Nanosilver in Biomedicine: Advantages and Restrictions

Nanosilver (in a range 1–100 nm) binds with thyol-, amino- and carboxy-groups of aminoacid residues of proteins and nucleic acids, thus providing inactivation of pathogenic multidrug-resistant microorganisms. Besides antibacterial, antiviral, antifungal and anti-cancer properties Ag-based nanomaterials possess anti-inflammatory, anti-angiogenesis and antiplatelet features. Drug efficacy depends on their stability, toxicity and host immune response. Citrate coated Ag nanoparticles (NPs) remain stable colloid solutions in deionized water but not in the presence of ions due to replacement of Ag+ by electrolyte ions, potential formation of insoluble AgCl, subsequent catalyzed oxidative corrosion of Ag and further dissolution of surface layer of Ag2O. Protein shells protect core of AgNPs from oxidation, dissolution, aggregation and provide specific interactions with ligands. These nanoconjugates can be used for immunoassays and diagnostics but the sensitivity threshold does not exceed 10 pg Cytotoxicity of AgNPs conjugated with proteins is associated with the rate of intracellular Ag+ release, a 'Trojan horse' effect, and exceeds one of Ag+ because of endocytosis uptake of NPs but not ions. Relatively toxic nanosilver causes immunosuppression of the majority of cytokines with a few exceptions (IL-1β, G-CSF, MCP-1) whereas AgNO3 additionally activates TNFα and IL8 gene expression.

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Introduction

Nanosilver refers to nanoscale Ag materials that have at least one dimension less than 100 nm, commonly in the form of silver nanoparticles (AgNPs). They remain the most used nanostructures in commercialized products, with approximately 320 tons of AgNPs manufactured yearly [1]. Currently, there are nearly 500 consumer products containing nanosilver. They are included in nanomedical devices, medical imaging, biosensing tools [2], diagnostics, wound dressings, long-term burn care products, and antibacterial cosmetic lotions [2]. Besides broad implementation in healthcare systems for diagnostic and therapeutic purposes, medical device coating, medical textiles, and contraceptive devices, Ag-containing nanostructures are also used in cosmetics, clothing, household, and food products.

The antimicrobial mechanisms of AgNPs include adhesion to cell surfaces altering membrane properties, the formation of free radicals damaging bacterial membranes and viral envelopes, interactions with DNA, and enzyme deterioration [4]. Oxidative stress induction and heavy metal ion release in aqueous solutions produce biologically active Ag+ [5]. Non-oxidative mechanisms have also been suggested for silver nanostructures [6]. The generation of reactive oxygen species (ROS) inhibits the antioxidant defense system, causing mechanical disruptions of viral envelopes and cellular membranes. Metal ions are slowly released from metal oxide and absorbed through cell membranes or viral envelopes, interacting directly with the functional groups of proteins and nucleic acids, such as mercapto (–SH), amino (–NH2), and carboxyl (–COOH) groups, damaging enzyme activity, changing their structure, affecting normal physiological processes, and ultimately inhibiting pathogens of different origins. Additional mechanisms of Ag+ antimicrobial action are becoming evident. Ag+ ions may react with phosphorus and sulfur groups of surface proteins of cellular membranes, bacterial cell walls, and virions after post-translation modification.

Ag+ binds to negative parts of membranes including viral envelopes, making a hole. Ag+ ions damage cytochrome of the electron transport chain, impasse and destroy RNA and DNA, and hinder DNA replication, preventing translation of protein due to damage of ribosomal 30S subunits. Ag+ ions are sources for the formation of ROS that harm both eukaryotic and bacterial cells. However, the impact of metal ions on the pH inside membrane-coated vesicles is small and has weak antimicrobial activity. Therefore, dissolved metal ions are not the main antimicrobial mechanism of AgNPs. Moreover, heavy metal ions can indirectly act as carriers of antimicrobial substances [6]. Disruption of cellular membranes, viral envelopes, and interactions with proteins and nucleic acids [6] are the major known processes of silver-induced disinfecting activity.

