Silver nanoparticles: aggregation behavior in biorelevant conditions and its impact on biological activity

Abstract

Purpose: The biomedical applications of silver nanoparticles (AgNPs) are heavily investigated due to their cytotoxic and antimicrobial properties. However, the scientific literature is lacking in data on the aggregation behavior of nanoparticles, especially regarding its impact on biological activity. Therefore, to assess the potential of AgNPs in therapeutic applications, two different AgNP samples were compared under biorelevant conditions.

Methods: Citrate-capped nanosilver was produced by classical chemical reduction and stabilization with sodium citrate (AgNP@C), while green tea extract was used to produce silver nanoparticles in a green synthesis approach (AgNP@GTs). Particle size, morphology, and crystallinity were characterized using transmission electron microscopy. To observe the effects of the most important biorelevant conditions on AgNP colloidal stability, aggregation grade measurements were carried out using UV-Vis spectroscopy and dynamic light scatterig, while MTT assay and a microdilution method were performed to evaluate the effects of aggregation on cytotoxicity and antimicrobial activity in a time-dependent manner.

Results: The aggregation behavior of AgNPs is mostly affected by pH and electrolyte concentration, while the presence of biomolecules can improve particle stability due to the biomolecular corona effect. We demonstrated that high aggregation grade in both AgNP samples attenuated their toxic effect toward living cells. However, AgNP@GT proved less prone to aggregation thus retained a degree of its toxicity.

Conclusion: To our knowledge, this is the first systematic examination regarding AgNP aggregation behavior with simultaneous measurements of its effect on biological activity. We showed that nanoparticle behavior in complex systems can be estimated by simple compounds like sodium chloride and glutamine. Electrostatic stabilization might not be suitable for biomedical AgNP applications, while green synthesis approaches could offer new frontiers to preserve nanoparticle toxicity by enhancing colloidal stability. The importance of properly selected synthesis methods must be emphasized as they profoundly influence colloidal stability, and therefore biological activity.

Keywords: antimicrobial activity; colloidal stability; cytotoxicity; green synthesis.

Figure 1

TEM images with corresponding size, aspect ratio and shape distributions, electron diffraction patterns, Characterization of silver nanoparticles stabilized (A) by citrate (AgNP@C) and (B) by green tea extract (AgNP@GT) consisting of TEM images with corresponding size, aspect ratio and shape distributions, electron diffraction patterns, UV-Vis spectra with characteristic surface plasmon resonance peaks, furthermore mean values for the average hydrodynamic diameter and zeta potential of the particles (pH ~7.2). Abbreviations: AgNP@C, citrate-capped nanosilver; AgNP@GT, green tea extract-stabilized silver nanoparticle; TEM, transmission electron microscopy.

Figure 2

The effect of pH on the aggregation behavior of the as-prepared silver nanoparticles with 10 mM NaCl background concentration. Notes: Average hydrodynamic diameter (Z-average) trend, zeta potential, and UV-Vis spectrum changes of (A) citrate-stabilized AgNP@C, (B) green tea-stabilized AgNP@ GT, observed over 24 hours. * marks a UV-Vis detection error during the measurements that should be disregarded. Abbreviations: AgNP@C, citrate-capped nanosilver; AgNP@GT, green tea extract-stabilized silver nanoparticle.

Figure 3

The effect of sodium chloride on the aggregation behavior of the as-prepared silver nanoparticles at pH ~7.2. Notes: Average hydrodynamic diameter (Z-average) trend, zeta potential, and UV-Vis spectrum changes of (A) citrate-stabilized AgNP@C, (B) green tea-stabilized AgNP@ GT, observed over 24 hours. * marks a UV-Vis detection error during the measurements that should be disregarded. Abbreviations: AgNP@C, citrate-capped nanosilver; AgNP@GT, green tea extract-stabilized silver nanoparticle.

Figure 4

The effect of cell culture medium components DMEM and FBS on the aggregation behavior of the as-prepared silver nanoparticles. Notes: Average hydrodynamic diameter (Z-average) trend, zeta potential, and UV-Vis spectrum changes of (A) citrate-stabilized AgNP@C, (B) green tea-stabilized AgNP@ GT, observed over 24 hours. * marks a UV-Vis detection error during the measurements that should be disregarded. Abbreviations: AgNP@C, citrate-capped nanosilver; AgNP@GT, green tea extract-stabilized silver nanoparticle.

Figure 5

The effect of nanoparticle aggregation (citrate-stabilized: AgNP@C, green tea-stabilized: AgNP@GT) on cytotoxicity toward A549 human lung cancer and MRC-5 human fibroblast cells. Note: Increasing aggregation grades were prepared using 150 mM NaCl for longer time intervals up to 24 hours. Abbreviations: AgNP@C, citrate-capped nanosilver; AgNP@GT, green tea extract-stabilized silver nanoparticle.

Figure 6

The effect of nanoparticle aggregation (citrate-stabilized: AgNP@C, green tea-stabilized: AgNP@GT) on the antimicrobial activity against C. neoformans, B. megaterium, and E. coli. Note: Increasing aggregation grades were prepared using 150 mM NaCl for longer time intervals up to 24 hours. Abbreviations: AgNP@C, citrate-capped nanosilver; AgNP@GT, green tea extract-stabilized silver nanoparticle.