Biophysical comparison of four silver nanoparticles coatings using microscopy, hyperspectral imaging and flow cytometry

Abstract

This study compared the relative cellular uptake of 80 nm silver nanoparticles (AgNP) with four different coatings including: branched polyethyleneimine (bPEI), citrate (CIT), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). A gold nanoparticle PVP was also compared to the silver nanoparticles. Biophysical parameters of cellular uptake and effects included flow cytometry side scatter (SSC) intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance (SPR), fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. The AgNP-bPEI were positively charged and entered cells at a higher rate than the negatively or neutrally charged particles. The AgNP-bPEI were toxic to the cells at lower doses than the other coatings which resulted in mitochondria being transformed from a normal string-like appearance to small round beaded structures. Hyperspectral imaging showed that AgNP-bPEI and AgNP-CIT agglomerated in the cells and on the slides, which was evident by longer spectral wavelengths of scattered light compared to AgNP-PEG and AgNP-PVP particles. In unfixed cells, AgNP-CIT and AgNP-bPEI had higher SPR than either AgNP-PEG or AgNP-PVP particles, presumably due to greater intracellular agglomeration. After 24 hr. incubation with AgNP-bPEI, there was a dose-dependent decrease in the G 1 phase and an increase in the G 2 /M and S phases of the cell cycle suggestive of cell cycle inhibition. The nuclei of all the AgNP treated cells showed a dose-dependent increase in nanoparticles following non-ionic detergent treatment in which the nuclei retained extra-nuclear AgNP, suggesting that nanoparticles were attached to the nuclei or cytoplasm and not removed by detergent lysis. In summary, positively charged AgNP-bPEI increased particle cellular uptake. Particles agglomerated in the peri-nuclear region, increased mitochondrial toxicity, disturbed the cell cycle, and caused abnormal adherence of extranuclear material to the nucleus after detergent lysis of cells. These results illustrate the importance of nanoparticle surface coatings and charge in determining potentially toxic cellular interactions.

Silver Nanoparticles Agglomerate Intracellularly Depending on the Stabilizing Agent: Implications for Nanomedicine Efficacy

“…Longer wavelengths of detected light (i.e., red shifts) observed in HSI are associated with an increase in nanoparticle size or, in this case, an increase in agglomeration. Shorter wavelengths (i.e., blue shifts) are indicative of a decrease in nanoparticle size, such as dissolution or de-aggregation, or partial degradation [ 51 , 52 ]. We show that Pos–AgNPs agglomerate intracellularly, whereas Neg–AgNPs and Neu–AgNPs tend to produce mixtures of single nanoparticles and small agglomerated nanoparticles independent of the scenario.…”

Detection of large extracellular silver nanoparticle rings observed during mitosis using darkfield microscopy

“…These molecules may be part of membrane structures that preferentially attracted extracellular nanoparticles from the medium. Incubation with the positively charged 80 nm branched polyethyleneimine (AgNP-bPEI) did not show this circular ring pattern [25].…”

“…When nanoparticles were observed with these other light microscopic contrast methods, they were imaging agglomerated particles with physical sizes above the microscope's diffraction limit of 220 nm. Darkfield microscopy was used previously to study nanoparticles in cells and on slides [13,15,17,[21][22][23][24][25]. Although the current study used 80 nm AgNP, smaller sizes of AgNP can also be observed using darkfield microscopy.…”

“…The distribution of AgNP inside the cytoplasm of ARPE-19 cells after in vitro treatment with AgNP has been described previously [13,15,17,25]. Initially, the AgNP traversed the cellular membrane into the cytoplasm, where the particles agglomerated around the nucleus or in the vicinity of the endoplasmic reticulum.…”

“…studied or evaluated in the scientific literature [25]. The circular structures consisted predominately of closely spaced nanoparticles which appeared to be the same size as individual extracellular particles on the slide.…”