. And prolonged exposure time would increase the amount of nanoparticles. Notably, they found that the penetration of AgNPs was achieved by the follicular penetration pathway and intercellular penetration pathway 323. Francesca et al. 324 attempted to use AgNPs to act on intact or damaged skin. They demonstrated a significantly higher penetration of AgNPs used on damaged skin as compared with intact skin, and they speculated that a small part of the particles would diffuse into the skin as silver ions. Radoslaw et al. 325 explored the cytotoxicity of AgNPs on epidermal keratinocytes (NHEK). The results showed that AgNPs inhibited cell proliferation and migration, induced activation of caspase 3 and caspase 7, and damaged DNA. In addition, by measuring the ATP content of cells treated with different concentrations (6.25, 12.5, 25 and 50 μg/ml), it was found that a high concentration of AgNPs significantly decreased the ATP production, and this phenomenon worsened with prolonging exposure time.

Eye Toxicity

AgNPs agent may cause concentration-dependent acute conjunctival irritation, but there is still no reliable evidence for toxicological effects. Pattwat et al. 326 dripped 50 ppm and 2,5000 ppm colloidal AgNPs into the eyes of guinea pig and explored whether there were acute eye irritation or corrosion throughout the 78 hours observation period. Although transient mild conjunctival irritation, i.e. blood vessel hyperemia in conjunctivae, was observed within 24 hours after 5000 ppm AgNPs treatment, neither low-dose nor high-dose colloidal AgNPs caused any acute toxicological effects in guinea pigs. AgNPs may have developmental toxicity in the eyes of early-stage individuals, which can eventually result in multiple types of eye defects. Yuan Wu et al. 327 studied the developmental toxicity of AgNPs by using Japanese medaka at early-life stages as experimental models, including embryonic, larval and juvenile stages. The Japanese medaka was exposed to 100-1000 mg/mL AgNPs for 70 days and various morphological malformations were described and analyzed, such as edema, visceral deformities, heart malformations, spinal abnormality, especially eye defects. AgNPs-treated group showed different eye defects, such as microphthalmia, exophthalmia, cyclopia and anophthalmia. Histopathological examinations of 2-day-old larvae showed increased thickness of retinal pigment epithelium and missing of the retina in inner segments. Interestingly, comparing with the high-dose groups, the types and numbers of eye malformations in the low-dose groups were significantly higher. These morphological abnormalities and non-linear dose-response pattern suggest that the developmental toxicity of AgNPs may exhibit complex toxicological mechanisms.

Respiratory toxicity

AgNPs can induce acute lung toxicity and therefore impair lung function, and the damage severity is related to particle accumulation and clearance. Akinori 328 et al. studied the pulmonary toxicity of nanometer particles in mouse models. Ultrafine particles may pass the air-blood barrier through the gap between alveolar epithelial cells, induce vacuolation and necrosis of bronchiolar epithelial cells, resulting in transient acute lung inflammation and tissue damage. The oxidative stress and apoptosis induced by ultrafine particles may contribute to lung damage. In addition, nanoparticles showed size-dependent pulmonary toxicity, i.e. the particles in smaller size exhibit higher capacity for inducing lung inflammation and tissue damage than larger size 36, 329. On the other hand, AgNPs may induce dose-dependent lung toxicity. Kaewamatawong et al. 330 demonstrated dose-dependent acute lung toxicity in mice induced by AgNPs using a single intratracheal instillation of 0, 10, 100, 1000 or 10000 ppm of colloidal AgNPs. And they observed moderate to severe bronchitis and multifocal alveolitis in 100, 1000 and 10,000 ppm AgNPs treated groups. Proinflammatory cytokines such as IL-1β and TNF-α released by alveolar macrophages and airway epithelial cells might involve in the inflammatory lesions in mice. The aggregation of AgNPs had a direct effect on the basement membrane, and disrupted equilibrium between the synthesis and degradation of the extracellular matrix, thus may cause pulmonary fibrosis. Similarly, they also speculated that AgNPs induced oxidative stress in the lung. Furthermore, they recognized that metallothionein (MT) expression induced by AgNPs might be regarded as one of the possible protective mechanisms of lung. Different concentrations of AgNPs, which induce lung damage, may also accumulate in peripheral organs and cause potential health risks. Joanna et al. 46 found that AgNPs disrupted the blood/alveolar epithelial permeability barrier, elicited oxidative stress, activated eosinophils and promoted the release of multiple cytokines. Most importantly, their results showed that AgNPs induced eosinophilic and neutrophilic inflammation, which was an important pathological change in asthma. This might suggest that exposure to AgNPs could trigger asthma.

