Silver nanoparticles: therapeutical uses, toxicity, and safety issues.

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Silver Nanoparticles: Therapeutical Uses, Toxicity, and Safety Issues CAROLINA ALVES DOS SANTOS,1 MARCELO MARTINS SECKLER,1 AVINASH P. INGLE,2 INDARCHAND GUPTA,2,3 STEFANIA GALDIERO,4 MASSIMILIANO GALDIERO,5 ANIKET GADE,2,6 MAHENDRA RAI2 1

Department of Chemical Engineering, Polytechnic School, University of Sao Paulo, Brazil Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India 3 Department of Biotechnology, Institute of Science, Aurangabad 431004, Maharashtra, India 4 Department of Pharmacy, CIRPEB, University of Naples, “Federico II” and Istituto di Biostrutture e Bioimmagini, CNR, Naples 80314, Italy 5 Department of Experimental Medicine, Division of Microbiology – II University of Naples, Via De Crecchio 7, 80138, Naples Italy 6 Department of Biology, Utah State University, Logan, Utah 84322 2

Received 11 February 2014; revised 7 April 2014; accepted 9 April 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24001 ABSTRACT: The promises of nanotechnology have been realized to deliver the greatest scientific and technological advances in several areas. The biocidal activity of Metal nanoparticles in general and silver nanoparticles (AgNPs) depends on several morphological and physicochemical characteristics of the particles. Many of the interactions of the AgNPs with the human body are still poorly understood; consequently, the most desirable characteristics for the AgNPs are not yet well established. Therefore, the development of nanoparticles with well-controlled morphological and physicochemical features for application in human body is still an active area of interdisciplinary research. Effects of the development of technology of nanostructured compounds seem to be so large and comprehensive that probably it will impact on all fields of science and technology. However, mechanisms of safety control in application, utilization, responsiveness, and disposal accumulation still need to be further studied in-depth to ensure that the advances provided by nanotechnology are real and liable to provide solid and consistent progress. This review aims to discuss AgNPs applied in biomedicine and as promising field for insertion and development of new compounds related to medical and pharmacy technology. The review also addresses drug delivery, toxicity issues, and C 2014 Wiley Periodicals, Inc. and the American Pharmacists the safety rules concerning biomedical applications of silver nanoparticles. Association J Pharm Sci Keywords: nanotechnology; silver nanoparticles; therapeutics; antimicrobials; toxicity; drug delivery; coating; synthesis; biomaterials

INTRODUCTION Metal nanoparticles in general and silver nanoparticles (AgNPs) in particular have attracted much attention in scientific research because of their versatility in different areas as engineering, medicine, chemistry, and physics.1–4 Now- a -days, with the fast development of microbial resistance against traditional antimicrobials and the difficult insertion of new drugs/compounds, there is a need to search for promising alternatives, which is an important question of public health safety.5 The development of antimicrobial-resistance mechanisms impact in public health system is responsible for excessive time in the treatment of diseases, longer duration of hospitalization, and due costs involved with equipment’s and facilities. The biocidal activity of AgNPs depend on several morphological and physicochemical (e.g., size, shape, and surface) characteristics that influence directly in the success of these compounds as antimicrobial agents. Enhanced antibacterial action of the AgNPs has been shown for the smallest particles within the nanometer size range that seems to improve the permeability of Ag ions in microbial cells facilitating the cell death.6 Moreover, the presence of certain capping agents also improve their biocidal effectiveness. Initially, it was believed that polymeric systems used were biologically inert and that they have the ability to (1) Correspondence to: Mahendra Rai (Telephone: +91-9422857196; Fax: +91721-2662135; E-mail: [email protected], [email protected]) Journal of Pharmaceutical Sciences C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

protect the drug, (2) increase the half-life of these in vivo components, and (3) facilitate their diffusion. However, this paradigm has been modified because of recent evidence that these polymeric materials can drastically change the cellular response of microorganisms.7,8 Application of AgNPs in biomedical devices should possess other features related to their interaction with the human body: they should be biofilm-inhibiting, noncytotoxic, and nonthrombogenic.9 The prevention of biofilm formation has been shown for catheters coated with AgNPs10,11 and their cytotoxicity against human cells is significantly lower than prokaryotic organisms.12,13 Unfortunately, many of the interactions of the AgNPs with the human body are still poorly understood; consequently, the most desirable characteristics for the AgNPs are not yet well established.14 Therefore, the development of nanoparticles with well-controlled morphological and physicochemical features for application in the human body is still an active area of interdisciplinary research. Many routes have been proposed for the synthesis of AgNPs, the most straightforward ones being those based on the chemical reduction of the Ag+ ions from aqueous solution. Citrate, D-glucose, ascorbic acid, aldehydes, amines,15 polysaccharides including chitosan,16 and leaf extracts17 have been used as reducing agents. The elementary steps in the formation of AgNPs are thought to be the generation of a neutral silver atom that subsequently forms positively charged precursors Ag2 + , Ag4 2+ , and larger clusters, which upon further aggregation, form the final nanoparticles.18 Challenges in chemical route are related Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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to avoid particles aggregation in the micrometer range and production of final particles free from impurities. The quality of nanoparticles obtained in ionic process is directly influenced by very small amount of absorbed water and chloride contamination, and leftover starting reagents will be destabilized and/or affect the morphology of metal nanoparticles that may affect the quality of particles.19 Several strategies have been applied to obtain stable dispersion of nanoparticles: (1) addition of a stabilizing agent that prevents aggregation and sometimes hampers molecular growth; (2) conducting the synthesis in two steps to separate the nucleation from the aggregation phenomenon4,20–22 ; (3) ripening driven processes to lead to uniform particle sizes or shapes23–25 ; and (4) microreactors enable robust reproducible synthesis of AgNPs with well-defined characteristics as they provide a better controlled hydrodynamics.26 Recently, emphasis has been made on the socalled green synthesis,15,27 which relies on benign reactants, solvents, and byproducts, producing particles with interesting characteristics for medicine application. Biogenic route is another important source of silver nanoparticles, which utilize microorganisms, such as bacteria and fungi to synthesize nanoparticles. In this route, the size of particles seems to be smaller than that produced by chemical routes and the stabilization of them is made by proteins produced as a result of microbial metabolism. In this process, the principal challenges are related with the growth of microorganism, contaminants, and the development of strains more specific for this use.28,29 Advances in the production of nanoparticles and their applications are also the consequence of the development in the characterization techniques that permit to analyze structural, morphological, compositional, and optical behavior using X-ray diffraction, scanning electron microscopy, energy dispersive Xray analysis, and UV–Visible spectroscopy.6 Apart from the antimicrobial application of AgNPs, they also have various applications in some other fields such as diagnosis and therapeutics and antiparasital efficacy and applications in drug delivery for life-threatening diseases. This review is mainly focused on antimicrobial and therapeutic uses of AgNPs, their role in drug delivery, toxicity issues, and most importantly the need of safety issues for the use of AgNPs.

