Nano silver is used as a multifunctional drug delivery system

Nanoparticles can overcome some of the essential problems of conventional small molecules or biomolecules (e.g., DNA, RNA and proteins) used in a number of diseases by enabling targeted delivery. and overcome biological barriers. Recently, silver nanoparticles have been exploited as carriers for therapeutic agents, including antisense oligonucleotides, and other small molecules.
Silver is the most profitable precious metal used in the preparation of nanoparticles and nanomaterials because of its antibacterial, antiviral, antifungal, antioxidant and physico-chemical properties that are enhanced any- times . often compared with bulk materials such as optical, thermal, electrical and catalytic properties.
Small silver nanoparticles offer many benefits as drug carriers, including adjustable size and shape, enhanced stability of surface-bound nucleic acids, binding of bile surface ligands high-altitude, transmembrane delivery without harsh infectious agents, protection of concomitant therapeutic agents from degradation, and the potential to improve controlled/temporal intracellular drug delivery.
Plant-mediated synthesis of silver nanoparticles is gaining interest as it is inexpensive, provides a cleaner working environment and protects human health resulting in reduced waste and safer products. .
This chapter presents the basic physicochemical properties, antibacterial and antitumor properties that silver nanoparticles obtained by plant intermediates possess, and their applications as drug delivery systems with a wide scope. important view of the potential toxicity to the human body. transmembrane delivery without potent transmission agents, protection of concomitant treatments from degradation, and improved/controlled intracellular drug delivery over time.


Nano silver used as a multifunctional drug delivery system

(Copyright belongs to NanoCMM Technology)

Customers who need 15000 ppm nano silver material for food use, please contact Hotline 0378.622.740 – 098.435.9664

1.   Introduction

Nanomedicine is a branch of medicine that uses nanomaterials and the application of nanotechnology in the prevention, diagnosis and treatment of diseases. [ 1].

The broad definition of nanomedicine involves nanoparticles (NPs) such as drug delivery systems (DDSs), medical nanosensors, biological spacecraft, insulin pumps, needleless injectors, and more.
The unique properties of NPs are related to their small size (typically 1 to 100 nm), large surface area, and surface features. “Nano” DDSs provide targeted optimal dosing with reduced side effects and toxicity. Furthermore, NPs solve problems related to drug solubility and bioavailability. These “nano” carriers can protect drugs from toxic environments as well as overcome biological barriers for drugs to penetrate targeted tissues and deal with drug resistance. They are of organic or inorganic origin and can be prepared from various polymers, metals, ceramics, etc.
Silver is the most profitable precious metal used for the preparation of NPs and nanomaterials. They are known for their antibacterial, antiviral, antifungal, and antioxidant properties and unusually enhanced physicochemical properties compared to bulk materials such as optical, thermal, electrical and catalytic properties. [ 2 , 3 , 4 , 5 ].

About 500 tons of silver nanoparticles (AgNPs), used in industries and daily life, are produced each year [ 6 , 7].

The increasing demand for silver nanomaterials necessitates the development of environmentally friendly synthesis methods. In general, nanosilver can be produced by chemical, physical and biological methods. Chemical protocols are mainly based on the reduction of Ag+ ions by organic and inorganic agents, such as sodium borohydride, sodium citrate, sodium ascorbate, elemental hydrogen, N, N-dimethylformamide, polymeric methods , Tollens, etc.

The reducing agent reduces Ag +  and leads to the formation of Ag 0 , metallic silver, which aggregates into oligomeric clusters. These clusters can form metallic silver colloidal particles. Various surfactants and polymers are used to prevent particles from further agglomerating and preserving their shape [ 8].

The most important physical methods are based on evaporation-condensation and laser combustion of bulk silver materials in solution. Both physical methods do not use chemical reagents that can be dangerous to the environment and the human body. Although these methods require expensive specialized equipment, physical methods provide an alternative to time-consuming and environmentally unfriendly chemical protocols.

The biological method, also known as the “green” method, does not use harmful chemicals in the preparation technique. Furthermore, these methods are based on using bacteria, fungi, algae and plants to obtain silver nanoparticles which are characterized by size and shape depending on optical, electrical and antibacterial properties [ 8 , 9 ].

They are based on the biosorption of Ag+ ions in aqueous media, where reducing agents are cited above biological sources.
AgNPs synthesis using live microorganisms (bacteria and fungi) can be carried out intracellularly or extracellularly [ 10]. Extracellular synthesis is cheaper, less time consuming and requires simpler manufacturing technology than intracellular synthesis. Studies have used supernatant cultures of pathogenic and non-pathogenic microorganisms such as A. flavus, B. indicus, B. cereus, Bacillus strain CS 11, E coli, P. proteolytica, P. meridiana, S. aureus, etc. … [ 10 , 11 , 12 ].

Limitations of bacterial nano silver synthesis are related to appropriate bacterial strain selection and culture, a mandatory purification stage, and poor understanding of the mechanisms regulating nanoparticle formation. hinder the process of scaling up industrial laboratories as well as the requirements of aseptic conditions and their maintenance. [ 13 ].

Plant-mediated nanosilver synthesis is gaining widespread popularity because of its environmental friendliness, accessibility, economy, simplicity of implementation, and large-scale production capabilities. Many studies have used different plant extracts such as Azadirachta indica, Crocus sativus L., Calliandra haematocephala, Neem leaf, Madhuca longifolia, grape seed extract, Andean blackberry fruit extract, maple leaf juice extract geraniums, marigolds, etc… [ 7 , 14 , 15 , 16 , 17 , 18 , 19 ].

