The Biological Fate of Silver Nanoparticles from a Methodological Perspective

We analyzed the performance and throughput of currently available analytical techniques for quantifying body burden and cell internalization/distribution of silver nanoparticles (Ag NPs). Our review of Ag NP biological fate data shows that most of the evidence gathered for Ag NPs body burden actually points to total Ag and not only Ag NPs. On the other hand, Ag NPs were found inside the cells and tissues of some organisms, but comprehensive explanation of the mechanism(s) of NP entry and/or in situ formation is usually lacking. In many cases, the methods used to detect NPs inside the cells could not discriminate between ions and particles. There is currently no single technique that would discriminate between the metals species, and at the same time enable localization and quantification of NPs down to the cellular level. This paper serves as an orientation towards selection of the appropriate method for studying the fate of Ag NPs in line with their properties and the specific question to be addressed in the study. Guidance is given for method selection for quantification of NP uptake, biodistribution, precise tissue and cell localization, bioaccumulation, food chain transfer and modeling studies regarding the optimum combination of methods and key factors to consider.


Chapter 1
We present a detailed review of studies on the biological fate of Ag NPs in most representative organisms used in environmental studies. Table S1 presents existing data on the Ag NPs body burden and Table S2 on the body distribution and cell/tissue internalization of Ag NPs in organisms. Table S1. Existing data on the Ag NPs body burden grouped according to the techniques used. Organisms are sorted according to taxonomic position.

HEDFM
Random distribution throughout the body. All Ag NPs were found in gill, gut lumina, mid-brain and liver parenchyma.
No Ag NPs were found in epidermis, spine, skeletal muscle, kidney or gonad.
Possible entry due to local tissue necrosis or via endocytosis. [69] Protozoan Authors suggest passive passage through cell wall, which may be enhanced when cells are exposed to a high concentration of Ag NPs. [2] Cress Arabidopsis thaliana Sigma-Aldrich (USA) 10 nm * TEM Ag NPs accumulate predominantly at the middle lamella and cell walls in root tissue and some Ag NPs can be translocated toward the leaves.
Authors suggest the intercellular passage of NPs through the cell wall. [58] Isopod Authors also suggest ionic uptake into the active region of the root tip and secondary formation of NPs. [78] Wheat Triticum aestivum L.
NanoAmor, PVP coated 52 ± 1 nm (determined by XRD) μ-XRF μ-XANES Ag NPs mostly adhered to the epidermis of roots and accumulated preferentially in discontinuities between root epidermal cells. No Ag 0 was found inside roots.
Transfer of NPs due to local damage of roots. Root hairs also considered as potential points of entry of NPs, due to thin cell walls and role in nutrient acquisition. Symplastic and apoplastic transfer of Ag + is possible. Secondary Ag NPs were also identified. Penetration through chorionic pores. [72] Nematode

DFOMS
Electron-dense material found around uterine area, but Ag was not confirmed.
Penetration through vulva to reach uterine area. [79] Zebrafish Danio reiro PlasmaChem GmbH (Berlin, Germany) 20 nm * hydrodynamic diameters > 100 nm SIMS Ag hot spots were found mainly around liver blood vessels and in the interstitial tissue between the intestine and the liver. Authors do not explicitly say that Ag NPs were detected, but refer to »Ag hot spots«.
Authors suggest that Ag NPs cross the skin of zebrafish larvae and accumulate in blood vessels. However, no discussion regarding the passage of dissolved Ag + is given. [80] Zebrafish

Chapter 2: Supplementary Description of Methods For NPs Body Burden, NPs Body Distribution and Cellular Internalisation
Studying interactions between nanomaterials and organisms is a multi-disciplinary approach where experts in biology, physiology and nanomaterials should be complemented with understanding of the performance of instruments and data processing. There are several review papers presenting techniques appropriate for nanomaterials detection in biological samples [84][85][86][87].
However, they are mostly focused on technical characteristics of those methods, but much less on biological nature of a sample and the purpose of using these techniques. In this section we prepared a detailed review of characteristics of the methods applied to study the biological fate of NPs. First, we briefly describe the basic principle of each method. The main focus is to describe the specificity of each method to separate NPs from ions. The limitations of each method are discussed along. These data were the basis to extract the summary of methods characteristics presented in the main body of the manuscript (Table 1, Figure 1).