THERAPEUTICAL USES OF AgNPs Nanotechnology is directly linked to physics, chemistry, biology, material science, and medicine. It finds application in multiple aspects of research and in everyday life. The availability of new nanocomposites and AgNPs has determined a rapid expansion of nanomedicine through their development and incorporation into a range of products and technologies. Their application can be broadly divided into diagnostic and therapeutic uses. We have witnessed an increased application of AgNPs in several fields such as molecular imaging, drug delivery, diagnosis, and the treatment of vascular diseases, wound healing, and the development of materials and medical devices with antimicrobial properties. The details of AgNPs and their mechanism of action against some pathogens are mentioned in Table 1. Therapeutical and Diagnostic Uses of AgNPs in Cancer Early diagnosis to any disease condition is vital to ensure that early treatment is started and perhaps resulting in a better chance of cure. This is particularly true for cancer. The deDos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

velopment of novel nanoparticles for the labeling, targeting, and therapy for tumors is great.49 The effectiveness of a therapeutic agent for cancer stands in its ability to reduce and eliminate tumors without harming the neighboring healthy tissues. Nanoparticles peripherally conjugated with targeting moieties offer major improvements in therapeutics through site specificity. Photodynamic therapy (PDT) is a kind of phototherapy using a combination of nonionizing radiation and lightsensitive (sensitizing) compounds, whereas radiotherapy (RT) employs high-energy-ionizing radiation (X-rays, gamma rays, electrons, protons, and light ions) that acts directly on DNA and cellular compartments without sensitizing substances. Both are conventional well-established cancer treatment modalities with their own advantages and disadvantages.50,51 More efficient photosensitizers are needed for PDT, whereas the ionizing radiation used in RT has the ability to penetrate much deeper into tissues depending on the type and energy of the radiation used (photons and charged particles), but is not selective and requires high accuracy in radiation delivery to increase energy deposition in the target tumor, minimizing damage to the surrounding normal tissues. To make RT more effective, it is necessary to apply the photosensitization concept employed in PDT. A photosensitizer/radiosensitizer would selectively absorb the radiation, thus increasing the amount of intracellularly generated free radicals. AgNPs could allow improvement of the therapeutic efficacy and minimization of negative effects on healthy surrounding tissues, thanks to their ability to absorb both ionizing and nonionizing radiation. Kleinauskas et al.52 have investigated novel carbon nanodots coated with a silver shell, which can be applied in both PDT and RT. Because of the high electron density in the coated metal, Transmission Electron Microscopy images of the C–Ag–PEG nanodots show that the silver forms no islands but a uniform layer. Boca-Farcau et al.53 used silver nanotriangles that were biocompatibilized with chitosan (bio) polymer, labeled with paraaminothiophenol Raman reporter molecule, and conjugated with folic acid. Conjugated particles were proved to be highly stable in aqueous and cellular medium. The targeted uptake of conjugated nanoparticles by human ovary cancer cells was confirmed by dark field microscopy and scattering spectra of the particles inside cells. Targeted cancer cell treatment conducted by irradiating the nanoparticle-treated cells with a continuous wave near-infrared laser in resonance with their plasmonic band has provided an efficient therapeutic response. Zhao et al.54 developed a multifunctional magnetic iron/silver nanocomposite particle functioning with cetuximab, a monoclonal antibody designed to target the epidermal growth factor receptor, an attractive target of many cancers, which is strongly associated with tumor metastasis, recurrence, and poor overall survival.55 The Fe(3)O(4)/Ag–cetuximab nanoparticle presented dose-dependent cytotoxicity with an IC50 concentration of 350 ± 3.14 :g/L; however, when used in combination with radiation at 30 mg/L (about 10% of the IC50 concentration), significant radiosensitization was achieved. Xu et al.56 investigated AgNPs as sensitizers for the treatment of radioresistant glioblastoma tumors and reported variable efficacy dependent upon nanoparticle size and concentration. The extent of the sensitizing capability was further enhanced using higher concentrations of the nanoparticles, while maximizing the surface area to volume ratio by opting for smaller-sized particles. The mode of sensitization was believed to depend on the release of Ag+ cations, which subsequently captured free DOI 10.1002/jps.24001

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Table 1.

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Details of AgNPs and Their Mechanism of Actions Against Different Organisms

Organisms

Size (nm)

Coating

Mechanism of Action

Gelatin Naked Naked Gelatin Chitosan Naked Chitosan Naked PEG Naked

Alteration of membrane permeability and respiration

S. epidermidis

9.68 1–10 13.5 9.68 55 and 278 1–10 55 and 278 13.5 11.23 14

S. typhi

55 and 278

Chitosan

1–10 14 11.23 1–10

Naked Naked PEG Naked

10 1–10 1–10 10 40 10 10 10 10–80 25 10 and 25

Naked Naked PVP Naked Mercaptoethanesulfonate Naked Naked Naked Polysaccharide – Uncoated and polysaccharide

Parasites F. hepatica L. tropica

4.66 10–40

Naked Naked

P. falciparum

35–55 100

Bacteria E. coli

P. aeruginosa

S. aureus

V. cholera Viruses HIV-1

HSV-1 HSV-2 HPIV-3 H1N1 influenza A virus MPV VACV TCRV

Fungus C. albicans

Alteration of membrane permeability and respiration Irreversible damage on bacterial cells

Inhibition of bacterial DNA replication, bacterial cytoplasm membranes damage, modification of intracellular ATP levels Inhibition of bacterial DNA replication, bacterial cytoplasm membranes damage, modification of intracellular ATP levels

Alteration of membrane permeability and respiration gp120 interaction

Competition for the virus binding to the cell Competition for the virus binding to the cell Inhibition of virus binding to the plasma membrane Reduction of cell apoptosis virus induced Block of virus host cell binding and penetration Inhibition of macropinocytosis Inhibition of viral replication

