Application of green synthesized silver nanoparticles in cancer treatment—a critical review

With the breakthrough in advance technologies, researchers are looking to devise novel approaches to control different types of deadly cancers. Progress in medicinal plants research and nanotechnology has drawn scientist’s attention toward green synthesis of metallic nanoparticles by exploiting plants secondary metabolites owing to its advantage over routinely used physical and chemical synthesis (simple, one step approach to reduce and stabilize bulk silver into silver nanoparticles (AgNPs), cost effectiveness, energy efficient, biocompatibility and therapeutic significance). Owing to control size, shape and functional surface corona, AgNPs hold considerable potentiality for therapeutic applications by opting different mechanistic pathways such as mitochondrial disruption, DNA fragmentation, cell membrane disruption, interruption of cellular signaling pathways, altered enzyme activity and reactive oxygen species (ROS) production leading to apoptosis etc In this review, we discussed the green synthesized AgNPs in the possible cancer treatment by harnessing phytochemicals present in plant extract. In addition, this review also provides recent advances and achievements in utilization of green synthesized AgNPs in cancer treatment and proposes mechanistic action for their anticancer and cytotoxic potential. By understanding the mechanistic action of AgNPs responsible for their therapeutic efficacy will help to devise customized therapies and treatment against cancer as a potential cancer therapeutic tool.


Introduction
Silver, being a noble metal, has been extensively harnessed since ancient times. Hippocrates suggested the use of silver to treat disease and for wound healing [1]. Silver is naturally occurring abundant metal possessing numerous physical, chemical and biological fascinating properties like electrical conductivity, optical activities, nonlinear catalytic effect, surface enhanced Raman scattering, elevated thermal response and various biochemical characteristics making silver most appropriate candidate having diverse biomedical potential i.e. as an antiseptic, integral component of medical and surgical devices, constituents of medicines, antimicrobial potential, significance in drug delivery, cosmetic products, biolabelling, optical properties, larvicidal applications and food preservation usage. Silver nitrate is applied on microbial infections in hospitals. Antimicrobial agents having silver ions are reported to disrupt the outer membrane of targeted cell. Silver ions react with thiol group of proteins and leads to bacterial inactivation. Application of silver results into impaired DNA replication owing to uncoupling of electron transport from oxidative phosphorylation. As a result it inhibits the enzymes of respiratory chain and disrupts the membrane permeability. However, this activity is found at concentrations which are 10 fold higher than those used for AgNPs. Hence, AgNPs are reported to have antimicrobial activities at low concentrations [2,3]. Moreover, silver ions in the form of silver salts, displayed considerably higher toxicity than AgNPs of any size [4]. Silver in ionic form such as AgCl and AgNO3 have

Trends on the application of Green Synthesized Silver Nanoparticles in Cancer Treatment
Silver nanoparticles are holding preeminent role in biomedical applications such as antimicrobial, antiviral, antidiabetic, antioxidant and anticancer. Presence of phytochemicals on the surface of green synthesized AgNPs is attributed to their antimicrobial and anticancer activities [24]. By keeping in view the expanding burden of cancer globally, several research groups have synthesized variety of metallic nanoparticles via green approach. Plant mediated AgNPs have been exhibiting potential anticancer activities against various types of carcinoma cells [25]. Such as banana leaf mediated AgNPs exhibited anticancer potential against A549 and MCF7 cell lines [26], Mangifera indica seed mediated AgNPs against Hela and MCF7 [27], AgNPs formulated by Heliotropium bacciferum showed anticancer potential against HCT-116 [28] and AgNPs using Zingiber officinale displayed anticancer potential against AsPC-1, PANC-1 and MIA PaCa-2 cell line [29].
AgNPs are reported to exhibit effective anticancer activity with great selectivity toward the cancer cells in dose dependent manner. The magnificent selectivity of AgNPs is due to biocompatibility of Ag0 and phytochemicals from plant source that attached on their source. Moreover, recent researches have revealed that plant mediated AgNPs shown their anticancer activity by decreasing the proliferation rate of cancerous cell via cell cycle arrest [30] The physiochemical properties of AgNPs have been confirmed by the immense and constantly increasing amount of literature data availability regarding synthesis of AgNPs (figure 1(A)) and their application in cancer treatment ( figure 1(B)). By keeping in view these critical concerns and growing applications of AgNPs in cancer treatment, this review article highlights the anticancer activities of AgNPs synthesized via green route.