Atomic absorption spectrometry (AAS) and inductively coupled plasma spectrometry (ICP-MS)
Basic principle: The basic difference between AAS and ICP-MS is that AAS relies on the atomic absorption process while ICP-MS is an atomic/ionic emission spectroscopic technique. A combustion flame or graphite furnace is typically used for AAS while ICP uses plasma for atomic or ionic species generation. Detection limit by ICP is more than three orders of magnitude lower than by AAS [88]. Specificity: All of the above techniques do not discriminate between ions and particulate matter as they are applied to decomposed samples. Both AAS and ICP-MS can provide data for tissue level if enough tissue is available for separate analyses of different body parts.
Limitations: Biological matrices contain abundant organic matter that must be oxidized prior to analysis to release the bound metal and reduce the physical interference from solid matter during analysis. For this purpose, acid digestion and microwave heating are usually used [89]. During this pre-analysis step, NPs are decomposed, and element of interest is released [90]. Discrimination of different chemical species is thus not possible.

Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS)
Basic principle: Liquid sample is introduced into the ICP-MS instrument by using a nebulization system, which produces an aerosol of polydisperse droplets. Once the droplets are in the plasma, the solvent evaporates forming solid particles, which in turn are vaporized and their elements atomized and ionized. Ions are extracted through the interface into the mass spectrometer, where they are separated according to their mass/charge ratio and detected [58,91]. The intensity of each pulse is proportional to the mass of the element (i.e. number of atoms) in each detected NP [91]. This method is suited for the analysis of body, organ/tissue NP burden and allows a simultaneous determination of elemental chemical composition, concentration, number concentration, size, and the number size distribution of NPs [91].
Specificity: SP-ICP-MS can differentiate the particle of interest from other incidental particles of the same size, but of different composition [92].
Limitations: SP-ICP-MS is not able to distinguish among particles, aggregates and agglomerates. It is particularly suitable for NPs consisting of one element only and sizes higher than 20 nm [92]. At the moment, the application of this method for biological samples is still under development and a number of questions need to be resolved.
An important aspect is how to store the samples prior to analysis to avoid NPs transformation. It needs to be considered that approaches required to digest and clean-up the samples may lead to misidentification of specific elements and may cause transformation of NPs (our personal experience, unpublished data).

Asymmetrical flow field-flow fractionation inductively coupled plasma mass spectrometry (AF4-ICP-MS)
Basic principle: AF4-ICP-MS is a separation technique where metal species are size-separated in a thin open channel with laminar flow under the influence of a perpendicular external field (separation force). Separation system is based on asymmetrical flow field of particles [92]. The elemental analysis is done in the ICP-MS. This technique is also suitable for body, organ/tissue burden analyses and has a better resolution than the SP-ICP-MS.
The enrichment of the nanoparticle fraction and simultaneous reduction of the ionic background via AF4 results in a clearly improved ICP-MS detection sensitivity, which enables a more refined identification and size characterization of the migrated ion species.
Specificity: It gives mass and element specific information and can detect smaller (2-50 nm) particles than SP-ICP-MS. As all ICP-MS, it has element specific capabilities, including mixed metals analysis [92]. Coupling of AF4 to SP-ICP-MS results in improved SP-ICP-MS sensitivity. AF4 and SP-ICP-MS are used to determine number and size of NPs in a mixture. In this combination, AF4 is essential for providing sample sub-streams that are sufficiently purified and simplified for the SP-ICP-MS analysis [93].
Limitations: There may be difficulties in sample separation due to particle aggregation within the channels and particle interactions with the membrane of the analyser.

Tracing labelled NPs
Basic principle: Nanoparticles can be labelled with radioactive isotopes, stable isotopes or florescent dyes. They are detected in tissue homogenates using spectrophotometric approach or by fluorescent or radioactive imaging [62,94]. Stable isotope labelling has been successfully used in conjunction with multiple-collector-inductivelycoupled-plasma-mass-spectrometry (MC-ICP-MS) [9,95].
Specificity: Labelling of NPs with radioactive or stable isotopes is a useful tool for the highly sensitive and selective detection of NPs in the environment and organisms, thus enabling the tracing of their uptake, distribution and clearance with high sensitivity [9,95,96]. Radiolabelled Ag NPs have been prepared using different approaches, including the following: (i) de novo synthesis using gamma radioisotope 110m AgNO3 as a precursor [62,64,97], (ii) the adsorption of 110m AgNO3 onto existing Ag NPs [59], or (iii) the neutron activation of Ag NPs [98]. Measurements can be done on tissue level and localisation can reveal the presence of Ag NPs at the cellular level (autoradiography).
Limitations: These methods can modify the NP surface chemistry and might alter its behaviour, but the general problem is that they are not able to differentiate between particulate or ionic dissolved forms of NPs after entering the tissue [12,64]. Radioactive labelling demands purpose-built laboratory and equipment. Stable isotope labelling is not beset with the problems of other methods such as fluorescent dyes or radioactive isotope labelling, where the label may be lost as a result of dissociation or radioactive decay [9].