The rich phytochemical composition of the extracts used implies its complex activity, for example, as reducing, stabilizing and capping agents. Therefore, the obtained nano silver can be exploited as DDS for various active pharmaceutical ingredients.
This chapter presents the basic physicochemical properties, antibacterial and antitumor properties, which nano silver obtained by plant intermediates possess, and their application as DDSs with an important point of view on toxicity. possible on the human body


2. Plant-mediated synthesis of silver nanoparticles

It is well known that plant extracts have a rich phytochemical composition including phenols, saponins, terpenoids, flavonoids, catechins, tannins, enzymes, proteins, polysaccharides,… All biomolecules This takes place by a very complex mechanism of Ag+ reduction and stabilization. ions to form silver nanoparticles. For example, Li et al. proposed a recognition-reduction-limited growth and nucleation model to explain the possible formation mechanism of AgNPs in Capsicum annuumL. quote [ 19 ]. According to the authors, proteins with amino groups play a reducing and controlling role in the formation of nanosilver in solution, and the secondary structure of the protein changes after reacting with Ag+ ions. In another study, Mirgorod and Borodina, based on surface-enhanced Raman spectroscopy data, stated that NPs are formed by a redox reaction between flavonoids and Ag+ ions as well as having flavonoids near the surface. of AgNPs, reacts complexly with Ag+ ions and with NP [ 20 ]. Ahmed and colleagues have detailed AgNP synthesis approaches and the different protocols used to synthesize them. [ 21 ].

It is important to note that technological parameters such as temperature, pH, concentration of Ag+ ions, time of acquisition, phytochemical composition of extracts used, mechanical agitation, support Microwave aids, etc., are very important for the preparation of nanoparticles and their characteristics and fate [ 6 , 7 , 14 , 15 , 16 , 17 , 18 , 19]. These parameters not only affect Ag+ deionization and metallic silver formation, but also affect its agglomeration into oligomeric clusters that can form colloidal particles with specific characteristics. Amin et al found that the reaction time, temperature and volume ratio of the methanol extract from Solanum xanthocarpumberry to AgNO 3  could accelerate the reduction rate of Ag+ ions and influence the size and shape of AgNPs [ 22 ]. The NPs were found to be about 10 nm in size, monodisperse and spherical in nature.

The surface functionality of nanomaterials is important for their applicability, compatibility and safety. In general, surface behavior determines how a nano-entity will interact with a biological system, environment, etc.. [ 23 ]. Nano silver are characterized with variable morphology — size, shape, surface area, purity/coating — and related electrochemical and electromagnetic properties — charge, zeta potential, redox potential , surface plasmon resonance and electrical conductivity [ 24 , 25]. A change or deliberate attempt to control these essential characteristics is an essential tool for tailoring AgNPs for specific purposes and can be highly sought after on several accounts: (1) increase stabilization; (2) increased selectivity; (3) increase the effectiveness of treatment or diagnosis; (4) enhanced catalytic activity; (5) reduced toxicity; and (6) reduced reactivity [ 23 , 25 ]. Surface functionalization of AgNPs can be determined by the chosen synthetic pathway (single-step functionalization) or additional processing after isolation (multi-step functionalization).

3. Surface properties of nano silver

3.1. Surface purity of “green” synthetic silver nanoparticles

The synthesis of “green” silver nanoparticles using plant extracts often not only leads to intentional surface functionalization but is also unavoidable because every component of the total plant extract contains water. (as a reducing agent, stabilizer or simultaneous components) has a certain affinity for the silver surface [ 16 , 24 , 26]. After isolation and purification, only the most strongly bound components are left on the surface that are “attached” to nano silver. Absorption, also known as “cohesion”, can occur as a result of a chemical (chemical absorption) or a physical (physical absorption) phenomenon. Chemical absorption, in the case of nanosilver, occurs through ionic, covalent or coordination-covalent chemical bonds. S-containing molecules (several amino acids, peptides, and proteins) have the highest affinity for the silver surface because of the strong Ag-S bond and are therefore the first to be considered for the interaction. [ 17 , 26 , 27 , 28]. Next, the N and O atoms from the amide, amino, hydroxyl, phenol, carboxyl, and carbonyl groups are targets for complexation with Ag+ ions and thus are also very likely to be adsorbed on the surface. [ 7 , 15 , 16 , 18 , 25 , 26 , 27 , 28 , 29 , 30 ]. The latter exists in most of the primary and secondary metabolites in plants (phenolic acids, polyphenols, flavanoid alkaloids, glycosides, protein polysaccharides, etc.) [ 7 , 15 , 16 , 17 ,18 , 24 , 28 , 31 ]. Physical absorption arises due to Van der Waals forces, and although much weaker than chemical absorption; it is nonspecific and can affect every polarization unit around AgNP. Knowing that the electrical potential of colloidal silver can be significant, this explains the important role of physical absorption for the surface function of the “green” synthesized nano silver. It should be noted that regardless of the mechanism, biomolecules involved in Ag+ deionization, are more likely to participate in interaction with the silver surface because of their initial intimate contact with the silver surface. particles arise [ 7 , 15 , 16 , 17 ,18 , 24 , 31 ].

One question that may arise is whether this non-allergenic and uncontrollable “impurity” on the surface of AgNPs after “green” synthesis with plant extracts is just an advantage or does it have any other side. which weak. In practice, this is highly dependent on the designation of the particles. The presence of tannins, proteins, polysaccharides, flavonoids and lipids has been shown to favor stabilization, increasing the catalytic, antibacterial and antioxidant activities of nanosilver and reducing toxicity by passivating the surface [ 7 , 14 , 15 , 16 , 21 , 31 , 32 , 33 , 34]. However, the “coating” of AgNPs reduces their size and agglomeration rate, and some researchers suggest that this may have an adverse effect on cytotoxicity [ 33 ]. Furthermore, for surface-selective analytical techniques (such as surface-enhanced Raman spectroscopy, SERS), where the use of AgNPs yields promising results as enhancers, it is necessary to has a “clear” surface allowing access to the target analytes [ 26 ]. In this regard, it may be preferable to use pure natural reducing agents (eg, the flavonoids quercetin, chrysin, apigenin, luteolin, etc.)[ 25 , 26 , 28 , 35]. However, if the presence of multicomponent components and unpredictable adherence on nano silver are unwelcome, the need for a “limiting” agent persists. Sugars and polysaccharides, proteins and proteoglycans such as glucose, galactose, mannose, chitosan, sodium alginate, glucans, gelatin, and others are commonly used as coatings for this purpose. [ 17 , 27 , 35 , 36 ]. These substances are usually introduced into the reducing medium during synthesis, while the mechanism of their attachment to the surface follows the principles described above. [ 27 , 35 ].