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Hepatobiliary System Toxicity

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Part of the ingested nanoparticles tend to be sequestered, degraded and accumulated in the liver, which means the liver may be responsible for the metabolism of nanoparticles as well as one of the most frequently attacked organs. On the other hand, the gallbladder collects, stores and excretes bile or biological waste to the intestine. Various metal nanoparticles, including AgNPs, are recognized to be exported from the liver through this pathway. Therefore, hepatocytes are widely studied in the liver toxicity of AgNPs. Maglie et al. 48 found that AgNPs induced severe hepatobiliary damage in mice, including significant hepatocyte necrosis and gallbladder hemorrhage. In this study, AgNPs exhibited size- and dose-dependent hepatobiliary toxicity, i.e. AgNPs in smaller size produced more serious toxic effects, and higher dose of AgNPs induced severer hepatobiliary damages. Camilla et al. 331 observed multi-system acute toxicities in mice with a single intravenous injection of AgNPs. First of all, significant hepatobiliary damages were recognized, including hepatocyte necrosis, micro-hemorrhage around the biliary tract, and portal vein injury. Secondly, they also observed that AgNPs could induce acute tubular necrosis and apoptosis, and moderate splenomegaly. The results of Mohammad et al. 332 showed that AgNPs penetrating via the skin induced time-dependent liver damage such as hyperemia, dilatation in central venous, swelling hepatocytes and increased inflammatory cells. Besides hepatocytes, Kupffer cells (KCs) are also responsible for the removal of AgNPs 333. KCs are macrophages that reside in the hepatic sinusoids and have the active ability of phagocytosis, maintaining the normal immune response and removing nanoparticles from organisms 333, 334. Therefore, KCs become the focus of research on liver toxicity and metabolism of AgNPs.

Central Nervous System Toxicity

The central nervous system consists of two parts: the brain and the spinal cord. Lots of supporting non-nervous cells, i.e. neuroglial cells fill the interneuronal space within the central nervous system. In recent years, some articles point out that AgNPs may penetrate the brain and subsequently induce neuronal death. Due to the limited self-repairing ability of nerve cells, the potential neurotoxicity of AgNPs is receiving more attention. Different exposure patterns can lead to the accumulation of AgNPs in the brain. Injected AgNPs cross blood-brain barrier (BBB) and then penetrate the brain, while inhaled AgNPs reach the central nervous system through the olfactory and/or BBB 335, 336. Due to the unique physicochemical properties of AgNPs, deposited AgNPs in nerve cells, astrocytes and extravascular lymphocytes may cause and aggravate neurotoxicity and inflammation, and increase the permeability of BBB. In the study of the cytotoxicity of AgNPs on rat cerebral astrocytes, Cheng et al. 337 investigated the neurotoxicological effects of AgNPs and Ag+ and compared the mechanisms. Both AgNPs and Ag+ exposure could internalize silver in astrocytes in dose- and time-dependent manners. The AgNPs had higher bioaccumulation ability than Ag+ after 24 h treatment. After the same treatment time, they found that AgNPs might induce intracellular ROS generation in rat cerebral astrocytes and caused cell apoptosis, however, there were undetectable alterations in Ag+ group. More importantly, they confirmed that AgNPs could increase the level of phosphorylated JNK, a kind of kinase involved in mediating apoptosis. The non-cytotoxic dose of AgNPs, rather than Ag+, might induce neuroinflammation by promoting the secretion of multiple cytokines of astrocytes, including CINC-2a/b, CINC-3, IL-10, IP-10, L-selectin and thymus chemokine. Liming et al. 38 investigated the neurotoxicity of AgNPs in the rat after intragastric administration of low-dose (1 mg/kg, body weight) or high-dose (10 mg/kg, body weight) for two weeks. They observed a variety of cell morphological changes in the nervous system, including neuron shrinkage, astrocytes swelling and extravascular lymphocytes. They also observed significantly increased inflammatory factors such as IL-4 in the serum. These data supported the proinflammatory effects of AgNPs in the nervous system. Then they focused on the possible mechanisms for AgNPs or Ag+ transporting across the blood-brain barrier. AgNPs or released Ag+ might cross through the blood-brain barrier via ionic pores or channels and subsequently damage the nerve cells. Besides, AgNPs could enter the central nervous system via vesicular transport of endothelial cells and subsequently induced neuroinflammation. At the same time, they demonstrated the increased permeability of the blood-brain barrier in a rat model after AgNPs treatment. They also observed that AgNPs might inhibit the antioxidant defense of astrocytes by increasing thioredoxin interacting protein, thus lead to the central neurotoxicity. AgNPs might induce ROS, inflammation and apoptosis through regulating the MAPK pathway, mTOR activity and Bcl-2 expression in astrocytes. AgNPs could cause severe ultrastructural changes in astrocytes, including mitochondrial contraction, endoplasmic reticulum expansion and nuclear atypia. Furthermore, AgNPs regulated the expression of multiple genes, inhibited metabolic and biosynthetic processes, thus affect astrocytes function and increase the neurotoxicity. More importantly, the impairing of learning, memory and cognition processes by AgNPs reduced the learning ability and cognition function of rats 338. AgNPs may induce neurological diseases such as Alzheimer's disease by altering gene expression. Chin et al. 339 reported that AgNPs could induce the expression of amyloid precursor protein (APP) gene in nerve cells. APP gene promoted the deposition of amyloid-β (Aβ) protein, a key pathological feature of Alzheimer's disease.