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)
Basic principle: The upper layer of a dry sample (whole organism or lyophilised slices) mounted on sample holder is sputtered by laser ablation and subsequently correlates the measured ion signals with one of the matrix elements by inductively coupled plasma mass spectrometry [99][100][101].

Specificity:
The advantage of this method is the tissue mapping for elemental distribution, but it does not distinguish between ionic or particulate form of investigated element. The quantitative imaging by LA-ICP-MS requires the external matrix-matched calibration which is not trivial [100].
Limitations: One disadvantage of LA-ICP-MS is the occurrence of nonstoichiometric effects in the transient signals, defined as elemental fractionation. The internal standards are needed for quantitative analysis [99].

Secondary ion mass spectroscopy (SIMS)
Basic principle: Secondary ion mass spectroscopy (SIMS) is well established as a surface analytical method for advanced material research of composition of solid surfaces and thin films by sputtering the surface of the specimen with a focused primary ion beam and collecting and analysing the ejected secondary ions. In generated charged molecules or molecule fragments their mass-to-charge ratios are measured. Recent advances in the instrumentation have made these techniques even more powerful and applicable to biological research as well [102]. It utilizes a tightly focused primary ion beam to desorb chemical species from a solid matrix. A variety of different SIMS instrumentation has been used for imaging at nanoscale, among them ToF-SIMS, FIB-SIMS and Nano-SIMS [103]. In case of TOF-SIMS the detached particles are then accelerated into a flight tube and their mass is determined by measuring the exact time at which they reach the detector [104]. The main advantage of Nano-SIMS over TOF-SIMS and other generations of SIMS is the ability of Nano-SIMS to operate at nanoscale resolution, whilst maintaining both excellent signal transmission and high spatial resolution [105].
Specificity: SIMS offers information on elements present in the sample, but detailed molecular information can also be gained with high sensitivity [106]. The information on elemental composition of the sample is provided in the form of a mass spectrum and as elemental maps. An approximate range of 30-50 nm was provided for lateral resolution for ToF-and Nano-SIMS [73,103].
Limitations: The analysis of the obtained data is complex. Generally, it does not produce quantitative data.
Quantification is possible with the use of standards. Optical capabilities are typically limited, which presents a difficulty for finding specific regions of interest for analysis. Charging may also be a problem in some samples [104]. Limitations of SIMS are complex sample preparation, difficult to differentiate NP from localized ions and ultrahigh vacuum required [73,103].

Dark-field single nanoparticle optical microscopy and spectroscopy (DFOMS)
Basic principle: Dark-field light microscopy, which captures scattered light from the sample, is ideal for identifying strongly scattering objects such as Ag NPs in low scattering matrices i.e. dark background [107]. DFOMS enables imaging on tissue level on slices or even in small organisms (mm range).
Specificity: It provides in vivo imaging in real-time [108].

Limitations:
With this method only very bright NPs can be detected -noble metals NPs, NPs with highest quantum yield (QY) of Rayleigh scattering. Very small particles cannot be detected [109]. DFOMS is known for limited spatial resolution [73].

Hyperspectral enhanced dark-field imaging (HEDFM)
Basic principle: Hyperspectral enhanced dark-field imaging is an optimized dark field microscopy. It allows tissue level or small organism (in the mm range) investigation. In case of NP imaging, an analysis is based on optical signals from resonant light scattering and a spectral signature library for NP of interest. Under enhanced dark field conditions, particles appear 150-fold brighter due to Koehler illumination and the critical illumination by a collimated light source at oblique angles [110]. The HEDFM spectrometer has the ability to acquire the optical spectrum for all points in a microscope image and couple it with specialized spectra [110]. In HEDFM, spectral information of each pixel of the image is added as a third dimension of values to the two-dimensional spatial image, generating a three-dimensional data cube, sometimes referred to as hypercube data or as an image cube. It is possible to use dark field microscopy for the detection of metallic nanoparticles, since due to their plasmonic properties NPs with dimensions larger than 50 nm scatter light strongly at a particular resonant wavelength [111].
Specificity: It is possible to distinguish between ions and NPs, because they produce different signals [69,112,113].

Limitations:
Detection of very small NPs (below 50 nm) is possible, but requires increasing illumination intensity.
Biological media may significantly affect the surface characteristics of NPs due to the formation of a corona, which in turn affects the signal [111]. Therefore, the effects of biological media on NPs must be well known to properly interpret the obtained spectral imaging signal.

Transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (TEM-EDX)
Basic principle: In transmission electron microscopy (TEM) a beam of electrons is transmitted through a specimen, it interacts with specimen and forms an image by emitting electrons. When a beam of electrons interacts with the sample being studied, characteristic X-rays are also emitted [48,79]. TEM can thus be used for intracellular imaging and as well as chemical analyses.
Limitations: Conventional TEM, although powerful in terms of lateral resolution, is limited by low signal intensity due to thin samples (low interaction volume) [115]. Also, electron dense deposits seen in the sample may be artefacts from osmium or uranium crystals formed during sample preparation [116] or any other electrondense cellular structure. It is therefore crucial, that EDX is applied to identify the nature of the observed spots. In the case of un-sectioned samples (for example bacteria) it is difficult to confirm that material is internalized and not adsorbed to the cell surface or any other extracellular material [117].

EDX)
Basic principle: FIB-SEM-EDX is a scanning microscope with an electron column and an ion column embedded in the same specimen chamber where both beams are aiming at the same point on the specimen surface. FIB-SEM-EDX is imaging and analytical technique which permits simultaneous sectioning, electron imaging of selected region and EDX analyses at any desired location [118,119].
Specificity: This type of microscopy allows imaging and elemental analyses of FIB exposed regions. The FIB-SEM investigation can be applied to bulk samples, prepared for conventional SEM at any chosen site or to bulk resinembedded specimens, prepared for conventional TEM. The FIB-SEM allows the 3D imaging of NPs in a single cell, albeit at lower spatial resolution than in TEM. As a result, the absolute amount of NPs per cell could be estimated which is not possible by any other tool [120].
Limitations: FIB-SEM-EDX cannot distinguish between ions and NPs.

Particle induced X-ray emission (PIXE)
Basic principle: PIXE technique provides a multi-elemental imaging for small biological samples. A beam of protons is accelerated to an energy of a few mega-electron-volts that excites characteristic X-rays in the atoms of the specimen [121]. It allows the investigation of samples up to 50 µ m thick.

Specificity:
The elemental distribution and concentration at the tissue level is provided with the lateral resolution in the micron range [70]. With this method, imaging and X-rays based identification of elemental composition is possible.

Limitations:
The system is not as sensitive for higher-Z elements, hence not all elements can be measured. It is also a destructive imaging technique which induces stronger beam damage to the specimen than other X-rayinduced methods described before [70,122]. Exessive damages can be avoided by using lower beam intensities.

X-ray absorption spectroscopy (XAS) and micro-X-ray fluorescence (XRF)
Basic principle: X-ray absorption micro spectroscopy (XAS) technique provides information on the chemical state of the metal-rich particles detected by micro-X-ray Fluorescence (XRF) mapping [123]. With both methods it is possible to generate chemical maps of an element in relation to its oxidation state and chemical bonding. X-rays are absorbed in matter and the energy of the X-rays is converted into fluorescent X-rays. The incident X-ray energy finally becomes the thermal energy of the absorber. The X-ray absorption spectra of condensed matter near the threshold energy have fine structures (X-ray absorption near edge structure spectroscopy (XANES)), observable at energies less than the threshold energy. Obtained spectra show both the line shape modification and chemical shift of the absorption edge or peak [124]. With XANES, complete maps of the chemical forms can be derived, sometimes revealing details which would remain hidden using only μ-XRF and μ-XAS. Additionally, irradiation durations and the elapsed time between the start and completion of data collection for each pixel are significantly reduced [125]. Sensitivity of these methods is very high if the light source is a synchrotron [126].
Synchrotron X-rays are often preferred to desktop-generated X-rays as they offer a significantly higher resolution, a better signal-to-noise ratio, short acquisition times and quantitative reconstructions and provide phase contrast in addition to absorption contrast imaging [127].
Specificity: Besides elemental characterization, this technique enables also the analysis of the chemical (e.g. oxidation) state of the element of interest [128]. The technique provides subcellular analyses.
Limitations: Beam damage of samples can occur. Effects of radiation damage can vary from subtle changes in spectra to, in extreme cases, total sample destruction [125]. Areas within a thin specimen, which are denser or thicker, will provide a more intense signal even if the element of interest is homogeneously distributed. In the elemental map, elements can appear co-localised, when in fact could be located at different depths within the sample [126]. Another limitation is the inability to distinguish between scattering atoms with little difference in atomic number. It is difficult to use for light elements [129].