, Volume 70, Issue 6, pp 1607–1618 | Cite as

Silver/silver chloride nanoparticles inhibit the proliferation of human glioblastoma cells

  • Mateus Eugenio
  • Loraine Campanati
  • Nathalia Müller
  • Luciana F. Romão
  • Jorge de Souza
  • Soniza Alves-Leon
  • Wanderley de Souza
  • Celso Sant’AnnaEmail author
Original Article


Glioblastomas (GBM) are aggressive brain tumors with very poor prognosis. While silver nanoparticles represent a potential new strategy for anticancer therapy, the silver/silver chloride nanoparticles (Ag/AgCl-NPs) have microbicidal activity, but had not been tested against tumor cells. Here, we analyzed the effect of biogenically produced Ag/AgCl-NPs (from yeast cultures) on the proliferation of GBM02 glioblastoma cells (and of human astrocytes) by automated, image-based high-content analysis (HCA). We compared the effect of 0.1–5.0 µg mL−1 Ag/AgCl-NPs with that of 9.7–48.5 µg mL−1 temozolomide (TMZ, chemotherapy drug currently used to treat glioblastomas), alone or in combination. At higher concentrations, Ag/AgCl-NPs inhibited GBM02 proliferation more effectively than TMZ (up to 82 and 62% inhibition, respectively), while the opposite occurred at lower concentrations (up to 23 and 53% inhibition, for Ag/AgCl-NPs and TMZ, respectively). The combined treatment (Ag/AgCl-NPs + TMZ) inhibited GBM02 proliferation by 54–83%. Ag/AgCl-NPs had a reduced effect on astrocyte proliferation compared with TMZ, and Ag/AgCl-NPs + TMZ inhibited astrocyte proliferation by 5–42%. The growth rate and population doubling time analyses confirmed that treatment with Ag/AgCl-NPs was more effective against GBM02 cells than TMZ (~ 67-fold), and less aggressive to astrocytes, while Ag/AgCl-NP + TMZ treatment was no more effective against GBM02 cells than Ag/AgCl-NPs monotherapy. Taken together, our data indicate that 2.5 µg mL−1 Ag/AgCl-NPs represents the safest dose tested here, which affects GBM02 proliferation, with limited effect on astrocytes. Our findings show that HCA is a useful approach to evaluate the antiproliferative effect of nanoparticles against tumor cells.


Cancer Glioblastoma Antiproliferative effect Metallic nanoparticles Silver/silver chloride nanoparticles High-content analysis 



This work was supported by the National Council for Scientific and Technological Development (CNPq), the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ) and the Coordination for the Improvement of Higher Education Personnel (CAPES).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10616_2018_253_MOESM1_ESM.docx (94 kb)
Supplementary material 1 (DOCX 94 kb)
10616_2018_253_MOESM2_ESM.tif (19.6 mb)
Supplementary Fig. 1. Representative images showing the effect of Ag/AgCl-NPs on the proliferation of GBM02 cells and astrocytes. Cells labeled with Hoechst 33258 were left untreated, or were treated with different concentrations of Ag/AgCl-NPs (0.1, 0.5, 1.0, 2.5 and 5.0 µg mL-1) for 72 h. Images are representative of 3 independent experiments (6 fields imaged per experimental condition, in each experiments). Scale bar: 50 µm. (TIFF 20027 kb)
10616_2018_253_MOESM3_ESM.tif (19.6 mb)
Supplementary Fig. 2. Representative images showing the effect of TMZ on the proliferation of GBM02 cells and astrocytes. Cells labeled with Hoechst 33258 were left untreated, or were treated with different concentrations of TMZ (9.7, 19.4, 29.1, 38.5 and 48.5 µg mL−1) for 72 h. Images are representative of 3 independent experiments (6 fields imaged per experimental condition, in each experiments). Scale bar: 50 µm. (TIFF 20027 kb)
10616_2018_253_MOESM4_ESM.tif (18.3 mb)
Supplementary Fig. 3. Representative images showing the effect of Ag/AgCl-NPs + TMZ on the proliferation of GBM02 cells and astrocytes. Cells labeled with Hoechst 33258 were left untreated, or were treated with different concentrations of Ag/AgCl-NPs + TMZ (0.1 + 9.7, 0.5 + 19.4, 1.0 + 29.1, 2.5 + 38.5 and 5.0 + 48.5 µg mL−1) for 72 h. Images are representative of 3 independent experiments (6 fields imaged per experimental condition, in each experiments). Scale bar: 50 µm. (TIFF 18706 kb)