Methods of AgNPs Synthesis
There are different techniques used for synthesis of nanoparticles such as 'top-down' and 'bottom up'. Topdown approach involves the synthesis of nanoparticles from bulk whereas bottom-up starts synthesis from  [23] nanoscaled material at atomic level ( Figure 2). Top-down approach is generally used during physical method of nanoparticles synthesis while the bottom-up approach is manipulated in chemical or plant mediated synthesis. Bottom up approach is useful to get monodispersed nanostructure with lesser flaws [31]. Physical method facilitates the nanoparticles synthesis without solvent and suitable for AgNPs of uniform size formation in contrast to chemical method [32]. Physical route and chemical synthesis of AgNPs includes various different procedures [33] as illustrated in (Figure 2). Chemical reduction process of AgNPs in the solution typically comprises of three main stages such as (1) metal precursors (2) reducing agents and (stabilizing or capping agents). There are various commonly used reducing agents such as N2H4, NaBH4, tri-sodium citrate (TSC), sodium citrate, polyols and N, N-dimethylfarmamide (DMF). Ascorbic acid as a reducing agent advocates the synthesis of flower like silver nano architecture with average size of 20nm at room temperature. There are some capping agents use to stabilize and prevent the AgNPs from agglomeration include sodium dodecyl sulfate (SDS), polyvinyl pyrrolidone (PVP), polymethacrylic acid and polymethyl methacrylate. PVP not only reported to prevent nano materials from  agglomeration but also favor the process of nucleation. In order to synthesize AgNps of uniform size, the simultaneous formation of nuclei is necessary for growth/ capping of nanoparticles [34].
Nanomaterials synthesis by using physical and chemical method restricts their usage to limited zone and their therapeutic applications also confined due to toxicity produced and noxious to environment [35]. However, now researchers focus is diverted toward cost effective and eco-friendly synthesis of AgNPs which can be of plant source or of microbial origin. [36,37]. Biosynthesis of AgNPs can be carried out using microorganisms such as bacteria, fungi, yeast, viruses' DNA, diatoms and plants (figure 3) [38].
Green synthesis, commonly referred as biogenic synthesis is another method for AgNPs synthesis and lead to emergence of novel domain of 'phytonanotechnology' involving green synthesis of metallic nanoparticles by utilizing plant resources followed by their optimization and characterization. The size and shape of AgNPs is determined by type of solvent, reduction and stabilization [39]. Green synthesis comprises the manipulation of natural constituents and environment friendly elements with handling safety which results in product formulation that is nontoxic to human and environment [40]. Table 2 Presents the phytochemicals associated with AgNPs formulation in particular plants. Plants being excellent source of secondary metabolites possess various activities and reduce the bulk metals into metal nanoparticles. In plants, naturally occurring potential to interact with metals and their conversion into innocuous form directed the scientists and nanotechnologists' attention toward the utilization of secondary metabolites in plants as a stabilizing and reducing agent [7,8]. The phytochemical constituents i.e. alkaloids, phenols, ketones, amines and enzymes are not only responsible for plant routine activities but also act as an outstanding deposit of chemicals which is employed to reduce metal in bulk to metal ions. The synthesis of nanoparticles can be optimized by adjusting the physiochemical parameters such as pH, temperature, salt concentration, amount of reducing agents and capping agents [41]. The biological materials utilization has supremacy over physical and chemical method in several ways such as biomass accessibility in excess, cost efficient, handling safety and nontoxicity. Physical method demands for high energy and force which increase the cost value of product and harmful to environment. Whereas chemical method demands costly chemicals which may be hazardous to workers and environment as well [42]. Green synthesis of nanostructures not only restrains the demand of dangerous chemicals but also leads to one step synthesis of nanomaterial [43]. In this review, we have thoroughly discussed the exploitation of plant resources in synthesis of AgNPs, synthesis mechanism and their therapeutic efficacy with respect to anticancer potential.  