3.2. Surface area of nano silver

The active surface area of silver nanoparticles is determined by their size, shape and rate of agglomeration. Reaction conditions such as pH, temperature, extraction volume and concentration, reactant ratio, and time determine the size and extent of crystal growth and thus affect the size and shape. of silver assemblages [ 16 , 24 , 25 , 31 ].

The polydispersity of the AgNPs formed is a drawback of “green” synthesis with plant extracts, possibly due to the uncontrolled deposition of various compounds on surface and heterogeneity of the reaction medium. In this regard, the use of the o/w microemulsion upgrade method gave good results [ 18 ]. Postsynthetic agglomeration can lead to expansion of aggregates and ultimately to colloidal instability. This is the role of the “cap” on the surface of AgNPs, aiming to overcome the attractive forces between particles and increase physical stability. Large surface area is desirable as it provides higher catalytic and antibacterial efficiency due to increased Ag+ release from the surface, which is the underlying mechanism of the antibacterial activity of nanosilver. [25 , 32 ]. However, this exact mechanism, which has been demonstrated by many, is also involved in increased oxidative stress and cytotoxicity. [ 33 , 35 ]. Furthermore, AgNPs smaller than 10 nm can pass through nuclear pores and interact with chromosomes and DNA. Thus, such particles are suitable for gene therapy and diagnostics, but are dangerous in relation to genotoxicity [ 33 ]. On the other hand, each intervention leading to the prevention of particle agglomeration and their size reduction is welcomed with regard to the stability and potency in catalysis, antibacterial therapy and diagnostics. On the other hand, the same intervention may have potential risks associated with increased toxicity of NPs obtained. [ 25 , 3335 ].

The shape of nanosilver has also been shown to have an impact on toxicity [ 34 , 37 ]. Ví dụ, AgNP hình dây cho thấy độc tính cao hơn so với NP hình cầu [ 37 ], trong khi một nghiên cứu khác chứng minh rằng tiềm năng độc hại của AgNP hình đĩa vượt quá khả năng gây độc của dây và hình cầu [ 34 ].

3.3. Tính chất điện hóa và điện từ của AgNPs

The charge and zeta potential of the silver nanoparticle present in the suspension are the main determinants of colloidal stability and are highly dependent on a combination of variables. Among them, the pH of the reaction medium and the type of coating are very important [ 31 , 35 ]. The Zeta potential (ζ) is the potential that occurs between the surface of AgNPs and the surrounding liquid phase and is an important measure of colloidal stability. Values ​​in excess of ζ = ± 30 mV are generally considered to be the strength requirement of the colloid [ 31]. Adjusting pH during synthesis is considered an electrostatic approach to stabilize colloids (by varying the type and amount of charge), while coatings aim to reduce gravitational forces in such a way that steel [ 31 , 35 ]. AgNPs obtained by reduction with plant extracts usually carry a negative charge [ 7 , 15 , 17 , 18 , 27 , 29 , 35 ]. The negative zeta potential can be considered an advantage because increased cytotoxicity and cytotoxicity are subsequently found for positively charged AgNPs. [ 23 , 33 ].

The presence of “capping” agents on the surface is essential for colloidal stability, but they also affect the so-called “redox potential” of AgNPs, i.e. the ability to obtain electrons and their reduction [ 38 ]. The low redox potential is required for surface oxidation and Ag +  release and thus promotes higher antibacterial activity and toxicity [ 33 ]. In some cases, immobilization of AgNPs in the lightly permeable “cap” can lead to loss of oxidizing capacity and antibacterial properties. [ 35 ].

Surface plasmon resonance (SPR) is a characteristic optical property of AgNPs due to resonant oscillations of electrons on the surface caused by light radiation [ 39 ]. This electromagnetic phenomenon produces an intense peak in the blue-violet region of the visible spectrum [ 7 , 15 , 18 , 24 , 26 ]. The latter is highly dependent on surface function (size, coating, etc.) and is considered evidence for successful AgNPs synthesis. [ 7 , 15 , 18 , 24 , 26 , 39 ].

3.4. AgNP in complex and distribution mode

Several attempts to incorporate synthetic “green” nano silver into the structures of liposomes, cyclodextrins, nanoemulsions and hydrogels have been reported. Such approaches offer opportunities for targeted delivery, better compatibility, and lower toxicity [ 35 , 40]. For example, one-step synthesis of stable liposomes using AgNPs has shown improved vesicle stability, compatibility, and antibacterial properties compared with  nanosilver alone, also providing an opportunity for cross-delivery. skin[ 40 ]. Other studies reported that the binding of AgNPs to β-cyclodextrin improved their catalytic activity [ 25 ], while the synthesized AgNPs kappa-carrageenan hydrogel particles “green” were detected. to deliver Ag + in the desired controlled manner [ 41 ].