These three independent mechanisms occur simultaneously with reversible equilibrium between AgNPs with permanent liberation of Ag+ ions and reverse deposition of AgNPs from recovered ions and nanoclusters in cells. Numerous mechanisms of action against infectious agents would require multiple simultaneous gene mutations for resistance to develop; therefore, resistance to silver-containing compounds and nanostructures is hardly possible [6].

Despite the increasing presence of Ag-containing products in the market and extensive reports on the antimicrobial activity of AgNPs, there is insufficient data currently available about the principal restrictions for nanosilver use as diagnostic and therapeutic agents. The rapid growth in manufacture and utilization inevitably follows an increased environmental and human exposure, whereas the potential acute and chronic toxicity has yet to be fully addressed.

Current research aims to analyze the stability, cytotoxicity, and immunomodulation potential of Ag+ ions and NPs.

Stability of Ag+ ions, citrate-coated AgNPs, and their nanoconjugates with proteins

AgNPs in the presence of ions and especially after the addition of EDTA are not stable due to oxidation, dissolution, and aggregation within a few hours. UV–visible spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM) revealed that citrate-coated AgNPs remain stable colloid solutions in deionized water at room temperature for decades but not in the presence of ions. Citrate-coated AgNPs with the surface Ag2O layer are not stable in the presence of phosphate buffer solution (PBS) (0.01 M Na2HPO4/KH2PO4, 0.15 M NaCl/KCl) for 1 hour at room temperature due to the replacement of Ag+ by electrolyte ions, potential formation of insoluble AgCl, subsequent catalyzed oxidative corrosion of Ag, and further dissolution of the surface layer of Ag2O [7, 8]. To prevent AgNPs dissolution and aggregation, various surfactants and polymers are introduced during or after synthesis [7]. Coating layers enhance electrostatic and steric repulsion. Adsorption of polymers or nonionic surfactants provides steric hindrances depending on the thickness of the adsorbed layer [7].

Nanosilver, like other NPs, becomes wrapped by serum and cellular proteins constituting the protein corona immediately after administration into the organism. These protein shells decrease the efficiency of targeting by directing the NPs to the reticuloendothelial system, by masking the active targeting moieties and decreasing their ability to bind to their target receptor, but may re-direct NPs towards endogenous targets. The NPs stability depends on the affinity of coating molecules to the particle surface, repulsion from neighboring molecules, loss of chain entropy upon adsorption, and nonspecific dipole interactions between the macromolecule, solvent, and surface. Protein corona protects AgNPs from dissolution and aggregation.

Citrate Coating

Citrate-coated AgNPs exhibit properties that influence their stability in various environments. While stable in deionized water, they are prone to destabilization in ionic solutions.

Polymer and Surfactant Coatings

To address stability issues, surfactants and polymers are often employed. These coatings can significantly enhance electrostatic and steric repulsion, aiding in preventing aggregation and dissolution.

Colloids of Ag possess high affinity for binding with serum albumins; their ability to bind with Staphylococcus aureus protein A is less efficient, whereas several proteins (for example, human immunodeficiency virus (HIV-1) envelope antigen) cannot attach to AgNPs at all. Despite known chemical affinity of sulfur atoms to precious metals, direct correlation between cystine disulfide bridge content and binding with AgNPs was not observed, perhaps because of strong bonds between two cysteines that stabilize protein conformation.

Protein Corona

Upon exposure to biological environments, a 'protein corona' forms around AgNPs, playing a vital role in their interaction with biological systems. This layer impacts the stability, cellular uptake, and overall functionality of the NPs.

AgNO3 and its water solutions should be stored in the dark because of the possible recovery of silver atoms with the formation of nanostructures. Fluorescent metal nanoclusters with sizes less than 2 nm consisting of a few silver atoms can be recovered from Ag+ in the presence of proteins (albumins, immunoglobulins of different classes, and origin) and NaBH4.