Kidney Toxicity

The kidney participates in balancing body fluid volume and pH, regulating osmotic pressure and electrolyte concentration, drug metabolism, and toxic emissions. Abnormal renal function may occur in AgNPs-treated mammalian kidneys. AgNPs exhibits a dose-dependent accumulation in most examined tissues, such as the brain, lung, liver, dermis, blood and testes. However, there is a gender-related difference in silver accumulation in the kidney. Wan et al. 340 observed that female rats treated with AgNPs had a twofold higher concentration of silver in kidneys than male rats. Ag enhancement staining of the kidneys showed that AgNPs preferentially accumulated in the basement membrane of the glomerulus as well as renal tubules, while mildly accumulated in the adrenal capsule and cortex. There were two possible mechanisms of gender difference in the accumulation of AgNPs: the gender difference in the expression of organic cation transporters, and hormonal regulation. Renal metallothionein and zinc-binding protein, a kind of transporter or binding protein molecules in the kidney, might contribute to the silver accumulation. While organic anions secreted by kidneys might influence the clearance and accumulation of silver 341. Mirta et al. 342 studied the uptake mechanism and potential cytotoxicity of AgNPs in porcine kidney (Pk15) cells in vitro. TEM results showed that there were aggregates in the lysosome and early endosomes. In addition to micropinocytosis, as an uptake pattern, clathrin- and caveolin-mediated endocytosis could also be the possible endocytotic mechanisms. AgNPs could decrease the number of viable Pk15 cells in vitro in a dose-dependent manner. Hua et al. 343 studied the distribution, accumulation and potential toxicity of AgNPs in different sizes in liver, lung and kidney of mice. They found that AgNPs could be taken up by vascular endothelial cells, then induced the generation of intracellular ROS and down-regulated the expression of vein endothelial cadherin. Therefore, AgNPs destroyed the conjunction between endothelial cells, allowing AgNPs to cross the endothelial layer and accumulate in organs. Besides, the leaking AgNPs could also result in peripheral inflammation in a size-dependent manner. Mice receiving single or multiple intravenous injections of AgNPs showed basement membrane injury in glomeruli.

Immune System Toxicity

Our immune system, a natural host defense barrier, is composed of immune cells, tissues and organs, can constantly interact with the internal environment and protect us from pathogens in the external environment, and provide the inherent knowledge to separate the friend and foe within our body 344. Seung et al. 345 found that AgNPs inhibited the proliferation and the production of cytokines, including IL-5, INF-γ and TNF-α, and induced cytotoxicity in peripheral blood mononuclear cells in a concentration-dependent manner. AgNPs may deposit in the immune organs and influence the number of immune cells and the production of cytokines. Wim et al. 45 investigated the effects of AgNPs on the immune system of rats by repeated intravenous administration of AgNPs with different sizes (20 nm and 100 nm) for 28 days. They found that AgNPs administered at the maximum dose (6 mg/kg) were still well tolerated by the rats. The size and weight of the spleen increased significantly, possibly due to the increased cell number of T cells and B cells. However, the cytotoxic activity of NK cells in the spleen was almost completely inhibited. For multiple immune-related cytokines in serum, levels of interferon-γ, IL-10, IL-6 and TNF-α declined, while levels of IL-1β, IgM and IgE increased. The number of neutrophilic granulocytes in peripheral blood also increased. Besides, brown and black pigments were observed in histopathological sections of spleen and lymph nodes, indicating the accumulation of AgNPs in these immune organs. This study suggested that the immune system was sensitive to the potential adverse effects of AgNPs. The spleen may be one of the main organs for the accumulation and elimination of AgNPs, and both processes are in a sex-dependent manner. Yuying et al. 346 observed the potential acute toxicity and biokinetics after repeated intravenous administration of AgNPs in mice. During the 14-day observation period, both the survival and behavior of the mice were normal. They found that AgNPs were widely distributed in tissues, especially in the spleen, followed by the liver. The biokinetics of AgNPs in the kidney and lung seem to show gender-related differences, i.e. the accumulation of silver in kidney and lung of female mice was higher than that of male mice, the longer elimination half-life and slower clearance of AgNPs in female mice than male mice. Besides, the KCs in the liver were mainly responsible for the retention and elimination of AgNPs. The silver content in the liver significantly decreased after one day. While in the spleen, the marginal zone and the red pulp macrophages contributed to the clearance of silver.