3.5. Functionalization by conjugation

The next level of surface functionalization is the conjugation of nanosilver with bioactive molecules. This approach, unlike all the ones mentioned above, is not only mutable but also leads to completely new functionalities. The conjugation of oligonucleotides to the surface of metal nanoparticles is widely studied for targeted gene therapy and biodiagnostics. However, the attachment of DNA sequences on the surface of silver nanoparticles has been difficult due to the lower stability of the complex. There have been very few successful reports over the years with disulfide or sulfhydry inserted DNAl [ 42 , 43 ].

An interesting area of research is the potential of nano silver as drug carriers [ 29 , 30 , 44 ]. It is hypothesized that nano silver can be used as a vehicle to deliver drug molecules to target regions and thereby improve therapeutic efficacy; moreover, exhibits synergism with synthetic antibiotics with regard to antibacterial properties. These assumptions have been tested by several scientists in the field, who report successful conjugation of tetracycline (multiple hydroxyl groups, phenols and amides), the glycopeptide antibiotic vancomycin (multiple amide groups, phenols). and hydroxyl) and the immunosuppressant azathioprine (basic S- and N-atoms in the heterocycle) [ 29 , 30 , 44 ].

4. Antimicrobial activity of silver nanoparticles

Since ancient times, elemental silver and its compounds have been used as antibacterial agents. Nano silver synthesized by different methods have been extensively tested and have been shown to be effective against more than 650 microorganisms including bacteria (both Gram-positive and Gram-negative), fungi, and viruses. 21 , 45 ]. Many mechanisms of antimicrobial action of nano silver have been considered, but most studies have simplified them to three main mechanisms: (1) the adhesion of nanosilver to the surface of cell walls and cell membranes; (2) entry of AgNPs into cells and destruction of intracellular structures (mitochondrial, vacuoles and ribosomes), and biomolecules (proteins, lipids and DNA); and (3) generation of reactive oxygen species (ROS), leading to induced cytotoxicity and oxidative stress [ 2145 , 46 ]. According to Prabhu et al. and Dakal et al., modulation of signal transduction pathways is also a distinct mechanism of antibacterial action of nano silver. [ 45 , 47 ].

The adhesion of the silver nanoparticles to the surface of the cell wall is facilitated by the positive surface charge of the silver nanoparticles, and an electrostatic attraction occurs between the AgNPs and the negatively charged cell membrane of the silver nanoparticles. organisms [48 ]. The interaction of Ag+ ions with sulfur-containing proteins, present in the bacterial cell wall, irreversibly disrupted the bacterial cell wall [ 49 ]. The destruction of cell membranes by AgNPs causes structural changes that make bacteria more permeable and disrupts respiratory function [ 45 , 46 ]. Morones et al. demonstrated the existence of silver in the membrane of treated bacteria as well as its interior by transmission electron microscopy (TEM) analysis [ 50]. The composition and thickness of the cell wall also affect the antibacterial potential of nano silver [ 45 , 48 ]. In Gram-negative bacteria such as E. coli, Pseudomonas, Salmonella, the cell wall consists of a lipopolysaccharide layer, followed by a thin layer of peptidoglycan (3–4 nm). The cell wall in Gram-positive bacteria such as Staphylococcus, Streptococcus, Bacillus is composed mainly of a thick layer of peptidoglycan (30 nm thickness) [ 48 , 51 ]. Therefore, AgNPs exhibit a higher antibacterial effect against Gram-negative bacteria regardless of their degree of resistance compared to Gram-positive bacteria [ 49 ]. It has also been suggested that the Ag+ ion enters the cell and interacts with the sulfur and phosphorus of the DNA, which can lead to problems in bacterial DNA replication and cell death. [ 47 ].

The antimicrobial potential of nanosilver is also associated with the generation of free radicals and ROS and thus an increase in oxidative stress in cells. Silver ions can interact with the thiol groups of many important enzymes, inactivating them and generating ROS. An excessive amount of free radicals is generated leading to direct damage to the mitochondrial membrane causing necrosis and eventually cell death [ 52 ].
The antibacterial efficacy of nanosilver depends on various parameters including size, shape, zeta potential, dosage and colloidal state discussed above [15 , 46 , 49 ]. AgNPs with sizes between 10–100 nm showed strong bactericidal activity against both Gram-positive and Gram-negative bacteria [ 50 , 51 ]. Depending on the size of the NPs, a large surface area is in contact with the bacterial cell to provide a higher interaction rate than larger particles [ 51 , 53 ].

The effect of shape on the antibacterial activity of AgNPs was investigated by Pal et al. [ 54 ]. Silver nanoparticles of different shapes (triangle, sphere and rod) were tested against E coli. According to the authors, triangular NPs are more active than spherical NPs, which are more active than rod-shaped AgNPs against E coli. This may be due to the surface area-to-volume ratio and structure. their crystallographic surface is larger [ 54 ]. Rout et al. synthesized AgNPs of different shapes (i.e. spheres, triangles and rods) using Mulberry (Morus rubra L.) leaf extracts and studied their antibacterial activities against  E colitrong both liquid and agar-based systems. The high reactivity of truncated triangular NPs was also observed compared with spherical and rod-shaped particles. [ 55 ].

Sondi and Salopeck-Sondi investigated the antibacterial activities of AgNPs against  E coli on Luria-Bertani agar plates and reported that the antibacterial activity of nanosilver was dose dependent [56 ]. Colloidal AgNPs, i.e. suspended nano-sized Ag particles, have shown enhanced antibacterial potential compared with AgNPs alone. Colloidal AgNPs generated by green synthesis are characterized with controlled size, high stability and improved antibacterial activity which have been tested in different studies by micro bacteria in direct contact with nano silver [ 45 , 57 ].

