Two distinct cellular pathways leading to endothelial cell cytotoxicity by silica nanoparticle size.

BACKGROUND: Silica nanoparticles (SiNPs) are widely used for biosensing and diagnostics, and for the targeted delivery of therapeutic agents. Safety concerns about the biomedical and clinical applications of SiNPs have been raised, necessitating analysis of the effects of their intrinsic properties, such as sizes, shapes, and surface physicochemical characteristics, on human health to minimize risk in biomedical applications. In particular, SiNP size-associated toxicological effects, and the underlying molecular mechanisms in the vascular endothelium remain unclear. This study aimed to elucidate the detailed mechanisms underlying the cellular response to exposure to trace amounts of SiNPs and to determine applicable size criteria for biomedical application. METHODS: To clarify whether these SiNP-mediated cytotoxicity due to induction of apoptosis or necrosis, human ECs were treated with SiNPs of four different non-overlapping sizes under low serum-containing condition, stained with annexin V and propidium iodide (PI), and subjected to flow cytometric analysis (FACS). Two types of cell death mechanisms were assessed in terms of production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress induction, and autophagy activity. RESULTS: Spherical SiNPs had a diameter of 21.8 nm; this was further increased to 31.4, 42.9, and 56.7 nm. Hence, we investigated these effects in human endothelial cells (ECs) treated with these nanoparticles under overlap- or agglomerate-free conditions. The 20-nm SiNPs, but not SiNPs of other sizes, significantly induced apoptosis and necrosis. Surprisingly, the two types of cell death occurred independently and through different mechanisms. Apoptotic cell death resulted from ROS-mediated ER stress. Furthermore, autophagy-mediated necrotic cell death was induced through the PI3K/AKT/eNOS signaling axis. Together, the present results indicate that SiNPs within a diameter of < 20-nm pose greater risks to cells in terms of cytotoxic effects. CONCLUSION: These data provide novel insights into the size-dependence of the cytotoxic effects of silica nanoparticles and the underlying molecular mechanisms. The findings are expected to inform the applicable size range of SiNPs to ensure their safety in biomedical and clinical applications.

Tissue distribution of gold and silver after subacute intravenous injection of co-administered gold and silver nanoparticles of similar sizes.

Gold (AuNPs, 12.8 nm) and silver nanoparticles (AgNPs, 10 nm), mixed or separate, were injected into the caudal vein of male Sprague-Dawley rats for 4 weeks. The rats were allowed to recover for further 4 weeks to examine the differences in AuNP/AgNP tissue distribution and clearance. The size distribution of injected AuNPs and AgNPs were not statistically different. The dose groups (five males per group for the administration and three males for the recovery) consisted of seven divisions, i.e., control, AgNPs (with a low dose of 10 µg/kg/day, and, a high dose of 100 µg/kg/day), AuNPs (with a low dose of 10 µg/kg/day, and, a high dose of 100 µg/kg/day), as well as mixed AgNPs/AuNPs (with a low dose of 10/10 µg/kg/day, and a high dose of 100/100 µg/kg/day). The AgNPs accumulated in a dose-dependent manner in the liver, spleen, kidneys, lung, brain, testis or blood. Au concentration increased also in a dose-dependent manner in the liver, kidneys, spleen and lungs, but not in the brain, testis and blood. Ag concentration in the tissues increased dose-dependently after 4 weeks of AgNP/AuNP mixed administration, but to a much lower extent than those observed when they were administered separately. Ag concentration in the tissues after 4 weeks of AgNP/AuNP mixed administration cleared dose-dependently after 4 weeks of recovery. Au concentration in the tissues increased dose-dependently after 4 weeks of AgNp/AuNP mixed administration, while Au concentration in the tissues did not clear as seen in Ag after 4 weeks recovery. Au concentration showed biopersistency or accumulation in the liver, kidneys, spleen and brain of the 4 weeks of recovery. In conclusion, AgNPs and AuNPs showed different toxicokinetic properties and the mixed administration of AgNPs with AuNPs resulted in mutual reduction of their tissue distribution which appeared to be due to competitive inhibition. Furthermore, this subacute intravenous injection study has suggested that these nanoparticles were distributed to the organs in particulate instead of ionic forms.