3.1. Chemical mechanism of green synthesis of AgNPs Large number of studies is performed to explore the potential of plants to produce nanoparticles of various sizes and shapes and to reveal their significant job in biological domain. Although physiological analysis of secondary metabolites which take part in nanoparticles synthesis need to be explored. The investigation of exact mechanism responsible for reducing bulk material into nanomaterial is critical to frame protocol for nanoparticles formulation of required size and shape. Different secondary metabolites present in plants are classified by various functional groups and these function groups confer surface modifications and functional capabilities to green synthesized nanoparticles and provide biomedical potential to nanoparticles ( figure 3). Various studies reported that hydroxyl, carboxylic and amines group are involved in reduction of silver salt [41]. Biogenesis of silver nanoparticles can be described by chemical mechanism proposed (figure 4). During synthesis the first step involves the oxidation of bioactive compound of plant source and release of electrons. These electrons lead of Ag+to Ag0. After reduction, Ag0 undergoes sequence of agglomeration known as nucleation (I) and (II) to form a nanoparticle. Nucleation of silver zero atoms lead to formation of nanoparticles i.e. AgNPs as shown in figure [58]. Presence of various significant bioactive compounds and bonding of their function groups with metallic salt provides stabilization to nanoparticles. Presence of phenolic compounds on the surface of nanoparticles is attributed to prevent their coalescence [59].

Cancer: A Global Threat
Cancer is reported as leading cause of death globally, 10 million people are estimated to have died from cancer and about 19.3 million fresh cancer cases reported in 2020 [60]. The global cancer burden will upsurge to 27 million fresh cancer cases by 2040 [61]. Onset of cancer and metastasis is due to uncontrolled cell division and its invasion into surrounding healthy cells and tissues [62]. Any mutation in proto oncogenes and tumor suppressor genes results into cancer [63]. It is estimated that 1 out of 6 deaths is caused by cancer and approximately 70% of all cancer deaths reported from middle and low income countries [64]. It is estimated that 1 out of 5 people develops cancer before age of 75 years and one out of ten would die in this age range suffering from cancer [65]. Higher growth rate of cancer is denoting that cancer occurrence will 60% increase by 2030 [66]. The cause of cancer can be categorized as external and internal factors. Viruses, radiations and chemical exposure are external cause while mutations, hormones and internal conditions are internal factors which can stimulate carcinogenesis [67,68].
Lung, thyroid, cervical, liver, stomach, colorectal, prostate and breast cancers are most common types of cancer diagnosed in humans. Most frequently occurring cancer among women and men are breast and prostate cancer respectively [63]. Surgical excision of tumor or cancerous part, chemotherapy to kill cancer cells, endocrine and radioactive therapy is used to treat cancer traditionally [69]. Hormone therapy and immunotherapy are lesser used secondary approaches for cancer treatment as they may cause abnormalities and side effects inside patient's body including damage to healthy cells and different organs due to decline in life quality [70]. Nonspecificity, less bioavailability, rapid clearance and toxicity are some other side effects [71]. Manipulation of chemotherapeutic agents may cause various types of toxicities. 5-fluorouracil is a common used therapeutic agent which results into myelotoxicity, cardiotoxicity and blood vessels constriction [72]. Moreover, cyclophosphamide and bleomycin are associated with bladder toxicity and cutaneous toxicity respectively [73]. Keeping in view the scenario, there is needed to investigate novel approaches and develop promising and systematic vehicle for effective cancer therapy with limited side effects.

The Interface of Phyto-Nanotechnology and Cancer
There is novel emerging technology utilizing cost effective therapeutic entities from natural resources i.e. plants, opposing to established approaches for cancer treatment [74].
Medicinal plants have open up new prospects and possibilities for cancer treatment. They are not only source of new compounds upon phytochemical screening but also help to develop novel approaches to treat cancer such as green synthesis of AgNPs [75]. In the recent years, paradigm shift in methodologies and approaches found concerning cancer treatment. Progress in medicinal plants research and nanotechnology developed a variety of new strategies toward various types of cancer treatment [76]. This coalescence of nanotechnology and plants for green synthesis of nanoparticles has drawn the attention of researchers for cancer treatment [77,78]. These green synthesized nanoparticles may overthrow the obstacles and complications of conventional diagnosis and treatment therapies [79]. Table 3 presents the anticancer results of green synthesized AgNPs from recently conducted studies.