  1. Allahverdiyev A, Abamor EŞ, Bagirova M et al (2011) Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light. Int J Nanomed 6:2705–2714. CrossRefGoogle Scholar
  2. Alves TR, Lima FRS, Kahn SA et al (2011) Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma. Life Sci 89:532–539. CrossRefPubMedGoogle Scholar
  3. Arvold ND, Reardon DA (2014) Treatment options and outcomes for glioblastoma in the elderly patient. Clin Interv Aging 9:357–367. CrossRefPubMedPubMedCentralGoogle Scholar
  4. AshaRani P, Hande MP, Valiyaveettil S (2009) Anti-proliferative activity of silver nanoparticles. BMC Cell Biol 10:65. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Assanga I (2013) Cell growth curves for different cell lines and their relationship with biological activities. Int J Biotechnol Mol Biol Res 4:60–70. CrossRefGoogle Scholar
  6. Balça-Silva J, Matias D, do Carmo A et al (2015) Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines. Biochim Biophys Acta Gen Subj 1850:722–732. CrossRefGoogle Scholar
  7. Beier D, Schulz JB, Beier CP (2011) Chemoresistance of glioblastoma cancer stem cells—much more complex than expected. Mol Cancer 10:128. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Brayden DJ, Cryan S-A, Dawson KA et al (2015) High-content analysis for drug delivery and nanoparticle applications. Drug Discov Today 20:942–957. CrossRefPubMedGoogle Scholar
  9. Buttacavoli M, Albanese NN, Di Cara G et al (2018) Anticancer activity of biogenerated silver nanoparticles: an integrated proteomic investigation. Oncotarget 9:9685–9705. CrossRefPubMedGoogle Scholar
  10. Chan GKY, Kleinheinz TL, Peterson D, Moffat JG (2013) A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays. PLoS ONE 8:e63583. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Devi TB, Ahmaruzzaman M, Begum S et al (2016) A rapid, facile and green synthesis of Ag@AgCl nanoparticles for the effective reduction of 2,4-dinitrophenyl hydrazine. New J Chem 40:1497–1506. CrossRefGoogle Scholar
  12. Diniz LP, Almeida JC, Tortelli V et al (2012) Astrocyte-induced synaptogenesis is mediated by transforming growth factor β signaling through modulation of d-serine levels in cerebral cortex neurons. J Biol Chem 287:41432–41445. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Durán N, Durán M, de Jesus MB et al (2016a) Silver nanoparticles: a new view on mechanistic aspects on antimicrobial activity. Nanomed Nanotech Biol Med 12:789–799. CrossRefGoogle Scholar
  14. Durán N, Nakazato G, Seabra AB (2016b) Antimicrobial activity of biogenic silver nanoparticles, and silver chloride nanoparticles: an overview and comments. Appl Microbiol Biotechnol 100:6555–6570. CrossRefPubMedGoogle Scholar
  15. Eugenio M, Müller N, Frasés S et al (2016) Yeast-derived biosynthesis of silver/silver chloride nanoparticles and their antiproliferative activity against bacteria. RSC Adv 6:9893–9904. CrossRefGoogle Scholar
  16. Faria J, Romão L, Martins S et al (2006) Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization. Differentiation 74:562–572. CrossRefPubMedGoogle Scholar
  17. Gavish A, Krayzler E, Nagler R (2016) Tumor growth and cell proliferation rate in human oral cancer. Arch Med Res 47:271–274. CrossRefPubMedGoogle Scholar