Differentiating gold nanorod samples using particle size and shape distributions from transmission electron microscope images.

Size and shape distributions of gold nanorod samples are critical to their physico-chemical properties, especially their longitudinal surface plasmon resonance. This interlaboratory comparison study developed methods for measuring and evaluating size and shape distributions for gold nanorod samples using transmission electron microscopy (TEM) images. The objective was to determine whether two different samples, which had different performance attributes in their application, were different with respect to their size and/or shape descriptor distributions. Touching particles in the captured images were identified using a ruggedness shape descriptor. Nanorods could be distinguished from nanocubes using an elongational shape descriptor. A non-parametric statistical test showed that cumulative distributions of an elongational shape descriptor, that is, the aspect ratio, were statistically different between the two samples for all laboratories. While the scale parameters of size and shape distributions were similar for both samples, the width parameters of size and shape distributions were statistically different. This protocol fulfills an important need for a standardized approach to measure gold nanorod size and shape distributions for applications in which quantitative measurements and comparisons are important. Furthermore, the validated protocol workflow can be automated, thus providing consistent and rapid measurements of nanorod size and shape distributions for researchers, regulatory agencies, and industry.

Electromechanical Properties and Spontaneous Response of the Current in InAsP Nanowires.

The electromechanical properties of ternary InAsP nanowires (NWs) were investigated by applying a uniaxial tensile strain in a transmission electron microscope (TEM). The electromechanical properties in our examined InAsP NWs were governed by the piezoresistive effect. We found that the electronic transport of the InAsP NWs is dominated by space-charge-limited transport, with a I ∞ V2 relation. Upon increasing the tensile strain, the electrical current in the NWs increases linearly, and the piezoresistance gradually decreases nonlinearly. By analyzing the space-charge-limited I-V curves, we show that the electromechanical response is due to a mobility that increases with strain. Finally, we use dynamical measurements to establish an upper limit on the time scale for the electromechanical response.

Silica nanoparticles inhibit brown adipocyte differentiation via regulation of p38 phosphorylation.

Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.

Size-dependent clearance of gold nanoparticles from lungs of Sprague-Dawley rats after short-term inhalation exposure.

Gold nanoparticles are known to be distributed to many tissues following their oral, inhalation, or intravenous exposure. Information on the biodistribution and clearance of gold nanoparticles from these tissues is, therefore, important to understand their behavior in vivo. To study the effect of size on the biodistribution of gold nanoparticles, Sprague-Dawley rats were exposed by inhalation to small gold nanoparticles (13 nm in diameter on average) at an exposure concentration of 12.8 ± 2.42 µg/m(3), and to large gold nanoparticles (105 nm in diameter on average) at an exposure concentration of 13.7 ± 1.32 µg/m(3). The experimental animals were exposed to the gold nanoparticles and the control animals to fresh air for 5 days (6 h/day), followed by a recovery period of 1, 3, and 28 days in fresh air. None of the exposed animals exhibited any toxic response to the gold nanoparticles. Despite the difference in size, both small and large gold nanoparticles deposited mainly in rat lungs. Their biodistribution from the lungs to secondary target organs was significantly higher with the small compared to the large gold nanoparticles. While the large gold nanoparticles were only found in the blood, the small gold nanoparticles were detected in the liver, spleen, brain, testes, and blood. In addition, the elimination half-life of the small gold nanoparticles from the lungs was significantly shorter than that of the large gold nanoparticles. The present data may, therefore, suggest that the smaller gold nanoparticles are able to translocate from the lungs, the primary exposure organ to extrapulmonary organs at a faster rate than the larger gold nanoparticles and thus confirming previous observations reported in the literature.

Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single-Molecule Diffusivity in the Membrane of Living Cells.

We present a single-molecule diffusional-mobility-shift assay (smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells. We found that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and ß-adrenergic receptors, as determined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.

Gold nanoparticle silica nanopeapods.