Reproductive System Toxicity

Biological barriers, such as the blood-testis barrier, placental barrier and epithelial barrier, can protect the reproductive system from infection and toxicity. AgNPs can cross the biological barriers to deposit in reproductive organs including testis, epididymis, ovary and uterus. Thus, AgNPs may damage germ cells and related cells, such as primary and secondary follicles, germline stem cells, Sertoli cells and Leydig cells 42, 347. Besides, AgNPs can also cause changes in sexual behavior by affecting the secretion of hormones within the reproductive organs and glands. Further studies confirmed that the reproductive toxicities of AgNPs are achieved by increasing inflammation, disrupting DNA structure, down-regulating gene expression, decreasing mitochondrial function, inducing ROS production and apoptosis. These toxicities of AgNPs to the reproductive system are size-, time- and dose-dependent 347, 348. Zhang et al. 347 investigated the effects of AgNPs with different sizes (10 nm and 20 nm) on male somatic Leydig cells and Sertoli cells, and found that cell viability was inhibited by AgNPs in size- and concentration-dependent manners. The 10 nm AgNPs showed more cytotoxicity than the 20 nm AgNPs. and cell proliferation was significantly decreased as the concentration of AgNPs increased from 0 to 100 μg/ml. AgNPs-treated Sertoli cells showed decreased mRNA levels of ZO-1 and Cx43, both are involved in encoding tight junction proteins which playing a crucial role in the formation of BTB. As well as AgNPs-treated somatic Leydig cells showed decreased mRNA levels of StAR, 3β-HSD and 17β-HSD, which are involved in the production of testosterone. It is widely acknowledged that spermatogonial stem cells (SSCs) can continuously proliferate, renew and produce sperms throughout male's postnatal life. Cytokines secreted by Sertoli cells play an important role in the proliferation and renewal of SSCs. In this study, AgNPs-treated Leydig cells secreted decreased level of testosterone, which was responsible for inducing spermatogenesis and maintaining normal functions of Sertoli cells. These results suggest that AgNPs can impair the function of Leydig cells and Sertoli cells, then worsen the function of SSCs, ultimately suppress male fertility. Cynthia et al. 42 evaluated the fecundity and development of Drosophila fed with AgNPs at various concentrations from 0 to 5 µg/mL. AgNPs decreased the viability and delayed the development of Drosophila in a dose-dependent manner. Germline stem cells (GSCs) and early germ cells were concentrated at the apical tip of the testis. Among different treated groups, a significantly increased ROS level was observed at this tip area of Drosophila treated with 5 µg/mL AgNPs. They also proved that AgNPs might disrupt GSCs maintenance by triggering precocious differentiation of GSCs, thereby decreased the number of sperms. Besides, the first generation of Drosophila fed with a higher concentration of AgNPs showed delayed eclosion and decreased male offsprings as compared to control or lower concentration group. The mating success of Drosophila and the number of their second or third generations decreased in AgNPs-treated groups than the control group. This might suggest that AgNPs accumulated in GSCs could be passed onto offspring and affect the development and fecundity of the offspring. Lafuente et al. 37 studied parameters of epididymal sperm of rat fed with different doses of PVP-AgNPs (50, 100 and 200 mg/kg/day), including sperm morphology, motility and viability. PVP-AgNPs induced sperm morphology abnormalities in a dose-dependent manner. In their study, 100 mg/kg/day of PVP-AgNPs significantly increased abnormal morphologies in epididymal sperms, such as banana head, tail bending, head loss and neck abnormalities. Abnormal sperm morphology reduced sperm motility and vitality. Some studies focus on the effects of AgNPs on female reproduction. Chen et al. 349 evaluated the potential toxicity of AgNPs and Ag+ on zebrafish oocytes. Vacuolation or swollen mitochondria, and condensed nucleus were observed in AgNPs- and Ag+-treated follicular cells. Zebrafish oocytes treated with AgNPs or Ag+ showed a decreased concentration of cAMP, which plays a key role in the maintenance of meiosis arrest, and results in meiosis resumption and subsequent oocyte maturation. Besides, AgNPs and Ag+ up-regulated caspase 3 and caspase 9, respectively, both of which play important roles in the initiation and execution of apoptosis, ultimately leading to apoptosis in ovarian follicle cells.

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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