Okafor et al. produced nano silver by synthesizing green color from extracts of aloe, geranium, magnolia, and black cohosh and studied their antibacterial activity against different bacterial species: three Gram-negative bacteria and three Gram-positive bacteria [ 58]. The overall results indicated that nanosilver showed antibacterial activity at doses of 2 and 4 ppm against Gram-positive and Gram-negative test bacteria. Aloe extract NPs showed the highest antibacterial activity, followed by black cohosh and geranium NPs with the lowest inhibitory activity. The high antibacterial efficacy of AgNPs generated from aloe may be due to the combination of AgNPs and aloe’s bioactive molecules (quinine and other aromatic compounds), which enhances the Inactivate or inhibit the growth of bacterial species. In another study, Zhang and colleagues also reported that NPs produced from aloe were able to inhibit high growth in E coli at low concentrations. [ 59 ].

Ahmed et al. silver nanoparticles synthesized by using Azadirachta indica water leaf extract and studying their antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E coli) bacterial strains compared with controls and plant extracts alone [ 7 ]. According to the authors, AgNPs show effective antibacterial properties compared to other substances due to their large surface area providing better contact with the cell wall of the microorganism. In addition, Bagherzade et al. AgNPs synthesis using saffron extract (Crocus sativusL.) [ 14 ]. The biosynthesized AgNPs showed significant antibacterial effects against E. coli, P. aeruginosa, K. pneumonia, S. flexneri, and  B. subtilis.
Gomathi et al. obtained spherical silver nanoparticle using Cacao poisonleaf extract and studied their antibacterial activity against E coli and S. aureus using well diffusion technique [ 32 ]. The authors reported that AgNPs exhibited greater antibacterial activity against E coli than S. aureus, due to variation in the cell membranes of these bacteria. In another study, spherical AgNPs with size 50-100 nm were observed using Alternanthera dentures water extract and tested against E coli, P. aeruginosa, K. pneumonia, and E faecalis by agar diffusion method [ 60 ].
The authors reported that the antibacterial effect of AgNPs was size and dose dependent and more pronounced for Gram-negative bacteria than for Gram-positive bacteria.
The antibacterial activity of nanosilver with different antibiotics was studied, and synergistic antibacterial effects were found. The bactericidal ability of nanosilver was synthesized from the leaf extract of Murraya koenigii  alone and in combination with antibiotics (gentamycin, ampicillin and streptomycin) against pathogenic bacteria, namely E coli, S. aureus, and P aeruginosa has been studied [ 61 ]. The authors reported that AgNPs combined with gentamycin showed maximum activity against E coli  with an increase in fold area of ​​4.06, while the combination of tetracycline with NP showed maximum activity against  S. aureus. The authors concluded that the activity of standard antibiotics was significantly enhanced in the presence of AgNPs and could be used effectively against antibiotic-resistant pathogens.

5. Nano silver as anti-cancer drug delivery system

Over the years, nanomedicine has created new horizons in the development of future anti-cancer strategies. Conventional cancer treatment such as chemotherapy, radiation therapy, or surgery has limitations regarding drug toxicity, unpredictable side effects, resistance issues, and lack of specificity. AgNPs overcome these disadvantages by reducing side effects and enhancing the effectiveness of cancer therapy. One of their distinguishing features is their ability to overcome various biological barriers and provide targeted drug delivery. Green synthesis of nano silver together with specific anticancer drug delivery to tumor tissues offers an innovative approach to improve cancer treatment. [ 62 ].

5.1. Anti-cancer activity of biosynthetic nano silver

The antitumor activity of biosynthetic AgNPs has been investigated using both in vitro and in vivo models. The reported results show that the cytotoxicity of AgNPs can be influenced by the particle size, shape and surface chemistry. Some authors have stated that increasing the concentration of AgNPs reduces the viability of tumor cells. [ 63 , 64 ].

Effects of time and concentration of nano silver on inhibition of cell viability and membrane leakage were evaluated by multiple methods. [ 65 , 66 ]. Usually, MTT assay, ROS quantification, RT-PCR and western blotting techniques are used to evaluate the ability of AgNPs to inhibit cell growth and mediate cell death. [ 65 , 66 , 67 , 68 ].In vitro Dose-dependent cytotoxic activity was estimated for green synthetic AgNPs from different plants—Vitex negundro L., Acalypha indica, Euphorbia nivulia, and Premna serratifolia[ 63 ] .MCF-7 (human breast carcinoma) AgNPs-treated cell lines were obtained using  Erythrina indica  and  Andrographis echioides extracts. In both cases, cancer cell growth was inhibited according to the relationship between concentration and response of AgNPs. [ 63 ].

Similar results were found in other studies [ 65 , 67 ]. Nano silverobtained using Artemisia marshalliana Sprengel extract and Reishi neo-japonicum Imazeki extract have confirmed cytotoxicity against human gastric cancer AGS cell lines and  MDA-MB-231 cells human breast cancer cells. The authors found that the cytotoxic activity of AgNPs was time and dose dependent as well as the size of the NPs and the temperature of the preparation.

Dependence on the antitumor activity of nanosilver on human cancer cell lines was found, according to the source of NP synthesis as well as on the type of cell line.[ 69 ].

Extracts from fruits, leaves, seeds and roots of Citrullus colocynthis produced silver nanoparticles with different sizes and variations at ID 50  in different cell lines. Toxicity assay of biosynthetic AgNPs using seaweed Ulva lactuca showed the cytotoxic potential of AgNPs against tumors. For human colon cancer, HT-29 cell line ID 50  is 49 μg/ml while its value reaches 12.5 μg/ml in human liver cancerHep G-2  cell lines.

One of the significant limitations of conventional anticancer therapy is drug-mediated toxicity in healthy cells. Synthetic plant-based AgNPs have the potential to avoid this problem by providing selective toxicity to cancer cells.nano silver produced using leaf extract of Podophyllum hexandrumRoyle are cytotoxic to cervical carcinoma cells. The reported results demonstrated that nano silver can selectively inhibit the cellular mechanism of HeLab by DNA damage and caspase-mediated cell death [ 70 ].