The cancer cells differentiate from normal healthy cells in the context of blood vessels proliferation (angiogenesis), reduced permeability of capillaries and lymphatic drainage system. This altered microenvironment of cancerous cells provides a platform to nanotechnologists to device suitable nanodrug having selective advantage to precisely target cancer cells [111]. Currently, research studies are paying focus on the synthesis of anticancer drugs and to device a tool which can specifically and precisely target cancer cells with optimized drug concentration and less adverse effects [112]. Cancer nanobiotechnology has promising capacity to improve the detection, diagnosis and treatment of cancer [113]. At present, AgNPs have been widely explored for cancer diagnosis and cancer therapy. Green synthesized AgNPs featured with phytochemicals coating furnish them increased biological actions than AgNPs synthesized via chemical method. Apart from potential application of green synthesized AgNPs in cancer treatment, AgNPs reported to have various other biomedical applications such as antimicrobial, antileshmanial, antiviral, wound healing, targeted drug delivery, antitubercular and antidiabetic potential [114,115].
Several studies have reported the anticancer property of plant mediated AgNPs against various cancer cell lines such as HEPG2 (human liver cancer cells), Hep-G2 (liver cancer), COLO 205 (colon cancer), MCF-7 (human breast cancer), PC3 (human prostate cancer), HCT-116, HCT-15 (human colon adenocarcinoma), VCaP (prostate cancer), AGS (human gastric carcinoma), SiHa (cervical cancer), Caco-2 (intestinal adenocarcinoma), HeLa (cervical cancer), Hek-293 (kidney cancer), H1299, A549 (lung cancer), PA1 (ovarian cancer), HL-60 (human promyelocytic leukemia cells), B16 (mouse melanoma cell line), A431 (epidermoid carcinoma) and BxPC-3 (pancreas cancer) [106,116,117] Nanoparticles possess site specific and targeted action which enhances the drug efficiency as nanoparticles move across impermeable membrane and could tackle the immune system response, so suitable for cancer treatment [118]. Green synthesized AgNPs revealed their efficacy against several cancers such as breast cancer, colon adenocarcinoma, Ehrlich ascites carcinoma and liver cancer. Green synthesized AgNPs application in different concentrations showcased the promising anticancer potential against lung cancer, liver cancer, cervical and carcinoma. However, there is need to explore the anticancer mechanism of green synthesized AgNPs. AgNPs synthesized from mint extract exhibited notable activity againt HCT116 colon cancer cells in human. These green synthesized AgNPs delayed the cell division in G1 phase revealing that the AgNPs can control the cell cycle and reduce their proliferation [119]. AgNPs syhtesized from leaf extract of Piper nigrum shown efficacy against MCF-7 and Hella cancer cell lines [120]. The green synthesized AgNPs have shown change in morphological parameters when apllied against MCF-7. Significant disruption in plasma membrane integrity and inhibition in cell growth was found. Moreover, there was shrinkage of cytoplasm and cells aggregation was recorded as compared to healthy cell with AgNPs treatment. AgNPs synthesized from Prosopis cineraria and Coriandrum sativum indicated potent anticancer activity against MCF-7 cancer cells [121].
Green synthesized AgNPs have shown promising toxic effects against A549 (human lung carcinoma cell) as compared to non-cancerous lung cells, revealing that AgNPs could induce toxicity specifically in target cells. This site specific toxicity might be due to particular acidic pH of cancer cells [122].
