Revista de Ciencias Tecnológicas (RECIT). Volumen 3 (1): 10-22.

Revista de Ciencias Tecnológicas (RECIT). Universidad Autónoma de Baja California ISSN 2594-1925

Volumen 1 (1): 8-11 Julio-Septiembre 2018 https://doi.org/10.37636/recit.v11811

ISSN: 2594-1925

Silver Nanoparticles as Nanoantibiotics:

A Comparative Analysis of their Toxicity on Biological

Systems of Different Complexity

Nanopartículas de plata como nanoantibióticos:

Un análisis comparativo de su toxicidad en sistemas biológicos

de diferente complejidad

Vázquez-Muñoz Roberto

1,2

, Borrego-Rivero Belén

, Juárez-Moreno Karla Oyuky

, García-

García Maritza Roxana

, Mota Morales Josué David

1,4

, Bogdanchinkova Nina

, Huerta-

Saquero Alejandro

Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Km 107,

Carretera Tijuana-Ensenada. CP 22860, Ensenada, Baja California, México.

Centro de Investigación Científica y de Educación Superior de Ensenada, Carretera Tijuana-Ensenada,

3918. CP 22860, Ensenada, Baja California, México.

Centro de Investigación en Sanidad Animal, INIA (National Research Institute for Agricultural and Food;

Technology), Carretera Algete el Casar s/n. 28130. Valdeolmos, Madrid, Spain.

Centro de Física Aplicada y Tecnología Avanzada. Universidad Nacional Autónoma de México,

Querétaro, Querétaro 76230, México.

Autor de correspondencia: Alejandro Huerta Saquero, Centro de Nanociencias y Nanotecnología, Universidad

Nacional Autónoma de México, Km 107; Carretera Tijuana-Ensenada. CP 22860, Ensenada, Baja California,

México. E-mail: saquero@cnyn.unam.mx. ORCID: https://orcid.org/0000-0002-0156-6773

Recibido: 30 de Noviembre del 2016 Aceptado: 18 de Diciembre del 2016 Publicado: 01 de Septiembre del 2018

Resumen. - Actualmente, las nanopartículas de plata (AgNPs) se estudian ampliamente para aplicaciones biomédicas, pero

a pesar de que los nanomateriales proporcionan muchos beneficios, recientemente su toxicidad comparativa apenas se ha

explorado. En el trabajo actual, la toxicidad de AgNPs en sistemas biológicos de diferentes niveles de complejidad se evaluó

de forma exhaustiva y comparativa. Los organismos incluyen virus, bacterias, microalgas, hongos, células animales y humanas

(incluidas líneas celulares cancerosas). Encontramos que el crecimiento de los sistemas biológicos de diferentes grupos

taxonómicos -in vitro, a nivel celular- se inhibe a concentraciones de AgNP dentro del mismo orden de magnitud (101 μg /

ml). Por lo tanto, la toxicidad de AgNPs no depende de la complejidad de los organismos. El hecho de que las células y los

virus se inhiban con una concentración de AgNP dentro del mismo orden de magnitud podría explicarse teniendo en cuenta

que la plata afecta las estructuras fundamentales de las células y virus por igual.

Palabras clave: Nanopartículas de Plata; Nanotoxicología; Complejidad de Sistemas Biológicos; Actividad Antimicrobiana.

Abstract. - Currently, silver nanoparticles (AgNPs) are extensively studied for biomedical applications, but although

nanomaterials provide many benefits, recently their comparative toxicity have barely been explored. In the current work,

AgNPs toxicity on biological systems of different levels of complexity was assessed in a comprehensive and comparatively way.

The organisms included viruses, bacteria, microalgae, fungi, animal and human cells (including cancer cell lines). We found

that growth of biological systems of different taxonomical groups –in vitro, at a cellular level- is inhibited at concentrations of

AgNPs within the same order of magnitude (101 μg/ml). Thus, the AgNPs toxicity does not depend on the complexity of the

organisms. The fact that cells and virus are inhibited with a concentration of AgNPs within the same order of magnitude could

be explained considering that silver affects fundamental structures for cells and virus alike.

Keywords: Silver Nanoparticles; Nanotoxicology; Biological Systems Complexity; Antimicrobial Activity.

Revista de Ciencias Tecnológicas (RECIT). Volumen 1 (1): 8-11.

ISSN: 2594-1925

1. Introduction

Silver nanoparticles (AgNPs) are among the most studied

nanomaterials due to all their applications. In the medical

field, AgNPs display antimicrobial and antiviral activity

[1]. Although their many benefits, their relative toxicity

has not been appropriately addressed. The few published

studies of toxic effects of AgNPs on biological systems,

i.e. viruses, bacteria or human cells, report different and

even contradictory results. The research of the AgNPs

comparative toxicity on different biological systems has

become a priority task.

It is generally assumed that the toxicity of AgNPs

decreases as the complexity of biological systems rises

[2–6]; but some studies suggest there is no such a

difference [7, 8]. Still, comparative analyses of the toxic

effects of a single silver nanoparticle on biological

systems of different level of complexity are scarce and

are not extensive [5, 9]. Also, antimicrobial tests for

AgNPs are not standardized leading to limited capacity

to assess the toxic effect of AgNPs on different taxa.

We performed a comparative analysis of the toxic effects

of a single type of AgNPs, on biological systems of

different cellular / structural complexity, from viruses to

human cancer cell lines. Also, the implications of such

analysis are discussed.