In another study, the cytotoxicity of AgNPs against cancer cells was estimated compared to human myeloid leukemia cells HL60  and cervical cancer cells HeLa  to mononuclear cells. in peripheral normal blood (PBMC) [ 66 ].Sargassum vulgare was used for green synthesis of AgNPs. It was found that HL60 cells were affected by AgNP-mediated toxicity while normal  PBMCs suffered less damage.

It has been demonstrated that biosynthesized nanosilver shows significant anticancer activity in a less toxic manner than particles whose preparation involves a number of toxic and expensive chemicals. . Production of AgNPs through a green chemistry approach through Cleome viscosac plant extract provides another solution for optimizing anticancer treatment. The old anticancer activity was in vitro assessed against the human cancer cell lines PA1 (Ovarian adenocarcinoma cell line) and  A549(Human lung adenocarcinoma) [ 68 ]. The results concluded that the synthetic green AgNPs can inhibit the growth of cancer cells and offer great potential in cancer treatment.

To determine the antitumor efficacy of biosynthetic AgNPs and to fully capture the mode of programmed cell death, three important parameters need to be considered: (1) DNA fragmentation; (2) structural changes in cell morphology; and (3) Annexin V binding and caspase activation. Regulation of apoptosis is only one of the possible mechanisms for the antiproliferative activity of biosynthesized AgNPs that has been demonstrated in many studies.

67 , 71 , 72 ]. AgNPs can induce cell death through ROS generation, membrane leakage, activation of caspases, and DNA damage. [ 65 , 66 , 72 ].

5.2. Nano silver for targeted drug delivery

AgNPs represent an alternative therapeutic strategy like DDSs in cancer cure because they can provide passive or active targeting of tumor tissue. Accumulation of drugs at desired sites increases the efficacy of in vivo anticancer therapy. Receptor-mediated intracellularity may facilitate cellular uptake of drugs. This type of active targeting relays based on molecular recognition. The proposed approach to optimize biological AgNP properties is either surface functionalization with specific targeting molecules or coating with biodegradable and biocompatible polymers. [ 73 , 74 ].

For example, nano silver were obtained using different concentrations of Setaria verticillata seed extract loaded with the hydrophilic antineoplastic drugs, doxorubicin (DOX) and daunorubicin (DNR). The remarkable load efficiency (80.50%) and capacity (40.25%) of DOX-AgNPs and DNR-AgNPs have introduced them as future new DDSs [ 64 ].
The delivery of drugs into cells by endocytosis depends on the size of the NPs. Spherical AgNPs are extracted from the Aerva javanica plant and combined with the anti-cancer drug gefitinib. The scanning transmission electron microscopy (STEM) image determined the average size to be 5.7 nm. The apoptotic potential of gefitinib-AgNPs has been compared with gefitinib alone. The reduction in viability of MCF-7 breast cancer cells treated with conjugated gefitinib-AgNPs was significant. Delivery of gefitinib using AgNPs optimizes its efficacy and reduces side effects[ 75 ].

The variety of green synthetic silver nanoparticles with anticancer activity offers new therapeutic opportunities. Their specific features as nanocarriers benefit the development of DDS with unique properties and biocompatible profiles.

6. Silver nanoparticles as activated drug delivery hosts

Nanoparticles can overcome some of the essential problems of conventional small molecules or biomolecules (e.g., DNA, RNA and proteins) used in a number of diseases by enabling targeted delivery. and overcome biological barriers [ 76 ]. Precious metal NPs have specific highly evolved photophysical properties that contribute to their potential as activated drug delivery hosts [ 77]. AgNPs have been widely used as biosensors utilizing plasmon resonance (PR) to enhance their ability to detect specific targets. The precious metal nanoparticle-based sensor benefits from the extremely high sensitivity of localized surface plasmon resonance (LSPR) spectroscopy to environmental changes. The application of metal nanoparticles is not limited to molecular detection. Recently, nanosilver has been used as a vehicle for the delivery of therapeutic agents, including antisense oligonucleotides and other small molecules. Small metal NPs offer many advantages as drug carriers, including adjustable size and shape, enhanced stability of surface bound nucleic acids, high density surface ligand binding, transmembrane delivery without a potent infusion agent, protection of co-administration from degradation, and the potential to improve controlled/timed intracellular release. The photophysical properties of AgNPs could bring these to the forefront of drug delivery, enabling targeted delivery, spatially controlled release (picture-) and confirmation of delivery across image [78 ].

AgNPs in the ~2–100 nm diameter range exhibit SPR spectra in the visible region, which are tunable and dependent on particle shape, size, medium, and interparticle spacing. AgNPs have unique properties that make them a desirable alternative particle in many cases. AgNP is the strongest light scatterer among precious metal particles, and it is reported that the light scattering cross section of AgNP is ~10 times larger than that of similar sized gold particles. The off-band (light absorption and scattering) of AgNPs is due to free conduction electron oscillations, and the motion of bound electrons also contributes to the spectrum..78 ].

Mie theory calculated the light absorption and scattering properties for silver nanoparticles of different sizes. For larger particle sizes (~50–60 nm), the scattering efficiency (Qsca) is higher (≈ 5). AgNPs in this size range scatter light at or on the solid metal surface, but the scattering efficiency increases even higher to 5.8 for the 70–80 nm size while maintaining the surface PR. face in UV to the visible range. This property is ideal for conventional and redshift photocatalysts commonly used as photocatalysts [ 78 ].
The generality of current nanoscale delivery systems is that they are macromolecular in nature. Studies on metal NPs have shown their suitability for delivery of various therapeutic agents including small molecules, antisense oligonucleotides and siRNAs. Nanoscale silver is one of the available optically active surface enhancing substrates. AgNP-based single delivery platforms combine solutions for intracellular detection and extrinsic control of surface-adhesive drug release via photothermal or photochemical activations [ 77 ].