Zureberek et al also provided evidence about engrossing capability of AgNPs to affect the cell viability specifically. They explored the status of respiration in oxidative stress induced by AgNPS. By keeping in view that cancer cell's key source of energy is glucose, researcher associated the glucose availability with nanoparticles toxicity. The production of H 2 O 2 by AgNPs application in dose dependent manner was observed [123]. Furthermore, less AgNPs induced toxicity found in cell with little glucose supply than cell with high glucose availability. This finding suggested that limited supply of glucose lead to upregulation of antioxidant defense system which ultimately regulated ROS production and AgNPs induced toxicity [124]. Kovacs et al reported the significant tumor killing activity of AgNPs in osteosarcoma cancer cells with tumor suppressing deficiency. They observed that AgNPs resulted into apoptosis in U2Os (wild type p53) and SOAS-2 cells with deficiency of p53, exhibiting their chemotherapeutic potential [125]. Similarly, in a comparative study AgNPs synthesized by using extract of 30 medicinal plants demonstrated pronounced cytotoxicity against lung cancer cells. Increased toxic effects observed on AgNPs incubation in culture media supplied with bovine serum albumin as it regulated the protein corona interactions [126]. AgNPs synthesized from tamarind fruit shell resulted into apoptosis in human breast cancer cells. Anticancer activity was observed in dose dependent manner as augmented ROS generation resulted into mitochondrial damage and DNA impairment [127]. In another study AgNPs synthesized from Nepeta deflersiana exhibited potent cytotoxicity against human cervical cancer cells. AgNPs induced oxidative stress which leads to mitochondrial impairment and cell cycle arrest followed by death of tumor cells [128]. AgNPs obtained from lotus extract exhibited cytotoxic effects against liver, gastric and prostate cancer cells [129]. AgNPs synthesized from fruit of Crataegus microphylla also showed significant impairment of gastric adenocarcinoma cells. 74 Kanipandian and kannan, reported the mitochondrial dependent death of lung adenocarcinoma cells upon exposure to nanosilver synthesized from cotton leaf [130].
AgNPs apart from their effects on cellular and subcellular structures, also remarkably work against tumor angiogenesis which is behind altered expression of growth factor and inhibited migration and proliferation of endothelial cells [131]. AgNPs not only cause apoptotic death in breast cancer but also restricted the transcription of hypoxia inducible factor-1 (HIF-1) and induced the expression of vascular endothelial growth factor-A (VEGF). Antiangiogenic activity of AgNPs reported by protection from tube formation in normal endothelial cells [132]. Similarly, the significant role of AgNPs reported in enhanced expression of caepase-3 and caspase-8 genes upon inoculation in chorioallantic membrane which leads to cellular apoptosis. AgNPs synthesized from red amaranth, reduced the number and length of blood vessels and demonstrated cytotoxicity against breast cancer cells [133].
Beside their magnificent anticancer efficacy, special focus was oriented toward development and analysis of novel silver based nanotechnology for improved chemotherapy and radiotherapy. A study in this context performed on sliver-gold nanoparticles coated by dopamine and exposed to near infrared radiation, evaluated for their photothermal induced cytotoxicity against colon cancer cells. In vitro and in vivo study provided evidence about nanoparticles induced photothermal therapy (PTT) via apoptotic and necrotic pathways [134]. He et al investigated the AgNPs core based multifunctional core-shell nanosystems and molecules with aggregation induced emissions. This complex nanosystem significantly improved the radiotherapy and regulated PTT and photo acoustic effect. They also showed more pronounce role as contrasting agent in tomography imaging and as a fluorescent [135].
Silver/magnetite nanoparticles along with PEG coating and folic acid, loaded with chemotherapeutic drug; Doxorubicin reported to have outstanding potentiality for PTT of cervical cancer. Apart from their chemotherapeutic as well as photothermal effect, this nanosystem also showed high selectivity for targeted cancer cells and imaging properties owing to their fluorescence and magnetic resonance [136]. Dual chemotherapeutic and photothermal effect resulted from laser irradiation of nanosystem based on Methotrexate conjugated graphene oxide (GO) and AgNPs lead to high cytotoxicity against malignant cells [137]. Similar results reported by using 5-fluorouracil loaded silver-gold nanoparticles coated with mesoporous SiO 2 [138].