30 31 32 30 33 31 33 32 34 35

33

31 35 34 31 36 37 38 39 40 39 39 36,41 42 43 44

Naked

Not available Inhibition of the proliferation and metabolic activity of promastigotes Not available

47

Naked

Alteration of membrane integrity

48

electrons, generating an oxidative agent. It further reduced adenosine triphosphate (ATP) production and increased the synthesis of intercellular reactive oxygen species (ROS).57,58 In addition to enhancing the generation of potentially damaging radicals, AgNPs have also been shown to negatively regulate the activity of DNA-dependent protein kinase, a key enzyme involved in DNA damage repair via nonhomologous end joining. This finding is particularly relevant, as the primary mode of radiation-induced cytotoxicity is the generation of potentially lethal double-strand breaks. Cheng et al.59 reported the synthesis of Au/Ag NPs conjugated with fluorescent nanodiamonds for cell labeling and photodermal therapy. Moreover, they demonstrated that this multifunctional material was easy to synthesize, noncytotoxic, and could be used in highly efficient photothermal therapy against cancer cells. Lin et al.60 reported AgNPs-based surfaceenhanced Raman spectroscopy in noninvasive cancer detection. This approach is highly promising and may prove to be an indispensable tool for the future. Tse et al.61 presented a novel DOI 10.1002/jps.24001

Irreversible damage on bacterial cells

Reference

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method to selectively destroy cancer cells. Human epidermoid cancer cell line was targeted with floated silver–dendrimer composite nanodevices and the labeled cancer cells were subsequently destroyed by the microbubbles generated through increased uptake of laser light energy by AgNPs. Wang et al.62 demonstrated that AgNPs (15 nm) can induce apoptosis and enhance radiosensitivity on cancer cells. They investigated the effect on rat glioma C6 cells upon the combination treatment of hyperthermia treatment. The particles showed dose-dependent cytotoxicity on C6 cells and heating cells could enhance the contents of cell uptake of AgNPs. From the survival curves, AgNPs showed the ability to enhance thermosensitivity on C6 cells. Guo et al.63 prepared AgNPs with various sizes and investigated their ability to inhibit the growth of acute myeloid leukemia (AML) cells’ cytotoxic effect on AML cells. They found that AgNPs could inhibit the viability of AML cells, causing the production of ROS, the losses of mitochondrial membrane potential, DNA damage, and apoptosis. Recently, Ranjitham et al.64 studied in vitro cytotoxicity of the Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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AgNPs against MCF-7 breast cancer cell line at different concentrations (viz., 50, 100, and 150 :g). The samples showed a considerable cytotoxicity against the MCF-7cell line. It was reported that the toxicity of AgNPs increase with increase in concentration. Samples having 150 :g concentration showed about 40% of cell toxicity; on the contrary, cell viability was found to be less (59%) at this concentration as compared with control sample (100%). The result concluded that the proliferation of MCF-7 cells was significantly inhibited by AgNPs with an IC50 value of 190.501 :g/mL of the concentration. Antibacterial Activity of AgNPs Silver has been widely used since antiquity as a therapeutic agent for many diseases. In fact, before the beginning of antibiotics therapy, silver was used for its antiseptic activity, specifically for the treatment of open wounds and burns. The potent antimicrobial activity against gram-positive and gramnegative bacteria,30,33 the low toxicity to mammalian cells at low concentrations,65 and the possibility of developing new generation antibiotics make AgNPs an attractive alternative to overcome the drug resistance problem. AgNPs have a high potential to solve the problem of multidrug-resistant bacteria because microorganisms are unlikely to develop resistance against silver as compared with antibiotics; in fact, silver attacks a broad range of targets in the microbes. In addition, AgNPs have also been proved to disrupt the biofilm formation.66 Biofilms are formed because of the attachment of bacteria to the solid surfaces resulting in the conglomeration of bacterial cells. Biofilms augment resistance to drug therapy, disinfectants, and aids pathogen to evade host immune responses and to establish chronic infections.67 Although the antibacterial effect of AgNPs has been extensively studied, the mechanisms underlying their action have so far been only partially elucidated with several hypotheses being proposed. Many studies reported the direct damage of cell membranes as a principal mechanism of action of AgNPs; in fact, their adherence on the microbial cell wall may cause structural changes in the cell membrane and increase cell permeability leading to a powerful toxic effect that is related to the uncontrolled transport across cytoplasmic membrane.30,31 Electrostatic interactions are possibly implicated in the binding between negatively charged bacterial cell membranes and nanoparticles, but these interactions and the effect on the membrane integrity are directly dependent on size,31,68 shape,69 and concentration57 of AgNPs. Sondi and Salopek-Sondi30 used Escherichia coli to evaluate the antibacterial activity of AgNPs. Shrivastava et al.35 suggested that the activity of AgNPs against gram-negative bacteria depends on their concentration.31 They showed that the concentration of AgNPs that prevents bacterial growth is different for each type of bacterium. Pseudomonas aeruginosa and Vibrio cholerae were found to be more resistant than E. coli and Salmonella typhi. However, at concentrations above 75 :g/mL, bacterial growth was almost completely abolished for all of them. Pal et al.69 demonstrated that the activities were also dependent on the AgNPs shape. They investigated the antibacterial properties of differently shaped AgNPs against the gram-negative bacterium E. coli and reported the higher activity of triangular nanoparticles. Kim et al.32 investigated the antimicrobial activity of AgNPs against E. coli and Staphylococcus aureus and demonstrated that E. coli was inhibited at low Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