A subnanometer gap-separated linear chain gold nanoparticle (AuNP) silica nanotube peapod (SNTP) was fabricated by self-assembly. The geometrical configurations of the AuNPs inside the SNTPs were managed in order to pose either a single-line or a double-line nanostructure by controlling the diameters of the AuNPs and the orifice in the silica nanotubes (SNTs). The AuNPs were internalized and self-assembled linearly inside the SNTs by capillary force using a repeated wet-dry process on a rocking plate. Transmission electron microscopy (TEM) images clearly indicated that numerous nanogap junctions with sub-1-nm distances were formed among AuNPs inside SNTs. Finite-dimension time domain (FDTD) calculations were performed to estimate the electric field enhancements. Polarization-dependent surface-enhanced Raman scattering (SERS) spectra of bifunctional aromatic linker p-mercaptobenzoic acid (p-MBA)-coated AuNP-embedded SNTs supported the linearly aligned nanogaps. We could demonstrate a silica wall-protected nanopeapod sensor with single nanotube sensitivity. SNTPs have potential application to intracellular pH sensors after endocytosis in mammalian cells for practical purposes. The TEM images indicated that the nanogaps were preserved inside the cellular constituents. SNTPs exhibited superior quality SERS spectra in vivo due to well-sustained nanogap junctions inside the SNTs, when compared to simply using AuNPs without any silica encapsulation. By using these SNTPs, a robust intracellular optical pH sensor could be developed with the advantage of the sustained nanogaps, due to silica wall-protection.

Anti-angiogenic effect of bare titanium dioxide nanoparticles on pathologic neovascularization without unbearable toxicity.

Local application requires fewer nanoparticles than systemic delivery to achieve effective concentration. In this study, we investigated the potential toxicity and efficacy of bare titanium dioxide (TiO2) nanoparticles by local administration into the eye. Mono-disperse, 20nm-size TiO2 nanoparticles did not affect the viability of retinal constituent cells within certain range of concentrations (~1.30µg/mL). Furthermore, local delivery of TiO2 nanoparticles did not induce any significant toxicity at the level of gene expression and histologic integrity in the retina of C57BL/6 mice. Interestingly, at the low concentration (130ng/mL) without definite toxicity, these nanoparticles suppressed in vitro angiogenesis processes and in vivo retinal neovascularization in oxygen-induced retinopathy mice when they are administered intravitreally. Taken together, our results demonstrate that even TiO2 nanoparticles can be safely utilized for the treatment of retinal diseases at the adequate concentration levels, especially through local administration. FROM THE CLINICAL EDITOR: In this paper the local application of titanium dioxide is described as a local treatment for retinal diseases associated with neovascularization. While these nanoparticles have known systemic toxicity, this work demonstrates that when applied locally in a mouse model, they can be used without observable toxicity even in their native forms.

Biopersistence of silver nanoparticles in tissues from Sprague-Dawley rats.

Silver nanoparticles are known to be distributed in many tissues after oral or inhalation exposure. Thus, understanding the tissue clearance of such distributed nanoparticles is very important to understand the behavior of silver nanoparticles in vivo. For risk assessment purposes, easy clearance indicates a lower overall cumulative toxicity. Accordingly, to investigate the clearance of tissue silver concentrations following oral silver nanoparticle exposure, Sprague-Dawley rats were assigned to 3 groups: control, low dose (100 mg/kg body weight), and high dose (500 mg/kg body weight), and exposed to two different sizes of silver nanoparticles (average diameter 10 and 25 nm) over 28 days. Thereafter, the rats were allowed to recover for 4 months. Regardless of the silver nanoparticle size, the silver content in most tissues gradually decreased during the 4-month recovery period, indicating tissue clearance of the accumulated silver. The exceptions were the silver concentrations in the brain and testes, which did not clear well, even after the 4-month recovery period, indicating an obstruction in transporting the accumulated silver out of these tissues. Therefore, the results showed that the size of the silver nanoparticles did not affect their tissue distribution. Furthermore, biological barriers, such as the blood-brain barrier and blood-testis barrier, seemed to play an important role in the silver clearance from these tissues.

Preparation of an aqueous suspension of stabilized TiO2 nanoparticles in primary particle form.