Phytonanotechnology and cancer treatment: a mechanistic approach
AgNPs due to their small size and potentiality to prompt cell death by various mechanisms are considered as phenomenal candidate for cancer treatment. AgNPs cause cell death by breaking double stranded DNA, chromosomal instability and oxidative stress. AgNPs of larger size (∼100 nm) produce these results more effectively than smaller size but AgNPs of smaller size (∼10 nm) induce cellular toxicity at greater level because they enter the cell and get localized inside nucleus smoothly. AgNPs are reported to induce cytotoxicity in mammalian cells via various mechanisms such as (a) disturbance in energy dependent cellular processes and impaired DNA replication induced by uptake of free silver ions (b) generation of reactive oxygen species (ROS) and free radicals (c) cell membrane damage owing to direct interconnection with AgNPs [139,140]. Avalos et al explored AgNPs of two different sizes i.e. 4.7nm and 42nm for their induced cytotoxicity in normal human dermal fibroblasts. Nanoparticles of smaller size were found to be more toxic than larger size particles observed through (3-(4, 5-dimethylthiazol-2-5-diphenyletrazolium bromide) (MTT) and lactate dehydrogenase assays [141].
Green synthesized AgNPS induce production of reactive oxygen species (ROS) which leads to cell death. ROS generation adversely strike the signal transduction pathways followed by cell apoptosis. Hydrogen peroxide generation affects the membrane potential of mitochondria resulting into uncoupling of respiration [142]. AgNPs after entry into the cell induce ROS generation and reduction of glutathione (SGH) level, activation of nuclear factor k B (NFk B) and tumor necrosis factor-alpha (TNF-α). The elevated superoxide radicals' level alters the mitochondrial transmembrane potential and disturbs the signal transduction pathway which triggers apoptosis and cell death [143,144]. The increased ROS production and decreased GSH evokes cellular components damage such as DNA fragmentation, peroxidation of lipid membrane and protein carbonylation (protein harmful oxidation). Figure 5 shows the possible mechanism for anticancer potential of green synthesized AgNPs. Furthermore, altered mitochondrial membrane potential lead to activation of caspases 3 and 9 leading to cellular apoptosis. Subsequently, it activates c-Jun NH 2 terminal kinase (JNK) which triggers formation of apoptotic bodies and DNA breaks results into cell cycle arrest [145]. New blood vessels formation (angiogenesis) is responsible for tumor formation as suggested by Folkmans's hypothesis. According to this hypothesis, blood supply is essential for growth and spread of tumor. These new blood vessels supply nutrients and oxygen to cancer cells helping them to invade surrounding tissues. Green synthesized AgNPs demonstrated efficacy against retinal neovascularization (RNV) like disease. AgNPs inhibited the RNV induced by vascular endothelial growth factor and blocked the activation of extracellular signal related kinase (ERK1/2) by regulating vascular endothelial growth factor receptor −2 phosphorylation. AgNPs due to these anti-angiogenic properties have been utilized in cancer treatment [146].
Apoptosis induced by caspase dependent and mitochondrial dependent pathways, cell cycle arrest in sub-G1 phase, generation of ROS and disturbance of cellular equilibrium, activation of p53 protein and casepase 3 upregulation, pH dependent release of silver ions and selective killing of cancerous cells and VEGF induced activities inhibition are proposed mechanism for anticancer action of green synthesized AgNPs [147]. Furthermore, green synthesized AgNPs induced p53 protein upregulation which leads to cellular toxicity or death is another suggested mechanistic pathway by nanotechnologists [148]. Similarly, another study reported the AgNPs induced p53 upregulation via Western blotting in PB16 cells. Furthermore, activation of apoptosis proposed to be responsible for upregulation of p53. Green synthesized AgNPs exposure induce oxidative stress and activates caspase-3 which are considered as key reason of cell death [149]. AgNPs treatment triggers the activation of caspase-3 expression. Now, it is experimentally proven that green synthesized AgNPs stimulates P53 upregulation by activation of apoptosis followed by cell death [150]. Gurunthan et al demonstrated that silver ions released by AgNPs were responsible for upregulation of caspase-3 leading to cell death [122]. Furthermore, acidic environment of cancerous cell attributed for release of phytochemicals from green synthesized AgNPs which boosts the anticancer effect of AgNPs [71].