  18. Glaser T, Han I, Wu L, Zeng X (2017) Targeted nanotechnology in glioblastoma multiforme. Front Pharmacol 8:166. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Goldlust SA, Turner GM, Goren JF (2008) Glioblastoma multiforme: multidisciplinary care and advances in therapy. Hosp Physician 1:9–23Google Scholar
  20. Hsin Y-H, Chen C-F, Huang S et al (2008) The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicol Lett 179:130–139. CrossRefPubMedGoogle Scholar
  21. Huang J, Li Q, Sun D et al (2007) Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology 18:105104. CrossRefGoogle Scholar
  22. Jain P, Aggarwal V (2012) Synthesis, characterization and antimicrobial effects of silver nanoparticles from microorganisms—a review. Int J Nano Mater Sci 1:108–120Google Scholar
  23. Jeyaraj M, Sathishkumar G, Sivanandhan G et al (2013) Biogenic silver nanoparticles for cancer treatment: an experimental report. Colloids Surf B Biointerfaces 106:86–92. CrossRefPubMedGoogle Scholar
  24. Kang Y, Jung J-Y, Cho D et al (2016) Antimicrobial silver chloride nanoparticles stabilized with chitosan oligomer for the healing of burns. Materials 9:pii:E215.
  25. Liang P, Shi H, Zhu W et al (2017) Silver nanoparticles enhance the sensitivity of temozolomide on human glioma cells. Oncotarget 8:7533–7539. CrossRefPubMedGoogle Scholar
  26. Locatelli E, Broggi F, Ponti J et al (2012) Lipophilic silver nanoparticles and their polymeric entrapment into targeted-PEG-based micelles for the treatment of glioblastoma. Adv Healthc Mater 1:342–347. CrossRefPubMedGoogle Scholar
  27. Locatelli E, Naddaka M, Uboldi C et al (2014) Targeted delivery of silver nanoparticles and alisertib: in vitro and in vivo synergistic effect against glioblastoma. Nanomedicine 9:839–849. CrossRefPubMedGoogle Scholar
  28. Louis DN, Ohgaki H, Wiestler OD et al (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109. CrossRefPubMedPubMedCentralGoogle Scholar
  29. MacEwan SR, Callahan DJ, Chilkoti A (2010) Stimulus-responsive macromolecules and nanoparticles for cancer drug delivery. Nanomedicine 5:793–806. CrossRefPubMedGoogle Scholar
  30. Naqvi SZ, Kiran U, Ali MI et al (2013) Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria. Int J Nanomed 8:3187–3195. CrossRefGoogle Scholar
  31. Piao MJ, Kang KA, Lee IK et al (2011) Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis. Toxicol Lett 201:92–100. CrossRefPubMedGoogle Scholar
  32. Raizer JJ, Fitzner KA, Jacobs DI et al (2015) Economics of malignant gliomas: a critical review. J Oncol Pract 11:e59–e65. CrossRefPubMedGoogle Scholar
  33. Ramery E, O’Brien PJ (2014) Evaluation of the cytotoxicity of organic dust components on THP1 monocytes-derived macrophages using high content analysis. Environ Toxicol 29:310–319. CrossRefPubMedGoogle Scholar
  34. Ramirez YP, Weatherbee JL, Wheelhouse RT, Ross AH (2013) Glioblastoma multiforme therapy and mechanisms of resistance. Pharmaceuticals 6:1475–1506. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Rosarin FS, Arulmozhi V, Nagarajan S, Mirunalini S (2013) Antiproliferative effect of silver nanoparticles synthesized using amla on Hep2 cell line. Asian Pac J Trop Med 6:1–10. CrossRefPubMedGoogle Scholar
  36. Rutberg FG, Dubina MV, Kolikov VA et al (2008) Effect of silver oxide nanoparticles on tumor growth in vivo. Dokl Biochem Biophys 421:191–193CrossRefGoogle Scholar
  37. Sharma S, Chockalingam S, Sanpui P et al (2014) Silver nanoparticles impregnated alginate-chitosan-blended nanocarrier induces apoptosis in human glioblastoma cells. Adv Healthc Mater 3:106–114. CrossRefPubMedGoogle Scholar