To prepare stabilized TiO2 nanoparticles (TiO2 NPs) in aqueous media as a suspension of the primary particles, we attempted to optimize the conditions for dispersing stable, aggregated TiO2 NPs (A-TiO2 NPs) in aqueous HCl/NaOH solutions or 5 mM pH buffered aqueous solutions. The A-TiO2 NPs with a hydrodynamic diameter (or DLS size) of 150 +/- 20 nm could be dispersed at high concentration (63.5 +/- 0.5 mg/ml) in a 5 mM phosphate buffer (PB) solution of pH 8, and a primary TiO2 (P-TiO2) NP suspension (1.2 +/- 0.3 mg/ml) with DLS size of 30 +/- 5 nm could be separated from the highly concentrated A-TiO2 NP suspension by sonication and subsequent centrifugation. It was observed by comparing the UV-Vis absorption spectra of the A-TiO2 and P-TiO2 NP suspensions that the extinction coefficient of the TiO2 NPs in the aqueous suspension depended on the degree of aggregation. The stabilized P-TiO2 NP suspension in aqueous solution can be used to study nanotoxicity as well as to characterize the physicochemical properties of TiO2 NPs.

Gene expression profiling of kidneys from Sprague-Dawley rats following 12-week inhalation exposure to silver nanoparticles.

The specific properties of silver nanoparticles (AgNPs), such as antimicrobial activity and electrical conductivity, allow them to be used in many fields. However, their expanding application is also raising health, environmental and safety concerns. Previous in vivo AgNP toxicity studies have indicated a gender-different accumulation of silver in the kidneys, with 2-3 times more silver in female kidneys compared to male kidneys. However, no other studies have further addressed this gender difference. Accordingly, the current study investigated the gender-dependent effect of AgNPs on the kidney gene level based on toxicogenomic studies of kidneys obtained from rats exposed to AgNPs via inhalation for 12 weeks. When compared with the fresh air control, the silver nanoparticle-exposed kidneys included 104 genes with a more than 1.3-fold expression increase. For the male rat kidneys exposed to a low or high dose of silver nanoparticles, 96 genes exhibited expression changes, where six genes changed with both the low and high dose; four increased and two decreased. Meanwhile, for the female rat kidneys exposed to a low or high dose of silver nanoparticles, 66 genes exhibited expression changes, where 11 genes changed with both the low and high dose; nine increased and two decreased. Gender-dependent gene expression changes of more than 2-fold were linked to 163 genes, with 79 genes in the male kidneys and 84 genes in the female kidneys, plus gender-dependent gene expression changes of more than 5-fold were linked to 21 genes. However, no genes involved in apoptosis or the cell cycle were activated by the 12-week silver nanoparticle inhalation exposure. Overall, the male rat kidneys showed a higher expression of genes involved in xenobiotic metabolism, while the female rat kidneys showed a higher expression of genes involved in extracellular signaling.

Chemical Analysis of an Isotopically Labeled Molecule Using Two-Dimensional NMR Spectroscopy at 34 µT.

Low-field nuclear magnetic resonance (NMR) spectroscopy, conducted at or below a few millitesla, provides only limited spectral information due to its inability to resolve chemical shifts. Thus, chemical analysis based on this technique remains challenging. One potential solution to overcome this limitation is the use of isotopically labeled molecules. However, such compounds, particularly their use in two-dimensional (2D) NMR techniques, have rarely been studied. This study presents the results of both experimental and simulated correlation spectroscopy (COSY) on 1-13C-ethanol at 34.38 µT. The strong heteronuclear coupling in this molecule breaks the magnetic equivalence, causing all J-couplings, including homonuclear coupling, to split the 1H spectrum. The obtained COSY spectrum clearly shows the spectral details. Furthermore, we observed that homonuclear coupling between 1H spins generated cross-peaks only when the associated 1H spins were coupled to identical 13C spin states. Our findings demonstrate that a low-field 2D spectrum, even with a moderate spectral line width, can reveal the J-coupling networks of isotopically labeled molecules.

Mesoporous Polydopamine-Encapsulated Fluorescent Nanodiamonds: A Versatile Platform for Biomedical Applications.