Light-responsive systems are of great interest in the field of drug delivery and gene therapy, due to their potential for extrinsic, spatial control over therapeutic delivery and activation alongside such systems. . Electromagnetic radiation triggers DDSs to respond to light, typically in the UV, visible, and near-infrared (NIR) ranges. These systems are based on photosensitive compounds that can be incorporated into a drug delivery vehicle or combined with the drug itself (the “caging” compound) and can be switched to an active or inactive state when the drug is delivered. electromagnetic irradiation in a specific frequency range. Cage compounds are powerful tools for physical spatial control of drug action in living systems. Groups of photosensors have been used to nest, or inactivate, various biomolecules, including nucleotides, proteins, and nucleic acids, for controlled, in situ imaging. Release via light irradiation allows rapid, spatial and temporal release of a biomolecule at the intended tissues or even within a specific intracellular compartment. [78 ].

Silver nanoparticles with a size of 60–80 nm decorated with thiol-terminal optical DNA oligonucleotides were used as photo-activated drug delivery hosts [77 ].In vitro tests showed an increased size. effective photoactivation of ISIS2302 antisense oligonucleotides superimposed on the internal optically separable linkers. These nanocarriers have several advantages such as protection against nucleases, photorelease efficiency and enhanced cellular uptake when compared with commercial transducers. The release of anti-sense oligonucleotides by light to silence ICAM-1 (intracellular adhesion molecule-1) has potential applications in wound healing, where inflammation is an important criterion such as in Crohn’s disease.

7. Evaluation of the toxicity of nano silver

Nanotechnology has grown rapidly with use in a wide variety of commercial products around the world. However, there is still a lack of information regarding increased human, animal and environmental exposure to NPs including nano silver and their potential risks related to short- and long-term toxicity. . However, some research has been done.

7.1. In vitro test

Nanosilver has emerged as an important nanomaterial for a wide range of industrial and medical applications with potential risks to human health. In vitro studies report that AgNPs induce toxicity targeting multiple organs including the lungs, liver, brain, vascular system, and reproductive organs. AgNPs induce expression levels of genes involved in cell cycle progression and apoptosis. Possible mechanisms of nano silver toxicity include ROS induction, oxidative stress, DNA damage, and apoptosis [ 79 ].
To understand the toxicity of NPs in vitro, different tests were evaluated. Testing of silver (Ag – 15 nm), molybdenum (MoO 3 –30 nm), and aluminum (Al – 30 nm) NPs on a rat spermatogenic cell line determined concentration-dependent toxicity to all NPs. all types. AgNPs are the most toxic (5–10 μg ml -1 ), and strongly reduce mitochondrial function and increase membrane leakage [ 80 ]. Similar conclusions were also reached when testing the toxic effects of the metal/metal oxide NPs mentioned above on a mouse liver-derived cell line (BRL 3A). The results showed that mitochondrial function was significantly reduced in cells exposed to AgNPs at (5–50 μg ml -1 ). Fe 3 O 4 , Al, MoO 3 , ​​and TiO 2  had no measurable effect at lower doses (10–50 μg ml -1 ), while having a significant effect at higher levels (100–250 μg ml -first ) [ 81 ].

In general, in in vitro assays, the mechanism of cytotoxicity mediated by AgNPs is mainly based on ROS induction. Notably, exposure to AgNPs induced decreased GSH, increased ROS levels, lipid peroxidation and increased expression of ROS-responsive genes; it also leads to DNA damage, apoptosis and necrosis. MTT reduction (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), Alamar Blue reduction (Invitrogen, Carlsbad, CA) and lactate dehydrogenase (LDH) leakage were used as parameters for cytotoxicity assessment, quantification, evaluation. The toxicity of different silver nanoparticles was compared with different concentrations of respective Ag+ ions.

Based on the IC50 values ​​determined by three cytotoxicity assays, AgNPs and Ag+ ions did not exhibit significant differences in cytotoxicity [ 82]. The cytotoxicity and genotoxicity of AgNPs depend on size-, concentration- and duration of exposure. Cell viability was determined by MTT and CB assays in macrophages (RAW 264.7, J774.1), lung epithelium (A549), renal epithelium (A498), liver (Hep G2), and neuron (Neuro 2A) cell line. AgNPs showed a concentration-dependent decrease in cell viability after 72 h incubation in all cell lines. A498  and  RAW 264.7 cells appeared to exhibit the highest sensitivity to the toxic effects of AgNPs. and showed significantly reduced cell viability at AgNP-1 and 3 μg/ml concentrations, respectively.

On the other hand, A549 cells were the least sensitive to the cytotoxic effects of AgNPs. Internal formation of NPs can induce stress response(s) by stimulating free radical production, which, in turn, stimulates inflammatory signaling pathways. Thus, the production of reactive nitrogen species (RNS), ROS and cytokines after exposure to nano silver was determined. AgNPs significantly increased nitrite release by RAWing 264.7 cells at the highest concentration after 72 h of incubation. Nano silver also stimulated concentration-dependent ROS production after 24 h of incubation. Pro-inflammatory cytokine production (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) was significant at 10 and 100 μg/ml while 1 μg/ml showed no effect. affect cytokine production. Free radical production has been shown to be directly correlated with the cytotoxicity of NPs. However, the involvement of other mechanisms cannot be ruled out. Therefore, to determine the contribution of free radicals in AgNP cytotoxicity, cells were incubated with AgNPs in the presence of different antioxidants. Surprisingly, the most potent antioxidants such as superoxide dismutase (SOD) and catalase did not show significant protection from the cytotoxicity of AgNPs.