concentration, whereas the growth inhibitory effects on S. aureus were less profound. Shameli et al.34 reported the antibacterial activity of different sizes of AgNPs in PEG against grampositive (S. aureus) and gram-negative bacteria (Salmonella typhimurium). They also concluded that the antibacterial activities of AgNPs in PEG can be modified by controlling the size of nanoparticles; in fact, the activity of AgNPs decreases with the increase in the particle size. A different mechanism of antibacterial action of AgNPs postulated the generation of ROS and the formation of free radicals, which induce membrane damage and have potent bactericidal activity.32 But of paramount importance is the ability of AgNPs to interfere with the bacterial replication process by adhering to their DNA or RNA. Ag+ interacts strongly with thiol groups in vital enzymes and with the phosphorus-containing bases of the DNA.31 The interaction with the DNA bases may prevent cell division and DNA replication, leading to cell death. This hypothesis is controversial; in fact, Hwang et al.70 did not evidence any DNA damage, whereby Klueh et al.71 hypothesized that the bactericidal activity is because of silver ions entering into cells and intercalating between the purine and pyrimidine bases of DNA, leading to the denaturation of the DNA molecule. Shrivastava et al.35 proposed that AgNPs modulate the phosphotyrosine profile of putative bacterial peptides that can affect cellular signaling, which leads to growth inhibition in bacteria. Klueh et al.71 supported the hypothesis of silver interaction with the thiol groups in enzymes. Antifungal Activity of AgNPs Apart from the antibacterial activity, antifungal activity of AgNPs was also extensively studied and these nanoparticles were found to be equally effective against wide range of plants as well human pathogenic fungi. Many reports are available in the literature but in the present article we have focused on some recent and significant studies. Gajbhiye et al.72 demonstrated the antifungal potential of biosynthesized AgNPs against some common fungal pathogens such as Phoma glomerata, P. herbarum, Fusarium semitectum, Trichoderma sp., and C. albicans. AgNPs were found to be effective against all these test fungi. In another study against plant pathogenic fungi, Roy et al.73 reported the antifungal activity of AgNPs against Aspergillus niger, A. foetidus, A. flavus, A. oryzae, A. parasiticus, and F. oxysporum. Furthermore, Candida species are highly pathogenic and involved in various infections including most important urinary tract infection. In addition, many species have been reported to be resistant to many commonly used antifungal agents. According to Rajarathinam and Kalaichelvan,74 AgNPs can be the appropriate agents for the control of three Candida species, namely, C. albicans, C. tropicalis, and C. krusei. Further, they also demonstrated its application as additive in commercially available dish and hand wash. Another study by Monteiro et al.75 has also proved the potential of AgNPs against C. albicans and C. glabrata. It was also observed in many studies that AgNPs showed synergistic effect when used in combination of commercially available drugs. Gajbhiye et al.72 reported the synergistic effect of AgNPs when used in combination with fluconazole against some plantpathogenic fungi. Similarly, Kandile et al.76 demonstrated the synergism between AgNPs and commercially available drugs. They reported that antifungal activity of AgNPs in combination with different heterocyclic compounds such as thiazolidine, DOI 10.1002/jps.24001

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phthalazine, pyrazolo, tetrazolo, hydrazide, and pyridazine derivatives was significantly increased against Aspergillus flavus and C. albicans. Georgea et al.77 reported the activity of AgNPs encapsulated with b-cyclodextrin against human opportunistic pathogens such as Aspergillus fumigatus, Mucor ramosissimus, and Chrysosporium species. They concluded that encapsulated AgNPs were more effective than the naked AgNPs because in case of encapsulated AgNPs slow release of nanoparticles occur for longer time. Moreover, there are reports concerning the antifungal activity of AgNPs against seed-borne pathogens such as Aspergillus, Alternaria and Rhizoctonia,78 A. niger, A. flavus, Rhizoctonia bataticola, Sclerotium rolfsii, Alternaria macrospora,79 C. albicans, C. parapsilosis, C. krusei, Cryptococcus neoformans, A. fumigatus, A. flavus, C. tropicalis, F. solani, Sporothrix schenckii,80 C. albicans and C. glabrata,81 C. albicans, C. kefyr, A. niger, C. tropicalis, C. krusei, A. flavus, A. fumigates,82 A. flavus, C. albicans,76 and so on. Antiviral Activity of AgNPs The antimicrobial efficacy of AgNPs have been widely analyzed and demonstrated during the last decade, but demonstration of their activity against viruses as a potential weapon is recent. As a result of common antivirals being prone to the development of drug resistance, AgNPs are emerging as one of the easiest option for the control of viral diseases because of their remarkable antiviral activity and the possibility of offering a unique broadspectrum treatment option. In fact, AgNPs are active on a broad range of viruses, regardless of each single specific virus characteristic, and present a lower possibility of developing resistance compared with conventional antivirals.83,84 Nanoparticles have strong antiviral potential and because of their multiple interactions with glycoprotein receptor and/or viral envelop,83 they can inhibit the viral multiplication inside the host cell by preventing the replication or blocking the entry of virus particles inside the host cell. Depending on the interaction and virucidal effect, they have huge potential not only to face the challenge posed by viral infections but also enhance the quality of existing antiviral therapies. Many successful attempts have been made to study the role of AgNPs without capping agent on the growth inhibition of viruses, such as, influenza virus,36,41 Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2),39,86 Coxsackievirus B3,87 tacaribe virus (TCRV),44 Vaccinia virus (VACV),43 human parainfluenza virus type 3 (HPIV-3),39 hepatitis B virus (HBV),85 and monkeypox virus (MPV).42 However, investigation of exact mechanism for the action of these nanoparticles is very difficult due to variations in their synthesis methods and sizes. It was reported that smaller the size of AgNPs more is the inhibition efficacy, which was confirmed for the study of Gaikwad et al.39 who demonstrated that AgNPs synthesized by fungi showed a significant reduction in the infectivity of HSV1/2 and HPIV-3. However, in case of cytotoxicity, bigger nanoparticles showed high level of cytotoxicity, while smaller nanoparticles demonstrated low level of cytotoxicity.85 Many possible explanations have been proposed for the antiviral activity of AgNPs, which mainly include- its interaction with DNA of viruses and direct binding of AgNPs with viral particles. The promising results obtained by Xiang et al.36 against H1N1 influenza A were further extended using different viral strains both in vitro and in vivo. AgNPs could significantly inhibit the growth of influenza virus DOI 10.1002/jps.24001