Fluorescent nanodiamonds (FNDs) are versatile nanomaterials with promising properties. However, efficient functionalization of FNDs for biomedical applications remains challenging. In this study, we demonstrate mesoporous polydopamine (mPDA) encapsulation of FNDs. The mPDA shell is generated by sequential formation of micelles via self-assembly of Pluronic F127 (F127) with 1,3,5-trimethyl benzene (TMB) and composite micelles via oxidation and self-polymerization of dopamine hydrochloride (DA). The surface of the mPDA shell can be readily functionalized with thiol-terminated methoxy polyethylene glycol (mPEG-SH), hyperbranched polyglycerol (HPG), and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS). The PEGylated FND@mPDA particles are efficiently taken up by, and employed as a fluorescent imaging probe for, HeLa cells. HPG-functionalized FND@mPDA is conjugated with an amino-terminated oligonucleotide to detect microRNA via hybridization. Finally, the increased surface area of the mPDA shell permits efficient loading of doxorubicin hydrochloride. Further modification with TPGS increases drug delivery efficiency, resulting in high toxicity to cancer cells.

Intermediate monomer-dimer equilibrium structure of native ICAM-1: implication for enhanced cell adhesion.

Dimeric intercellular adhesion molecule-1 (ICAM-1) has been known to more efficiently mediate cell adhesion than monomeric ICAM-1. Here, we found that truncation of the intracellular domain of ICAM-1 significantly enhances surface dimerization based on the two criteria: 1) the binding degree of monomer-specific antibody CA-7 and 2) the ratio of dimer/monomer when a mutation (L42→C42) was introduced in the interface of domain 1. Mutation analysis revealed that the positively charged amino acids, including very membrane-proximal 5°5R, are essential for maintaining the structural transition between the monomer and dimer. Despite a strong dimer presentation, the ICAM-1 mutants lacking an intracellular domain (IC1ΔCTD) or containing R to A substitution in position 505 (5°5R/A) supported a lower degree of cell adhesion than did wild-type ICAM-1. Collectively, these results demonstrate that the native structure of surface ICAM-1 is not a dimer, but is an intermediate monomer-dimer equilibrium structure by which the effectiveness of ICAM-1 can be fully achieved.

Development of multiplexed analysis for the photocatalytic activities of nanoparticles in aqueous suspension.

A multiplexed assay technique to measure the photocatalytic activity (PCA) of nanoparticles (NPs) in aqueous suspension was developed based on the observation of TiO(2) NPs-photocatalytic oxidation rate of NADH by monitoring the fluorescence intensities. 96 sample solutions of a small volume (<150 µL) could be assayed in a single run without separation of NPs within 15 min. PCA values can be measured with high sensitivity and low experimental uncertainties through the observation at various concentrations of photocatalyst, substrate, aqueous protons and pH buffer ions in a short measurement time.

Synthesis and characterization of a photoluminescent nanoparticle based on fullerene-silica hybridization.

Bright lights: Fullerene-silica hybrid nanoparticles have bright photoluminescence, high photostability, and low cytotoxicity, which are assets for bioimaging agents. The origin of the photoluminescence of the nanoparticle is the C-O-Si bond (see picture).

A reliable approach for assessing size-dependent effects of silica nanoparticles on cellular internalization behavior and cytotoxic mechanisms.

BACKGROUND: The size of nanoparticles is considered to influence their toxicity, as smaller-sized nanoparticles should more easily penetrate the cell and exert toxic effects. However, conflicting results and unstandardized methodology have resulted in controversy of these size-dependent effects. Here, we introduce a unique approach to study such size-dependent effects of nanoparticles and present evidence that reliably supports this general assumption along with elucidation of the underlying cytotoxic mechanism. METHODS: We prepared and physically characterized size-controlled (20-50 nm) monodispersed silica nanoparticles (SNPs) in aqueous suspensions. Then, a variety of biochemical assessments are used for evaluating the cytotoxic mechanisms. RESULTS: SNP treatment in three cell lines decreased cell viability and migration ability, while ROS production increased in dose- and size-dependent manners, with SNPs <30 nm showing the greatest effects. 30- and 40-nm SNPs were observed similar to these biological activities of 20- and 50-nm, respectively. Under the conventionally used serum-free conditions, both 20-nm and 50-nm SNPs at the IC50 values (75.2 and 175.2 µg/mL) induced apoptosis and necrosis in HepG2 cells, whereas necrosis was more rapid with the smaller SNPs. Inhibiting endocytosis impeded the internalization of the 50-nm but not the 20-nm SNPs. However, agglomeration following serum exposure increased the size of the 20-nm SNPs to approximately 50 nm, preventing their internalization and cell membrane damage without necrosis. Thus, 20-nm and 50-nm SNPs show different modes of cellular uptake, with smaller SNPs capable of trafficking into the cells in an endocytosis-independent manner. This approach of using non-overlapping size classes of SNPs under the same dose, along with serum-induced agglomeration analysis clarifies this long-standing question about the safety of small SNPs. CONCLUSION: Our results highlight the need to revise safety guidelines to account for this demonstrated size-dependent cytotoxicity under serum-free conditions, which may be similar to the microenvironment after tissue penetration.