  38. Sherley JL, Stadler PB, Stadler JS (1995) A quantitative method for the analysis of mammalian cell proliferation in culture in terms of dividing and non-dividing cells. Cell Prolif 28:137–144CrossRefGoogle Scholar
  39. Silva HFO, Lima KMG, Cardoso MB et al (2015) Doxycycline conjugated with polyvinylpyrrolidone-encapsulated silver nanoparticles: a polymer’s malevolent touch against Escherichia coli. RSC Adv 5:66886–66893. CrossRefGoogle Scholar
  40. Sriram MI, Kanth SBM, Kalishwaralal K, Gurunathan S (2010) Antitumor activity of silver nanoparticles in Dalton’s lymphoma ascites tumor model. Int J Nanomed 5:753–762. CrossRefGoogle Scholar
  41. Stengl A, Hörl D, Leonhardt H, Helma J (2017) A simple and sensitive high-content assay for the characterization of antiproliferative therapeutic antibodies. SLAS Discov 22:309–315. CrossRefPubMedGoogle Scholar
  42. Stojiljković A, Kuehni-Boghenbor K, Gaschen V et al (2016) High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture. Nanoscale 8:16650–16661. CrossRefPubMedGoogle Scholar
  43. Stupp R, Hegi ME, Mason WP et al (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10:459–466. CrossRefPubMedGoogle Scholar
  44. Urbańska K, Pająk B, Orzechowski A et al (2015) The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells. Nanoscale Res Lett 10:98. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Usaj M, Styles EB, Verster AJ et al (2016) High-content screening for quantitative cell biology. Trends Cell Biol 26:598–611. CrossRefGoogle Scholar
  46. Vigneshwaran N, Ashtaputre NM, Varadarajan PV et al (2007) Biological synthesis of silver nanoparticles using the fungus Aspergillus flavus. Mater Lett 61:1413–1418. CrossRefGoogle Scholar
  47. Villanueva-Ibáñez M, Yañez-Cruz MG, Álvarez-García R et al (2015) Aqueous corn husk extract—mediated green synthesis of AgCl and Ag nanoparticles. Mater Lett 152:166–169. CrossRefGoogle Scholar
  48. Wen PY, Kesari S (2008) Malignant gliomas in adults. N Engl J Med 359:492–507. CrossRefPubMedGoogle Scholar
  49. Zanella F, Lorens JB, Link W (2010) High content screening: seeing is believing. Trends Biotechnol 28:237–245. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Mateus Eugenio
    • 1
    • 2
  • Loraine Campanati
    • 3
  • Nathalia Müller
    • 1
  • Luciana F. Romão
    • 6
  • Jorge de Souza
    • 7
  • Soniza Alves-Leon
    • 7
  • Wanderley de Souza
    • 2
    • 4
    • 5
  • Celso Sant’Anna
    • 1
    • 2
    • 4
    Email author
  1. 1.Laboratory of Microscopy Applied to Life Science - LamavNational Institute of Metrology, Quality and Technology - InmetroDuque de CaxiasBrazil
  2. 2.Post-Graduation Program on Translational Biomedicine – BiotransDuque de CaxiasBrazil
  3. 3.Laboratory of Cellular MorphogenesisFederal University of Rio de JaneiroRio de JaneiroBrazil
  4. 4.National Institute of Science and Technology for Structural Biology and BioimagingRio de JaneiroBrazil
  5. 5.Laboratory of Cellular Ultrastructure Hertha MeyerFederal University of Rio de JaneiroRio de JaneiroBrazil
  6. 6.Biomedical ScienceFederal University of Rio de JaneiroDuque de CaxiasBrazil
  7. 7.University Hospital Clementino Fraga FilhoFederal University of Rio de JaneiroRio de JaneiroBrazil

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