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in Madin–Darby canine kidney cells and reduce cell apoptosis induced by H3N2 influenza virus at three different treatment conditions. They also demonstrated by transmission electron microscopy that H3N2 influenza viruses treated with AgNPs were able to interact with each other, resulting in destruction of morphological viral structures in a time-dependent manner in a time range of 30 min to 2 h. Moreover, intranasal administration of AgNPs in mice significantly enhanced survival after infection and lower lung viral titer levels with minor pathological lesions in lung tissue. A comparative study to test 10 nm versus 25 nm particles was conducted against TCRV of the Arenaviridae family.44 AgNPs of 10 nm showed a superior antiviral activity, especially interacting with TCRV before cellular exposure leading to a block of viral particle internalization by interfering with cellular receptor binding. The authors have also hypothesized that the enhancement of internalization of the AgNP complex with the virus may produce an inhibitory effect on viral replication interfering with the TCRV RNA-dependent RNA polymerase. Above reports proved that smaller nanoparticles were more effective in reducing infectivity for wide range of viruses and also for reducing cytotoxicity of host cells. Different capping agent(s) may be added to naked AgNPs (polyvinylpyrrolidone, citrate, polyethylene glycol, etc.) preserving the ability to reduce infectivity and increase biocompatibility. It was reviewed that capping/coating of AgNPs makes them less effective in case of some viruses. Coating of AgNPs with polysaccharide, not only protects the cell from the toxic effects of the AgNPs, but it also reduces its activity against TCRV.44 While in case of MPV and some other viruses, AgNPs coated with polysaccharide found to be more effective.42,43 There are some other studies on antiviral potential of different capped AgNPs against human immunodeficiency virus type-1 (HIV-1).37,38,86,88 These studies proposed that antiviral potential of capped AgNPs depend on the factors like nature of capping agent, size and dose of AgNPs. According to Trefry and Wooley43 AgNPs capped with poly(N-vinyl-2-pyrrolidone) (PVP) and size range of 1–10 nm was most effective to inhibit the replication of HIV. Many other strains of HIV such as laboratory strains, clinical isolates, macrophage [M]-tropic and T lymphocyte [T]-tropic strains, etc. were all found to be susceptible to the PVP capped AgNPs.88 PVP-coated AgNPs were also found to have microbicidal activity against topical vaginal microbes, which are responsible for transmission of HIV-1 infection.38 Similarly, polyurethane condoms coated with AgNPs were also developed to inactivate infectious microorganisms.89 The clear role of AgNPs in inactivation of HIV is not completely understood, but there are hypotheses explaining the direct interaction of AgNPs with surface glycoproteins of HIV, which minimizes the binding and fusion ability of viral penetration into susceptible cells. Moreover, AgNPs were also found to inhibit post entry stages of the HIV-1 life cycle. AgNPs have shown a potent anti-HIV activity in cells that had been previously infected. It may be due to inhibition of post entry stages of infection by inactivating the functional HIV-1 proteins, or reducing reverse transcription or proviral transcription rates by directly binding to the RNA or DNA molecules. On the contrary, most other viruses seem to be inhibited at an early stage of the infectious process; therefore, early infection might be the general time frame where AgNPs exert their antiviral activity impacting the rest of the viral replication cycle.39,40,43 Twenty-five nanometer AgNPs were able to mediate a consistent reduction Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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in VACV entry at noncytotoxic concentrations.78 A reduced effect of AgNPs on their antiviral potential was observed in cells where a vital component of macropinocytosis (Pak1) had been knocked down, suggesting that macropinocytosis is an essential step for the AgNPs inhibitory activity. Furthermore, Western blot analysis suggested that AgNPs bind directly to the entry fusion complex of VACV revealing a potential virucidal mechanism.43 RSV were potentially inhibited by PVP-coated AgNPs.90 Here also the exact mechanism of antiviral activity of AgNPs is not clear. Studies carried out proposed the binding of AgNPs to the viral surface glycoproteins present on the envelope of the RSV virion. As mentioned earlier, different capping agents found to have different mechanism of action. AgNPs capped with mercaptoethanesulfonate40 inhibit HSV-1 infection because it helps to avoid the attachment of virus to the cell thereby the entrance of the virus into the cells. Another reason was the mercaptoethanesulfonate capped AgNPs have ability to mimic heparan sulfate (the cellular primary receptor for HSV) and hence these AgNPs compete for the binding of the virus to the cell. In conclusion, different nanoparticles have different properties as a result of their production method (size, shape, capping agent, and level of dispersity) and further studies are warranted to elucidate their mechanism of action, which may render possibly the exploitation of this novel nanomaterial in the clinical setting against viral infections. Therapeutical Uses of AgNPs in Parasitic Infections Apart from the therapeutical uses of AgNPs in cancer, bacterial, and viral diseases, AgNPs have also showed an important role against parasitic diseases. The parasitic diseases such as malaria, leishmaniasis, and trypanosomiasis are lifethreatening diseases and represent a significant global burden. The control and management of these diseases represent a challenge for scientists working in the field of drug discovery because of their devious nature and the ineffectiveness of available drug therapies. Allahverdiyev et al.46 studied the antileishmanial activity of AgNPs against Leishmania tropica parasites. Leishmaniasis is increasing rapidly worldwide, and unfortunately antileishmanial drugs have several disadvantages. Therefore, AgNPs could be the alternative to treat leishmaniasis. In another study by Panneerselvam et al.,47 a successful attempt was made to study the antiplasmodial activity of AgNPs against Plasmodium falciparum. The parasitic property was analyzed by IC50 values which were found to be 26 ± 0.2% at 25 :g/mL and 83 ± 0.5% at 100 :g/mL, confirming significant antiplasmodial activity of AgNPs. Fascioliasis is another type of parasitic infection caused by Fasciola hepatica, commonly known as liver fluke. It has the ability to infect liver of various mammals, including humans. Gherbawy et al.45 studied the antifasciolasis properties of AgNPs produced by Trichoderma harzianum and also compared AgNPs activity with the commercially available effective drug triclabendazole. The study concluded that AgNPs have considerable antifasciolasis activity compared with triclabendazole. Santhoshkumar et al.91 for the first time demonstrated the comparative larvicidal activity of Nelumbo nucifer plant extract (aqueous and methanolic) and AgNPs synthesized from this plant against Anopheles subpictus and Culex quinquefasciatus, which are responsible for transmitting diseases such as Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

malaria and filariasis. The obtained results suggested that the methanolic and aqueous extracts of N. nucifera showed moderate activity but AgNPs have a remarkable efficacy and can be used as an eco-friendly approach for the control of the A. subpictus and C. quinquefasciatus. Recently, Soni and Prakash92 also studied the larvicidal activity of AgNPs against the larvae and pupae of Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti. The larvae of C. quinquefasciatus showed 100% mortality in the presence of AgNPs after 1 h of exposure, whereas the larvae of A. stephensi and A. aegypti were found less susceptible. The pupae of A. aegypti was highly susceptible as compared to pupae of C. quinquefasciatus and A. stephensi.