Nanosilver in immunodiagnostics

Physicochemical features of nanosilver determine possible implementation in diagnostics. Typical size range of AgNPs 30–80 nm provides high surface to volume ratio. Binding of AgNPs with NH2- and SH-groups of proteins is weaker compared to AuNPs, but protein corona can be formed with the majority of proteins including the main blood proteins. However, leaking Ag+ cations may damage proteins of envelopes. Extinction, light scattering, surface plasmon resonance (SPR), and SERS of AgNPs exceed those of AuNPs by 10 and 100 times, respectively. Relatively low price is also an advantage of nanosilver.

Immunoconjugates

The stable nanoconjugates of AgNPs with immunoglobulins of different origin, classes, and specificity, including both polyclonal and monoclonal antibodies, were constructed by:

1. Direct binding of AgNPs with purified IgG or IgM [8];
2. Nanoprecipitation of proteins from their solutions in fluoroalcohols [9];
3. Physisorption of proteins on the AgNPs surface treated with poly(allylamine)s;
4. Encapsulation of AgNPs into SiO2 envelope and functionalization with organosilanes. Adsorption of proteins on surfaces of AgNPs is reversible, and up to 70% of the attached proteins can be eluted.

Binding Specificity

AgNPs possess high affinity for binding with immunoglobulins but not with any protein. SiO2 layer on surfaces of metal NPs is suitable for silanization and covalent attachment of any protein. Protein corona prevents AgNPs from oxidation, dissolution, and aggregation. The developed methods of fabrication of AgNPs with protein shells permit to attach any protein at different distances from the metal core to avoid possible inactivation of proteins, to reduce fluorescence fading and to stabilize the nanoconjugates [8].

Cytotoxicity of Ag ions and nanoconjugates of AgNPs with major blood proteins

The common mass-only dose metric model employed in toxicology for traditional substances is not convenient for engineered nanomaterials. Alternative dose metrics include particle number, ion release (kinetics, equilibrium), and the total particle surface area. Nevertheless, polydisperse particle suspensions, ambiguity in the surface area, and concentrations will obscure the analysis. Therefore, the Organisation for Economic Cooperation and Development recommended that particle number, surface area, and mass should all be measured when possible to enable calculation of alternative dose metrics. For AgNPs, both surface area and ion release have been reported as effective alternative dose metrics for nanotoxicological assessment.

Silver in Different Forms

Silver in ionic, nanoparticulate, and bulk forms behave very differently. Due to large surface area, AgNPs are capable of rapid oxidation, dissolution, reactive capacity, and binding with biomolecules [10]. When metallic silver is reduced to a nanometer scale, it can enter cells and cause adverse health effects [10]. AgNPs enter eukaryotic cells either by endosomal uptake or by diffusion. They can penetrate living organisms via several routes, including inhalation, oral ingestion, intravenous injection, and dermal contact.

The American Conference of Governmental Industrial Hygienists has established threshold limit values for metallic silver (0.1 mg/m3) and soluble compounds of silver (0.01 mg/m3). Long exposure of humans to nanosilver from cations to NPs through oral and inhalation routes can lead to argyria, or skin discoloration, and argyrosis, or discoloration of the eyes, as soluble silver incorporates into the tissues with permanent damage to skin microvessels and eyes [11]. Studies in vivo with experimental animals have revealed AgNPs accumulation in their liver, spleen, and lung. Similarly, AgNPs-mediated cytotoxicity in mammalian cells depends greatly on the nanoparticle size, shape, surface charge, dosage, oxidation state, and agglomeration condition as well as the cell type. Smaller AgNPs cause more toxicity than larger ones owing to their larger surface area and reactivity [11].

AgNW Toxicity

Currently available data about toxicity of silver nanowires (AgNW) (micron-range long with diameters <100 nm) remain contradictory [11]. For both short (1.5 mm) and long (10 mm) AgNW after inhalation, lung inflammation at day 1, disappearing by day 21 has been described, and in bronchoalveolar lavage fluid, long AgNW cause neutrophilic and macrophagic inflammation [12].