Cation-assisted laser desorption/ionization for matrix-free surface mass spectrometry of alkanethiolate self-assembled monolayers on gold substrates and nanoparticles.

We propose a new scheme of matrix-free laser desorption/ionization with cation assistance for surface mass spectrometry of self-assembled monolayers (SAMs) of alkanethiolates on gold substrates and gold nanoparticles (NPs). In a proof-of-concept experiment, a simple treatment using an aqueous salt solution such as NaI(aq) was shown to lead to a significant laser desorption/ionization, producing the characteristic (disulfide) ions of alkanethiolate molecules from the monolayers. Further efforts to understand the mechanism were also given, including laser power and salt concentration dependence studies. In the power dependence study, the characteristic ions were found to be produced at low laser power where no gold substrate species was seen. At high laser power, the generation of gold species, Au(+)-Au5(+), resulted in a saturation behavior in the characteristic mass peak for alkanethiolate molecules. In addition, characteristic ions with gold adducts were not observed at any laser power. With increasing salt concentration, the characteristic mass peak was gradually increased. The results suggest that the adduct formation of a cation with alkanethiolates in the monolayers provide a facile pathway to supply a charge to UV laser-desorbed secondary neutrals for mass spectrometric detection. This cation-assisted laser desorption/ionization (CALDI) mass spectrometry was further examined with the SAMs and mixed SAMs with various terminals such as -OH, -OCH3, -NH2, -ethylene (-CH=CH2), and -acetylene (-C[triple bond]CH). The CALDI method was also successfully applied to surface mass spectrometry of monolayer-protected gold NPs (approximately 16 nm diameter) with OH- and COOH-terminated SAMs. The unique advantages of the matrix-free CALDI method may extend our capability in investigations of interfacial chemistry at SAMs as well as mass spectrometric applications using biochips and nanoparticles.

Interference of engineered nanomaterials in flow cytometry: A case study.

Nanotechnology is regarded as the enabling technology of the 21st century. However, only a relatively small number of nano-enabled medical and healthcare products finally made their way to the market. There are several reasons why such innovative approaches fail in translation, with one key factor being the uncertainty surrounding their safety assessment. Although well described, interference reactions of engineered nanomaterials (ENM) with classical cytotoxicity assays remain a major source of uncertainty. Flow cytometry is a powerful, widely used, in vitro technique. Its readout is based on the detection of refracted laser light and fluorescence signals. It is therefore susceptible to ENM interference. Here we investigated possible interferences of ENM in the Annexin V/propidium iodide (PI) assay, which quantifies apoptotic and necrotic cell populations by flow cytometry. Two case studies were conducted using either silica or gold nanoparticles differing in size, specific surface area and surface chemistry. Both ENM types were found to cause distinct interference reactions at realistic concentrations. Silica particles induced false-positive signals; however only in the absence of a protein corona and in conjunction with a particular fluorophore combination (FITC/PI). In contrast, gold particles led to complex quenching effects which were only marginally influenced by the presence of proteins and occurred for both fluorophore combinations analyzed. We present a versatile spike-in approach which is applicable to all ENM and cell types. It further allows for the identification of a broad range of different interference phenomena, thereby increasing the reliability and quality of flow cytometry and ENM hazard assessment.