NANOTOXICOLOGICAL ISSUES OF AgNPs From the discussion to this stage, it is very clear that there is huge advancement in nanotechnology, especially the nanotechnology of silver. But as mentioned in the next section of the present review, the study of toxicological aspect is also needed. It is a general observation of the whole scientific community that reasonably less information is available about the hazard associated with their use. Now-a-days, many research studies are also giving special attention toward the study of toxicological aspects of the nanomaterials prior to their application. For many of such studies, in vitro and in vivo models are apparently being used. One of those studies include, 28-day systemic toxicity effect of 20–100 nm-sized AgNPs on rats using intravenous administration. The AgNPs showed the severe increase in the spleen size with increased population of T and B cells population. The histopathological study of the affected tissues showed the accumulation of AgNPs in spleen, liver, lymph nodes, and other organs. The clinical chemistry revealed increased phosphatase, alanine transaminase, and aspartate transaminase, which all signified the liver damage.93 According to the study conducted by Trop et al.,94 AgNPs can enter in the body via ingestion and it get absorbed from stomach duct and enters the portal vein. Later, it enters the liver and thus exerts the toxic effect on liver cells. To find the details of live damage, Xia et al.95 and Hussain et al.96 have attempted to find nanoparticles toxicity effects on mouse liver cells. Notably, they observed the abnormal size of mitochondria in the exposed liver tissues. Additionally, they observed the irregular cell shape and their cleavage because of the action of nanoparticles. The liver is the organ with high metabolic activity. Therefore, like any other drugs, AgNPs cause harm to liver tissue after excessive accumulation. Moreover, the absorbed nutrients are usually processed in the liver and can also be stored and supplied to the rest of the body whenever needed. Thus, the liver damage is mostly occurring phenomenon in toxicological studies. The same reason also applies to the kidney as it is also one of the highly metabolic organs. As summarized in Figure 1, many studies confirmed that AgNPs decrease cell viability through different cellular mechanisms. One of those mechanisms include the induction of apoptosis-related genes and the activation of mechanism leading to apoptosis.97,98 Further, they have shown to cause DNA damage,99 increased mitochondrial membrane permeability,100 and accumulation of ROS.101 The ROS produced herein reacts with protein and oxidizes them. This process leads to the partial or permanent loss of structure and/or function of cellular DOI 10.1002/jps.24001

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Figure 1. Schematic representation of interrelations between various cellular responses to AgNPs-induced toxicity mechanisms.

protein. Independently or in combination, all of the aforesaid activities inside the cell induces the cellular apoptosis, causing decrease in cell viability.102 The exact mechanism by which AgNPs shows its effect on biological system is a matter of long-lasting debate. But most of the studies assumed that AgNPs are toxic depending on their size, shape, and concentration. A study by Navarro et al.103 favored the theory that mechanism of AgNPs action is because the release of silver ions from AgNPs. But the exact factors responsible for the dissolution of AgNPs are not clarified and it is assumed that particle shape and size might govern the dissolution. Whereas, according to the study led by Xiu et al.,104 if the AgNPs do not get ionized, their activity will be reduced. Further, they claimed that AgNPs do not kill bacteria by direct contact, whereas the silver ions released in the vicinity of the bacteria do complete the task. Further, they also claimed that AgNPs demonstrated the negligible toxicity as they are 7665 times less toxic than that of the silver ions. Although considering the mechanism of their action, lactate dehydrogenase assay revealed that silver ions impair the cell membrane integrity because of structural changes of membrane (Fig. 1). Moreover, silver ions have more tendency to interact with thiol groups of various enzymes and phosphorous-containing bases.105 Although interfacing with the bacterial membrane, it interacts with the surface respiratory chain enzymes.106,107 Such respiratory chain enzymes are also present in the mitochondria and therefore in the eukaryotic system, silver ions can disturb the normal functioning of the mitochondria. These studies warrant that more focus is needed to find the extent of silver ions contributing their activity. Hence, to have safe use of the AgNPs, various processes should be developed that can contribute in controlled release of ions from nanoparticles. Another way to identify the possible toxicity of AgNPs is the exploitation of attractive animal models. Zebrafish (Danio rerio) is being preferably used for such studies as its embryos are transparent, possess high degree of homology with the humans,

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and is easy to nurture in the laboratory condition. Use of such model organisms will definitely help us to make nanomaterials with minimal or no toxicity. This fish on exposure to the 3-, 10-, 50-, and 100 nm-sized AgNPs shown to generate size-dependent malformations and produced almost 100% mortality at 120 h after fertilization. In comparison to the control zebrafish embryo, the experimental data suggest that the toxicity of AgNPs is mainly because of nanoparticles.108 Metal nanoparticles in general and AgNPs have also been used as antimicrobial coating to prevent the infection in bone implants. But it is also required to study whether the same nanoparticles are safe for patient’s tissue where the bone is implanted. In this context, Pauksch et al.109 investigated the influence of AgNPs on bone cell metabolism. For this study, they exposed the primary human mesenchymal stem cells (MSC) and osteoblasts (OB) with the AgNPs. The study concluded with the remarks that after 21-days exposure, the AgNPs cause time- and dose-dependent impairment of MSC and OB at the concentration of 10 :g/g. Therefore, AgNPs below 10 :g/g have the capacity to be used for therapeutical purposes but above this limit it will be cytotoxic to bone tissues. Moreover, Silva et al.110 assessed the size, surface charge, and concentrationdependent toxicity of the citrate-coated AgNP (citrate-AgNP), polyvinylpyrrolidone-coated AgNP (PVP-AgNP), and branched polyethyleneimine-coated AgNP (BPEI-AgNP) against E. coli and Daphna magna. The study remarkably mentioned that Ag+ ions are more toxic as compared with all types of AgNPs, whereas among the aforesaid types of AgNPs, BPEI-AgNPs was found to be more toxic and the PVP-AgNPs was the least toxic. By using general linear model, the study claimed that toxicity of AgNPs was differing depending on their size and surface charge. An interesting study conducted by Levard et al.,111 revealed that the toxicity of AgNPs was shown to decrease by their sulfidation. The authors claimed that AgNPs readily reacts with sulfide to form Ag(0)/Ag2 S core–shell particles. This sulfidation has shown to decrease nanotoxicity of AgNPs against four