Exposure and Organ Accumulation

Exposure to different forms of silver leads to distinct outcomes. Whereas elemental silver exposure is not associated with health effects, soluble silver is associated with lowered blood pressure, diarrhea, respiratory irritation, and fatty degeneration in the liver and kidneys. Furthermore, after different routes of administration including intravenous, intraperitoneal, and intratracheal, the AgNPs can cross the brain-blood barrier in vivo and tend to accumulate in liver, spleen, kidney, and brain [9].

Entry Routes

Respiratory tract, gastrointestinal tract, skin, and female genital tract are the main entry portals of nanosilver into the human body through direct substance exchange with the environment. Additionally, systemic administration is also a potential route of entry, since colloidal silver nanoparticles have been exploited for diagnostic imaging or therapeutic purposes. Inhalation and instillation experiments in rats showed that low concentration but detectable ultrafine silver (14.6 ± 1.0 nm) appeared in the lung and was subsequently distributed to the blood and other organs, such as heart, liver, kidney, and even brain. Nanosilver accumulates in blood, liver, lungs, kidneys, stomach, testes, and brain. AgNPs less than 12 nm affect early development of fish embryos, causing chromosomal aberrations and DNA damage.

Animal and human studies indicate that it is difficult to remove silver completely once it has been deposited in the body; however, nanosilver can be excreted through the hair, urine, and feces.

Inhibition of innate immunity with the nanosilver

Cytotoxicity of nanosilver evidently determines host defense such as apoptosis, necrosis [2], and NETs formation [16]. Moreover, AgNPs, but not Ag+ ions, decrease viability and the cytotoxic potential of natural killer (NK) cells secreting cytokines and killing damaged cells [12].

Nanosilver possesses anti-inflammatory properties in both animal models and in clinics. AgNPs inhibit the expression of pro-inflammatory cytokines transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α. Nanosilver administration attenuates nasal symptoms of allergic rhinitis in mice and inhibits immunoglobulin IgE, IL-4, and IL-10. In clinical studies, wound dressing containing AgNPs promoted the healing of chronic leg ulcers due to antibacterial effect in the wound and by decreasing inflammatory response. The ability of nanosilver to reduce cytokine release and production of matrix metalloproteinases, decrease lymphocyte and mast cell infiltration, and induce apoptosis in inflammatory cells may explain their anti-inflammatory mechanisms [17].

The authors declare no conflict of interest.

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AgNPs have been reported to be toxic to human cell lines [11]. Cellular uptake of AgNPs takes place either via diffusion (translocation), endocytosis, or phagocytosis. Upon entering the cytoplasm, AgNPs themselves or Ag+ ions can generate ROS, leading to DNA damage, protein denaturation, and apoptosis. AgNPs of different sizes and shapes tend to accumulate in the mitochondria, thereby inducing mitochondrial dysfunction, i.e., a reduction in mitochondrial membrane potential (MMP), and promoting ROS creation [11].

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Cellular uptake of AgNPs takes place either via diffusion (translocation), endocytosis or phagocytosis. Upon entering the cytoplasm, AgNPs themselves or Ag+ ions can generate ROS, leading to DNA damage, protein denaturation, and apoptosis. AgNPs of different sizes and shapes tend to accumulate in the mitochondria, thereby inducing mitochondrial dysfunction, i.e., a reduction in mitochondrial membrane potential (MMP), and promoting ROS creation [11]. AgNPs' cytotoxicity in mammalian cells depends on the NPs sizes, shape, surface charge, dosage, oxidation state, agglomeration condition, and cell type. They induce a dose-, size- and time-dependent cytotoxicity, particularly for NPs with sizes less 10 nm.

Surface charge of AgNPs stabilized with citrate anions or protein envelopes is a parameter responsible for cellular uptake. In particular, high-level toxicity of positively-charged nanoconjugates versus negatively-charged coatings has been reported. It can be caused by the adhesion of AgNPs onto the negatively charged cell membranes, their consequent entry to the cell, potential release of Ag+ inside the cell, damage of cellular proteins and nucleic acids, and other cytotoxic effects. For instance, the following coatings possess surface charges: (1) positive: polyethylenimine, chitosan, poly-L-lysine, and cetyltrimethyl