Purpose: Silver nanoparticles (AgNPs) have been widely applied in various fields as excellent antibacterial reagents over the past decades. Although the particle size is considered as the most crucial factor influencing cellular uptake, transportation, and accumulation behaviors, there are still many controversies regarding the correlation between size and uptake of AgNPs. In this study, size-dependent cellular uptake of AgNPs with different diameters was investigated in B16 cells. Methods: The uptake of AgNPs was investigated by inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopic (TEM) imaging in B16 cells. Results: Twenty nanometer and 100 nm AgNPs had the lowest and highest uptake efficiency at both 12 hours and 24 hours, respectively. Smaller AgNPs crossed the plasma membrane faster with uniform distribution: 5 nm AgNPs were detected in both cytoplasm and nucleus at 0.5 hours after incubation. Larger AgNPs were extremely difficult to migrate: 100 nm AgNPs were detected in the nucleus at 12 hours after incubation. Internalization of AgNPs was directly observed, mainly within membrane-bound structures, such as intracellular vesicles and late endosomes. The uptake of all four-sized AgNPs (5 nm, 20 nm, 50 nm, 100 nm) decreased significantly after the pre-treatment with chlorpromazine hydrochloride, which can specifically inhibit the clathrin-mediated endocytosis. The internalization efficiencies of AgNPs (5 nm, 20 nm, 50 nm) were markedly reduced by methyl-β-cyclodextrin, a specific caveolin-mediated endocytosis inhibitor, whereas 5-(N-ethyl-N-isopropyl) amiloride as an inhibitor of macropinocytosis inhibited the uptake of larger sizes of AgNPs (50 nm and 100 nm). Conclusion: The results suggest that the size of AgNPs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. The clathrin-mediated endocytosis may be the most common endocytic pathway for AgNPs in B16 cells, and AgNPs at each size were likely to enter cells by a major internalization pathway.

Silver nanoparticles (AgNPs) have tremendous potentials in medical devices due to their excellent antimicrobial properties. Blood compatibility should be investigated for AgNPs due to the potential blood contact. However, so far, most studies are not systematic and have not provided insights into the mechanisms for blood compatibility of AgNPs. In this study, we have investigated the blood biological effects, including hemolysis, lymphocyte proliferation, platelet aggregation, coagulation and complement activation, of 20 nm AgNPs with two different surface coatings (polyvinyl pyrrolidone and citrate). Our results have revealed AgNPs could elicit hemolysis and severely impact the proliferation and viability of lymphocytes at all investigated concentrations (10, 20, 40 μg/mL). Nevertheless, AgNPs didn’t show any effect on platelet aggregation, coagulation process, or complement activation at up to ~40 μg/mL. Proteomic analysis on AgNPs plasma proteins corona has revealed that acidic and small molecular weight blood plasma proteins were preferentially adsorbed onto AgNPs, and these include some important proteins relevant to hemostasis, coagulation, platelet, complement activation and immune responses. The predicted biological effects of AgNPs by proteomic analysis are mostly consistent with our experimental data since there were few C3 components on AgNPs and more negative than positive factors involving platelet aggregation and thrombosis.

Protein coronas provide the biological identity of nanomaterials in vivo. Here we have used dynamic light scattering (DLS) and transmission electron microscopy (TEM) to investigate the adsorption of serum proteins, including bovine serum albumin (BSA), transferrin (TRF) and fibrinogen (FIB), on gold nanoparticles (AuNPs) with different surface modifications (citrate, thioglycolic acid, cysteine, polyethylene glycol (PEG, M _w = 2 k and 5 k)). AuNPs with PEG(5 k) surface modification showed no protein adsorption. AuNPs with non-PEG surface modifications showed aggregation with FIB. AuNPs with citrate and thioglycolic acid surface modifications showed 6–8 nm thick BSA and TRF coronas (corresponding to monolayer or bilayer proteins), in which the microscopic dissociation constants of BSA and TRF protein coronas are in the range of 10⁻⁸ to 10⁻⁶ M.

Introduction: Increased use of silver nanoparticles (AgNPs) has raised concerns that AgNPs may induce toxic effects. In vitro studies of cell monolayers and in vivo studies have produced conflicting results. The inconsistency of these results has been mainly due to limitations of two-dimensional (2D) monolayer cell systems. Methods: A three-dimensional (3D) epidermal model called EpiKutis^®, which exhibits good tissue viability and barrier function was developed. The cytotoxicity of AgNPs against EpiKutis was compared to that against 2D keratinocytes at equivalent AgNPs doses (0.035, 0.07, 0.14, 0.28, and 0.56 ng per cell). The amount and distribution of AgNPs in the 3D EpiKutis and 2D keratinocytes after exposure were determined. The toxic mechanisms of AgNPs, such as oxidative stress and production of pro-inflammatory cytokines, were investigated. Results: The results demonstrated that cell viability was greater than 80% and lactate dehydrogenase (LDH) release did not increase even at the highest dose of AgNPs in EpiKutis. In contrast, treatment of 2D keratinocytes with AgNPs resulted in dose-dependent decrease in cell viability from 63% to 11%, and a dose-dependent increase in LDH release from 8% to 16%. Cytotoxicity of AgNPs in 2D keratinocytes was related to oxidative damage and inflammation, as evidenced by increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), IL-1α, IL-6, and IL-8. In addition, levels of superoxide dismutase (SOD) were decreased. EpiKutis treated with AgNPs did not exhibit increased oxidative damage or inflammation, which may have been due to the barrier properties of the 3D structure, resulting in reduced penetration of AgNPs. At equivalent per cell doses, total silver penetration into EpiKutis was 0.9 ± 0.1%, and total silver penetration into 2D keratinocytes was 8.8 ± 0.6% detected by ICP-MS. The penetration and distribution of AgNPs in 2D keratinocytes were confirmed by the TEM-EDS analysis, which was not found in the 3D EpiKutis. These results showed that AgNPs penetrated EpiKutis to a lesser degree than they penetrated 2D keratinocytes, which suggested that EpiKutis exhibited significant barrier function. Discussion: The results of this study showed that AgNP toxicity should be evaluated using 3D epidermal models, which may provide better estimates of in vivo conditions than 2D models. The EpiKutis model may be an ideal model for assessment of nanotoxicity.

The biological safety of nanomaterials is a worldwide concern considering the extensive usage of nanoparticles in daily life as well as in medical care. However, toxicity evaluation of nanomaterials has met difficulties because of unique properties of nanomaterials. Here, we have investigated the interference of nanoparticles on the toxicity evaluations. Silver nanoparticles (AgNPs) with different sizes and surface coatings were used as model materials. Several widely used assays, such as lactate dehydrogenase (LDH) release, MTS assay and nitric oxide (NO) measurement, have been investigated in our study. Our results showed that 20 nm PVP-coated AgNPs with a concentration of 42.8 μg mL⁻¹ affected LDH detection by about 50%, while citrate-coated 20 nm AgNPs with a concentration of 42.8 μg mL⁻¹ affected LDH detection by about 70%. Moreover, 20 nm AgNPs with a concentration of 42.8 μg mL⁻¹ disturbed MTS assay and NO measurement by less than 20% and 10%, respectively. Based on our results, nanoparticles with higher concentrations gave more interference. Therefore, for accurate toxicity evaluation of nanoparticles, it is very necessary to limit particle concentrations or choose other approaches free from the interference.