Thus, two membrane ROS scavengers — Trolox (a water-soluble vitamin E analog) and tempol (a broad-spectrum antioxidant and SOD mimic) — were investigated. Along with the observations in SOD and catalase-treated cells, Trolox and tempol also failed to protect cells from the cytotoxicity of AgNPs. On the other hand, weak antioxidants such as N-acetylcysteine ​​(NAC), methionine and cysteine ​​abrogated the cytotoxic effects of AgNPs. The relative ineffectiveness of potent antioxidants suggests that free radical-dependent mechanisms do not significantly affect the cytotoxicity of AgNPs [weak antioxidants such as N-acetylcysteine (NAC), methionine and cysteine ​​abolished the cytotoxic effects of nanosilver. The relative ineffectiveness of potent antioxidants suggests that free radical-dependent mechanisms do not significantly affect the cytotoxicity of AgNPs [weak antioxidants such as N-acetylcysteine (NAC), methionine and cysteine ​​abrogated the cytotoxic effect of AgNPs. The relative ineffectiveness of potent antioxidants suggests that free radical-dependent mechanisms do not significantly affect the cytotoxicity of AgNPs. [83 ].

Other studies showed that p53 protein expression levels increased within 4 h after cells were exposed to AgNPs. The upregulated expression patterns of p53 protein in two types of mammalian cells exposed to AgNPs suggest that p53 could be an excellent molecular marker for the assessment of genetic nanotoxicity. The results show that different surface chemistry of AgNPs has different effects on genotoxicity [ 84 ]. Beer and associates. concluded that the free Ag+ ions in the AgNPs preparations play a significant role in the toxicity of the AgNPs suspension [ 85]. While the contribution of free Ag+ ions to the measured toxicity of AgNPs suspensions is an essential determinant of toxicity, the combined effect of Ag+ ions and AgNPs appears on ion concentrations. Ag+ is lower. These data indicate that the amount of Ag+ ions in nano silver preparations should be routinely measured and reported in toxicology studies. They recommend that the supernatant of the AgNPs suspension should be used as an additional standard control to make reliable statements about AgNPs toxicity and to distinguish between Ag ions toxicity. and toxicity caused by nano silver [ 85 ].

7.2. In vivo test

The most important issue to understand is the actual impact of AgNPs on human and animal health. There are a number of in vivo studies on cytotoxicity and genotoxicity of AgNPs reported. Due to the ultra-small size of AgNPs, they are highly mobile in different environments and are easily exposed by humans through routes such as inhalation, ingestion, skin, … nanosilver transposable from the pathway of contact with other vital organs and cell entry.

Inhalation toxicity of nanosilver was studied in Sprague-Dawley rats over a period of 28 days. The results showed that male and female rats did not show any significant changes in body weight compared to AgNPs levels during the 28 days of the experiment. There were also no significant changes in hematological and blood biochemical values ​​in male or female rats. Meanwhile, some investigators have reported that the lung is the primary target tissue affected by prolonged inhalation exposure to AgNPs [ 86 ]. Lee et al. reported that nano silver exposure regulates the expression of several genes involved in motor neuron disorders, neurodegenerative diseases, and immune cell function, suggesting potential for neurotoxicity and toxicity Immunity associated with exposure to AgNPs [ 87]. Minimal pneumonitis or cytotoxicity of mice was found 10 days after exposure to nano silver. Gastrointestinal toxicity from gastrointestinal exposure to AgNPs (60 nm) was also tested over a 28-day period in Sprague–Dawley rats. The results showed that male and female rats did not show any significant changes in body weight compared to the AgNPs dose during the 28 days of the experiment. Some significant dose-dependent changes were found in alkaline phosphatase and cholesterol values ​​in male or female rats, which seem to suggest that exposure to more than 300 mg of AgNPs can lead to mild liver injury. The results showed that AgNPs did not induce genotoxicity in the bone marrow of male and female mice in-vivo[  88 ].

Ahamed et al. indicated that AgNPs induce reproductive impairment, developmental malformations, and morphological malformations in several non-mammalian animal models. Common causes of AgNPs-induced toxicity include oxidative stress, DNA damage, and apoptosis [ 79 ].
Collectively, very few papers on the in vivo toxicity of AgNPs have been found, so further investigation in this area is needed to accurately assess the true impact of AgNPs in commercial products on humans. people and animals.

8. Conclusion

Plant-mediated silver nanosynthesis has revealed new horizons in drug delivery. On the one hand, this nanoparticle preparation method is preferred due to its economy, ease of access, environmental friendliness and simplicity of implementation. On the other hand, the rich phytochemical composition of plant extracts performs a multifunctional role in AgNPs synthesis such as reducing, stabilizing and surfactant agents. Therefore, the obtained AgNPs are often characterized by small size, single dispersion and colloidal stability because of the capping properties of some biomolecules in the extract. Despite their excellent antibacterial, antiviral, antifungal, anticancer, antioxidant and physico-chemical properties compared to bulk materials, AgNPs can be used as vehicle for transporting drug molecules (such as oligonucleotides, DNA, siRNA, etc.) to the target tissues and cells and thus to improve the therapeutic effect. Furthermore, nanosilver may exhibit synergies with different antibiotics with respect to enhanced antibacterial properties. In this regard, AgNPs can be used as multifunctional drug transporters with great potential for targeted drug delivery, minimizing side effects and improving treatment efficacy. However, there is still a lack of information regarding the increased human, animal and environmental exposure to nanosilver and the potential risks associated with their short- and long-term toxicity. Further studies are needed to safely include them as DDS in commercially available products for the prevention and treatment of life-threatening diseases.

References: Silver Nanoparticles as Multi-Functional Drug Delivery Systems

By Nadezhda Ivanova, Viliana Gugleva, Mirena Dobreva, Ivaylo Pehlivanov, Stefan Stefanov and Velichka Andonova