AgNPs synthesized from Mentha arvensis demonstrated the activation of caspase-9-dependent apoptosis in MCF-7 cells and resulting into toxicity against breast cancer cells. These nanoparticles also exhibited cytotoxicity against Hep2 cells via activation of caspase-9-dependent apoptosis [151]. Green synthesized AgNPs also showed potent cytotoxicity during cell cycle analysis by exhibiting significant increase in sub-G1 cell proliferation. Mao et al reported the relation between enhanced populations of cancer cells in sub-G1 phase and activation of caspase-3, which lead to apoptosis [152]. Similarly, Chang et al reported the connection between sub-G1 phase arrest of cancer cells and apoptosis by showing sub-G1 arrest in cancer cells treated by curcumin [153]. So, this suggests that green synthesized AgNPs induced death of cancer cells might be caused by increased sub-G1 arrest of cancer cells which is interrelated with apoptosis stimulation.
Death of cancer cells is displayed by release of silver ions from green synthesized AgNPs. Hence, selective killing of cancerous cells is direct by concentration of silver ions released inside cells. Release of silver ions in normal cell lines differ from cancer cell lines in different pH. Electrostatic interaction between normal cells and cancer cells also determines the silver ions release [154]. Selective killing of cancer cells also depends on wide ranging electrostatic interaction between normal and cancer cells [155]. Exorbitant release of silver ions strikingly found at acidic pH. AgNPs on incubation in buffers with pH 5 and pH 7.4, reported to release more silver ions at low pH that proves the selective killing of cancer cells at acidic pH. Green synthesized AgNPs are also proposed as anti-angiogenic agent by some analysts [156]. Biosynthesized AgNPs demonstrated their antiangiogenic properties by hampering cell proliferation induced by vascular endothelial growth factor (VEGF). AgNPs after entering cell inhibited the VEGF and 1L-1β induced vascular permeability through Src-dependent pathway [121]. Green synthesized AgNPs owing to their anti-angiogenic efficacy suggested as could provide a new gateway for cancer treatment. Autophagy induced cell degradation which leads to cell death are reported as another mechanism proposed for anticancer potential of AgNPs. Additionally, green synthesized AgNPs promotes autophagy because of autophagolysosomes accumulation in cancer cells and their increased level subsequently leading to cell death [85].

Toxicity of Green Synthesized AgNPs
Nano biotechnology has been flourishing with wide range applications of their commercialized products globally. However, there is need to investigate the consequences of increased exposure of animals, human and environment to nanoparticles particularly AgNPs and their potential hazards in terms of acute and chronic toxicity [157]. In vivo and in vitro potential hazards of green synthesized AgNPs to cells and their possible risks are reviewed here. Various studies have reported acute as well as chronic in vivo toxicity of different nanomaterials containing silver also [158,159]. Bioavailable silver ions reported as toxicity inducing agent in zebrafish embryos [160]. However, more in vivo cytotoxicity and genotoxicity of chemically produced AgNPs found than green synthesized AgNPs which suggests green synthesized AgNPs are less toxic and biologically compatible than chemically synthesized one [161]. Undetectable level of toxicity observed in healthy volunteers upon oral administration of AgNPs prepared commercially [162].
Overdose of AgNPs was found toxic and resulted into various health issues in animals and humans [163,164]. Furthermore, latest studies demonstrated the neurotoxicity, hepatotoxicity, cytotoxicity, pulmonary inflammation and genotoxicity which are outcome of over dosage of AgNPs of different shapes and sizes [165]. AgNPs application resulted into impaired mitochondria function and leakage via cell membrane in vitro which induce toxicity in mouse germ line stem cells. It was also demonstrated that AgNPs resulted into toxic response on expression of cytokines and proliferation through peripheral blood mononuclear cells (PBMC) production. Overdose of AgNPs reported to induce toxic effects on reproductive system in males. A research study reported the cytotoxicity of AgNPs on rats in vivo. Histopathological studies revealed the more frequent bile duct hyperplasia, pigmentation and fibrosis associated with excessive use of AgNPs [166].