2. Methodology

2.1. Virus, microorganisms, cells and culture

conditions

For the systematic, comparative analysis, we used the

following biological models: Rift Valley Fever Virus

(RVFV) MP12 strain from the National Research

Institute of Agricultural and Food Technology, Spain;

The bacteria Escherichia coli DH5α (Gram-negative)

and Staphylococcus aureus (Gram-positive), from the

Centro de Nanociencias y Nanotecnología – Universidad

Nacional Autónoma de México; the microalgae

Rhodomonas sp., from the Faculty of Marine Sciences of

the Universidad Autónoma de Baja California; The fungi

Candida albicans ATCC SC5614 (dimorphic yeast) and

Fusarium oxysporum, Race III, (filamentous), from the

Centro de Investigación Científica y Educación Superior

de Ensenada; the mammalian cells were Dendritic Cells

from Murine models and Vero cells (ATCC CCL-81); the

human cancer cells lines HeLa and MDA-MB-231

from the ATCC.

2.2 AgNPs preparation and characterization

The AgNPs are stabilized with

Polyvinylpyrrolidone (PVP). These PVP-AgNPs

were obtained from Vector Vita Ltd® (Russia).

AgNPs were analyzed by UV-Vis

spectrophotometry (Multiskan Go, Thermo

Scientific), λ= 200-800 nm. Also, an FT-IR

spectroscopy analysis was performed λ=4000-400

-1

(Nicolet 6700; Thermo Scientific). The

morphology was examined by Transmission

Electron Microscopy in a Jeol JEM 2100. AgNPs

dilutions were prepared in a range from 0.001 to 100

μg/ml of silver.

2.3 Toxicological assays

For the virus infection assays, 0.3 to 3x10

plaque-

forming units (PFU) of RVFV were incubated with

AgNPs. After, viruses were inoculated onto Vero

cells grown in MW6 plates for 1 h, washed; and then

semi-solid medium with agar was added. Plates were

incubated until infection plaques were clearly

developed, then were fixed and stained with crystal

violet dye. For bacteria and fungi, the M09 and M27

microdilution assays, respectively, from the Clinical

Laboratory Standard Institute were used, with some

modifications (YPD culture media for fungi).

Microbial cultures were exposed to AgNPs.

Inhibition was measured by UV-Vis

spectrophotometry. Microalgae inoculum was

adjusted to an optical density of 0.065 at λ= 670 nm

in a Jenway 6505 UV-Vis spectrophotometer.

Microalgae were cultured in F2 media, and exposed

to AgNPs, for 24 h, at room temperature, and under

continuous light conditions. Murine bone marrow

derived dendritic cells were grown in RPMI culture

media, supplemented with 10 % FBS, 1% of

streptomycin-penicillin G, at 37 °C with 5% CO

atmosphere. Cytotoxicity was evaluated with the

dual fluorescein diacetate /ethidium bromide test

after 24 h of cell incubation with AgNPs. Vero cells

were seeded in 96-multiwell plates with DMEM

media, and 24 h later, the AgNPs were added to the

medium, then incubated for 24 h more, at 37 ºC.

Viability was evaluated by the MTS Cell

Proliferation Assay (Promega). HeLa and MDA-

Revista de Ciencias Tecnológicas (RECIT). Volumen 1 (1): 8-11

ISSN: 2594-1925

MB-231 cancer cell lines were cultured in RPMI-

1640 or DMEM media supplemented with 10% FBS,

1 % penicillin-streptomycin, 1 % L-glutamine and

1.5 g/l sodium bicarbonate. Cells were maintained at

37 °C and 5 % CO

. Cell viability was assessed by

the MTT method reported by Mosmann [10].

3. Results and Discussions

3.1 AgNPs characterization

AgNPs are spheroids of 35 ± 15 nm in diameter

(fig. 1a). UV-Vis analysis shows a peak at 410

nm, which is typical for metallic silver

nanoparticles (fig. 1b). FT-IR measurements of

lyophilized AgNPs presented a profile similar to

the PVP standard.

Figure 1. AgNPs characterization. TEM micrography and UV-Vis profile.

3.2 Toxicological assays

Plaque-forming units of RVFV were reduced by 98

% at a concentration of 12 μg/ml of silver. The

minimum inhibitory concentration (MIC) for the

bacterial strains E. coli and S. aureus, was 12 μg/ml

of Ag. The Effective Lethal Concentration for the

microalgae Rhodomonas sp. was 4 μg/ml of Ag. In

fungi, the MIC was 42 μg/ml for C. albicans, and, 20

μg/ml for F. oxysporum. The Lethal Dose in all

animal and cancer cells ranged from 10 to 12 μg/ml

of Ag. In summary, AgNPs exerted inhibitory effects

in all biological models tested in vitro (figure 1). We

also performed an exhaustive literature revision

regarding the toxicity of AgNPs in organisms of

different taxonomic groups (included in figure 2).

We only considered those studies with an adequate

characterization of the AgNPs. AgNPs toxicity is not

related to the complexity of the cell (structural or

physiological) [11].

Figure 2. Inhibition concentration range (green slim bars)

of the AgNPs, as reported in different works. Most studies

show an inhibitory concentration of 10

μg/ml of silver –

AgNPs- (orange boxes). The blue dots show the

concentration values determined in our study, using the

same nanomaterial (PVP-AgNPs) for all the biological

systems tested. Modified from Vazquez-Muñoz et al [11]

Revista de Ciencias Tecnológicas (RECIT). Volumen 1 (1): 8-11

ISSN: 2594-1925

4 Conclusions

To the best of our knowledge, no other single

nanomaterial has been tested in such a wide spectrum of

BS of different levels of cellular/structural complexity,

ranging from virus to human cell lines. Our experimental

analysis showed that viruses and cells of different

complexity are inhibited in vitro at similar

concentrations of silver (10

μg/ml). Also, the meta-

analysis supports our results. Despite the differences

between the different AgNPs, the lethal concentration in

the majority of the studies for both viruses and cells

occurs within narrow concentration range around 10

μg/ml of Ag.

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