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types of aquatic and terrestrial eukaryotic organisms, namely, zebrafish (Danio rerio), killifish (Fundulus heteroclitus), Nematode worm (Caenorhabditis elegans), and the aquatic plant “least duckweed” (Lemna minuta). The main reason for this decreased toxicity was claimed to be the decrease in Ag+ concentration in the suspension medium. The lower release of Ag+ was because of the lower solubility of Ag2 S relative to Ag (Ag0 ). The study further concluded that even chloride ions in exposure medium can also affect the toxicity of AgNPs. This study, therefore, pointed out that environmental transformation of nanoparticles should also be considered to accurately measuring the risk associated with them. On similar pathway, Pokhrel et al.112 studied the effect of environmental transformation of citrate-AgNPs toxicity. The study revealed that the transformation of citrate-AgNPs, by thiol (SH) and carboxylate (COO) attenuates its toxicity, whereas dissolved organic carbon promotes the AgNP toxicity. This study came to the conclusion similar to that of Levard et al.,111 mentioning that organic ligands present in the receiving water can modify the AgNPs differentially and thereby affect their environmental persistence and toxicity. As that of other nanoparticles, AgNPs can also enter inside the human body via inhalation. Therefore, in this perspective, Ramirez-Lee et al.113 has assessed the effect of AgNPs on smooth muscle (ASM) cells. ASM cells control the airway contractility by various mediator, namely, acetylcholine (ACh) and nitric oxide (NO). AgNPs on exposure to these cells, at the concentration of 10 and 100 :g/mL cause the induction of ACh-dependent prolonged cytotoxicity and decreased cellular proliferation mediated by muscarinic receptor-induced nitric oxide synthase pathway. The unnecessary production of NO also results as a part of AgNP cytotoxicity. In recent innovative study, the effect of commercially available 10 and 32 ppm AgNPs was evaluated in healthy volunteers.114 The concerned researchers determined the silver serum and urine contents. The study finally revealed no morphological changes in the lungs, heart, and abdominal organs. They also did not find any significant changes in pulmonary ROS or proinflammatory cytokine generation. Furthermore, they came to the conclusion that oral exposure to these AgNP solution does not result in the clinically significant changes in human metabolic, hematologic, urine, physical findings, or imaging morphology. More of such studies are needed to get a comprehensive idea about the exact effect of AgNPs on human body itself. Consequently, with respect to the use of AgNPs for any therapeutic purposes, it can be said that the “4Ss,” namely, shape, size, structures, and the surface chemistry, govern their passage in vivo and in vitro. These parameters decide their transportation pathway, their distribution throughout the body, and ultimately their target in therapeutic procedures. But in future much research is needed to elucidate the exact mechanism of AgNP toxicity to the human and environment.

NEED OF SAFETY ISSUES FOR THE USE OF AgNPs The broad range of applications shown by AgNPs is mainly because of (1) large surface area and (2) small size. electron transport, manifested in phenomena such as Coulomb blockade,115 as well as the catalytic and thermodynamic properties of structures can be customized while designing the materials on this length scale. The unique physicochemical properties of Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

nanoparticles different from the bulk material have attracted the attention of several workers to harness the multiple functionalities of nanoparticles.116 New technological advances in reducing silver compound chemically to nanoscale-sized particles have enabled the integration of this valuable antimicrobial into a larger number of materials, including plastics, coatings, and foams as well as natural and synthetic fibers. Nanosized silver provides a more durable antimicrobial protection, often for the life of the product. According to a market research report,117 AgNPs are emerging as one of the fastest growing product categories in the nanotechnology industry. Still, the remarkably strong antimicrobial activity is a major direction for the development of nanosilver products. A wide category of silver nanoproducts available in medical arena includes wound dressings, contraceptive devices, surgical instruments, and bone prostheses either coated or embedded with nanosilver.118–121 The "Project on Emerging Nanotechnologies (PEN) is dedicated to helping ensure that as nanotechnologies advance, possible risks are minimized, public and consumer engagement remains strong, and the potential benefits of these new technologies are realized.” According to PEN, AgNPs ranks first among the most prevalent nanomaterial in consumer products. The updated Nanotechnology-based Consumer Products Inventory now contains 1628 consumer products, which were introduced to the market in 2005.122 Researchers have found that several nanomanufacturing processes are expensive and not environmental friendly. As a result to support sustainable production processes, the US Environmental Protection Agency (EPA) is working with the international community through the Organization for Economic Cooperation and Development (OECD) and pushing for green manufacturing of nanoproducts, which is a more environmentally benign process.123 The green synthesis of metallic nanoparticles help to improve and/or protect the environment by decreasing the use of toxic chemicals and eliminating biological risks in pharmaceutical and biomedical applications. Three of the main green chemistry areas in manufacturing that will be investigated by EPA include: solvent choice, the use of an environmentally benign reducing agent, and the use of a nontoxic material for nanoparticle stabilization. Biosynthesis of nanoparticles is coming in a big way because of its green approach.124 In biosynthesis, water is commonly used as an environmentally benign solvent, replacing toxic organic solvents. Recently, biomolecules have been reported to serve as both reducing and stabilizing agents for biosynthesis of metallic nanoparticles.125 Currently, all of these biomolecules are widely employed as tissue-engineering scaffolds and in drug delivery applications (as micelles, particles, or hydrogels) as described in a review.126 These biomolecules are generally considered biocompatible polymers. The biosynthesis of AgNPs is reported by using bacteria,127 fungi128,129 including endophytic fungus,130 plants,131–133 algae,134 and actinomycetes.135 Because of the enormous use of AgNPs in the consumer products, it has led to several questions in the minds of common people: Is it safe for me and my family? Is it safe for the environment we are living in? What could be the economic impact of nanotechnology on consumers and producer? To answer all these questions, research is needed to determine the key physical and chemical characteristics of nanoparticles that determine their hazard potential and awareness among common people. DOI 10.1002/jps.24001

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SAFETY ISSUES CONCERNING HUMAN

SAFETY ISSUES CONCERNING ENVIRONMENT

Although inhalation is considered the most important route of exposure for nanoparticles,136 little is known about the environmental and health risks of aerosolized nanosilver.137 There are a few reports in the literature on pulmonary studies of nanosilver toxicity in rats.138,139 Therefore, inhalation and toxicity assessment of AgNPs is of prime importance. With the increasing use of engineered nanomaterials, it is expected to increase the occupational exposure to nanomaterials in the workplace. The exposed nanomaterials may enter and accumulate in our body, potentially causing injury or death to humans. Especially, workers can be directly exposed to nanomaterials for a long time. However, the researches on the exposure assessment of nanomaterials to human and environment are just beginning step. Therefore, exposure assessment based on the real-time monitoring of exposed nanoparticle is one of the critical issues for EHS (environment, health, and safety). According to National Institute for Occupational Safety and Health (NIOSH),48 nanomaterial-enabled products such as nanocomposites, surface-coated materials, and materials comprising nanostructures, such as integrated circuits, are unlikely to pose a risk of exposure during their handling and use as materials of noninhalable size. However, some of the processes used in their production may lead to the exposure to nanomaterials, and the cutting or grinding of such products could release respirable-sized nanoparticles.48 There are naturally occurring nanoparticles, whereas other nanoparticles are created by man incidentally140 but the greatest causes of concern are engineered nanoparticles—those that are intentionally created by man.141 The critical steps for the risk assessment of engineered nanomaterials are similar to those used for the risk assessment of other types of chemicals,142 which includes:

Metal nanoparticles in general and silver nanoparticles in particular are emerging causes of concern for the environment regulators and consumer advocates, because of the increasing incorporation into consumer products such as pesticide, toothpaste, toothbrush, socks, washing machine, filters, creams, air purifier, mobile, and so on,122 because of their antimicrobial properties. AgNPs release from the consumer products are expected to enter terrestrial ecosystems, but their fate and transformations that these nanoparticles will undergo in real, complex environment during long-term aging and their impact on the environment are largely unknown. Even the common people are worried about what will happen to aquatic life forms when AgNPs get into water. Another concern is that silver nanoparticles do not distinguish between good microbes and harmful microbes. Most of the knowledge about the toxicity of AgNPs to different aquatic and terrestrial organisms is drawn from the controlled laboratory experiments carried on the single strain of bacteria or fungi. There are reports demonstrating the toxicity of AgNPs to bacteria, fungi,144 annual grass, Lolium multiflorum,145 green alga, Chlamydomonas reinhardtii,146 vertebrates (zebrafish),147 and earthworms.148 AgNPs are evaluated by the EPA under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). The study by Ma et al.149 indicates that Ag present in municipal wastewater or added in nanoparticles or bulk form transformed significantly into Ag2 S during wastewater treatment process. They could not observe any AgNPs in sludge leaving the waste water treatment or in the biosolids resulting from heat and lime treatment. To understand the fate and toxicity of AgNPs in the environment, detailed investigations of absorption, distribution, metabolism, and excretion of AgNPs need to be performed on species from the major phyla, although there are some reports on fish. The environmental risk assessment of engineered nanomaterials could be performed using the existing tiered approach and regulatory framework, but with modifications to methodology including considering their characterization and properties.

1. Hazard identification, that is, identification of properties of engineered nanomaterials that may be causing hazards to health. 2. Hazard characterization, that is, defining dose response to critical organ(s) and cell(s) and identifying their mode of action. 3. Assessment of exposure, that is, validation of analytical methods for their detection and measurement in bulk sample and workplace air. 4. Risk characterization, that is, assessing the likelihood of occurrence of a given hazard in certain exposure situation. Engineered nanomaterials complicate the issue of safety and risk assessment because of their novel properties at nanoscale as compared with bulk, lack of data, nonuniformity in the group of substances, and remarkable diversity. The diversity among the engineered nanomaterials is mainly because of the different types of solvents, reducing and capping agents used in the process. Schulte et al.143 have reported the presence of more than 50,000 different types of carbon nanotubes based on different raw materials, production process, and catalyst used. The government agencies from different countries along with scientists in academia, public, and private sectors are working together to answer the questions, collect more data, and develop strategies to counteract the problems faced for the risk assessment of the engineered nanomaterials. DOI 10.1002/jps.24001

CONCLUSIONS AND FUTURE PERSPECTIVES Nanotechnology has kept the promise to deliver the greatest scientific and technological advances in several areas. Nanostructured compounds have numerous applications in the field of engineering. The development of biocompatible and environmentally favorable nanostructures can minimize damage to the ecosystem, generating rational use of resources and economy of reagents in the synthesis of many processes. Application in the field of bioengineering favors the employment and development of equipment for diagnostics, more sensitive, potent, and specific in their therapeutic applications. However, one of the most interesting applications for these compounds is in the field of nanomedicine. There is an increasing need to develop a therapy that fits the requirement of each patient and to anticipate the damage caused by many chronic diseases are yet to be elucidated. The possibility of the development and application of nanocomposites for in situ monitoring of vital functions and applications as nanocarrier for drugs, genomes, and proteins, is a great hope for the treatment and diagnosis of many Dos Santos et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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diseases that present numerous complexities in their mechanisms, and therefore,require tools to elucidate at molecular level. The application of nanomedicine as a site-specific treatment, decreases the possibility of toxicity. For example, in cancer therapy, these compounds allow placement directly at the site of action, using smaller amounts of drugs and less adverse effects. Due to the versatility of the nanostructured compounds, in terms of size, physical characteristics, and ability to interact and incorporate into other compounds, their applicability in different areas are immeasurable; however, these characteristics that make them so attractive also raises important issues like toxicity to human and animal cells, safe use, exposure over extended periods and environmental safety. Effects of the development of technology of nanostructured compounds seem to be so large and comprehensive that probably it will impact on all fields of science and technology. However, mechanisms of safety control in the application, utilization, responsiveness, and disposal accumulation still needs to be further studied indepth to ensure that the advances provided by nanotechnology are real and liable to provide solid and consistent progress. Nanomaterials, particularly AgNPs, have the ability to interact with a broad range of microorganisms (e.g., bacteria, fungi, and viruses) and different parasites such as plasmodium responsible for malaria and hence can be used as broad-spectrum antimicrobial as well as antiparasital agents. Similarly, their applications in the field of drug delivery make them an ideal vehicle for the carrier of drugs in the diseases such as cancer. While studying the positive sides of nanotechnology, one must focus on their toxicity and safety issues.

ACKNOWLEDGMENTS The authors are thankful to Department of Science and Technology (DST), University Grants Commission (UGC), New Delhi, and Rajiv Gandhi Science and Technology Commission, Mumbai, for providing financial support for the present ˜ research work. The financial support provided by Fundac¸ao ˜ Paulo (Fapesp de Amparo a Pesquisa do Estado de Sao n◦ 2010/51933-9 and 2012/03731-3) and Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPQ) is gratefully acknowledged. I.R.G. is thankful to Council of Scientific and Industrial Research, New Delhi, for financial support in the form of Junior Research Fellowship to carry out this study. Authors have no conflict of interest.

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DOI 10.1002/jps.24001

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