Various studies reported that green synthesized AgNPs induce ROS generation and oxygen species (OS) which results into cytotoxicity and genotoxicity [148]. There is relation between ROS production and cytotoxicity. Exorbitant production of ROS leads to enhanced toxicity of green synthesized AgNPs which in turn conferring them more toxic effects [167]. Over production of ROS cause oxidative stress and in turn cell becomes unable to carry out routine physiological activities. The critical damage to physiological functioning and cell development result into protein oxidation which produce protein radicals and initiates lipid peroxidation , DNA's strand breakage, inflammatory response regulation via multiple pathways, regulation of gene expression by activating different transcription factors, cytotoxicity, genotoxicity , cell membrane disruption by increased ion permeability and subsequently inducing cell death through apoptosis [168]. The adverse effects induced by ROS generation from green synthesized AgNPs can be reduced. In the given context, a study reported that alkaloids, tannins and polyphenols are potent ROS scavengers, thus nanoparticles with their coating can inhibits the ROS generation and avoiding cellular damage [169]. Additionally, AgNPs synthesized from leaf extract of Butea monosperma exhibited effective cytotoxic response against cancer cells as compared to non-cancer cells. This response is attributed to the phytochemicals found in plant extract i.e. aldehydes, ketones, flavones, terpenoids, amides, quinones and carboxylic acid that are involved in reduction, capping and stabilization of AgNPs.
[174]. These findings from latest studies provides a new way for AgNPs utilization in clinical trial for cancer treatment in near future [98,99,144]. However, there is need to explore toxicological attributes together with pharmacodynamics and pharmacokinetics of metal nanoparticles apart from strengthening them as potent anticancer tool. Taking into account the less toxicity of green synthesized AgNPs, plant mediated AgNPs could be advocated as potent futuristic candidate for potential biomedical application owing to their significant specificity and lesser toxicity.

Future Perspective
In spite of recent advancements in the treatment of cancer, cancer is still leading cause of deaths worldwide. At the same time we are also familiar with side effects of traditional treatment approaches. While at present AgNPs has attain much attention in pharmaceutical sector due to their non-toxicity, cheap and environment friendly approach. AgNPs owing to their clearance potential and high biodegradability nature prevents from the effects of long term toxicity. Green synthesized AgNPs displayed outstanding efficacy as anticancer agent in vitro. However, clinical trials of green synthesized AgNPs based nanomedicines are needed to find the future direction of their application. Currently, biodegradability, dose and AgNPs route of administration involving studies are most significant concerns to be tackled out in clinical trials. Keeping in view the excellent anticancer activities of these AgNPs it is believed that green synthesized AgNPs will be applied as potential anticancer agent in upcoming are of cancer treatment. Moreover, business experts have anticipated that global market of nanotechnology has bright future.

Conclusions
The biogenesis of nanomedicine possess a considerable scope in 21st century to treat various diseases by devising dynamic drug delivery tools to deliver drugs effectively to selective targeted sites. After looking at the outstanding and significant research outcomes of green synthesized AgNPs it is anticipated that AgNPs have therapeutic efficacy to treat different deadly diseases owing to their pharmaceutical applications. AgNPs have physical as well as chemical advantage of ROS production, fine absorption and smooth penetration inside cell's cytoplasm and entry to nucleus to produce their deleterious result on cancerous cells.
The significant anticancer potential of green synthesized AgNPs come up with breakthrough in metastatic cancer treatment. We also reviewed the most recent research studies about toxicology activity of AgNPa at cellular level which presents it as effective anticancer agent with pronounced therapeutic efficacy. By keeping in view the excellent potential of AgNPs and biocompatibility at the same time, it is expected that green synthesized AgNPs could be used for development of commercialized nanomedicine for cancer treatment in near future. However, this advance silver nanoapproach is facing hurdles in usage as therapeutic agent in vivo in term of toxicity. To resolve this issue and for their preclinical in human beings, the green synthesized AgNPs should be nontoxic and free from side effects. There is also need to understand the various physiochemical parameters such as pH, temperature, concentration of silver salt to that of plant extract and incubation time to optimize the synthesis of nanoparticles of controlled size, shape and biochemical nature. Green synthesis of silver nanoparticles is still in its beginning and such efforts will refine the nanoparticles synthesis at massive level to produce nanoparticles of potential biomedical applications effective for customized cancer therapies.

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No new data were created or analysed in this study.

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Conflict of Interest
The authors declare no competing interests.

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