Dynamic light scattering (DLS), through detection of light scattering from matter, allows the measure of shape, size and size distribution profile, of suspended particles or polymers under Brownian motion. DLS measures the hydrodynamic diameter of nanoparticles, which includes the core particle, its solvation shell, and any adsorbed molecules. The technique is highly sensitive to aggregation, as larger aggregates scatter light more intensely, influencing size distribution results. DLS provides a rapid, non-destructive measurement but is less accurate for polydisperse or non-spherical particles.

The scattering fluctuations are then used to estimate the diffusion coefficient (D) applying the Stokes-Einstein equation (Equation 1) to calculate the hydrodynamic diameter (d _H ) of a particle. This calculation depends on the viscosity of suspending media (η), the absolute temperature (T), and the Boltzmann constant (k) as follows: d H = k T / 3 π η D (1)

The obtained diffusion coefficient (D) relies on the assumption that particles are spherical in shape and that the viscosity of suspension is known. But, particles in suspension exhibit constant movement and are seldomly spherical. The hydrodynamic particle diameter determined by DLS represents the diffusion of particles in suspension, which means that the obtained diameter provides only an approximate indication of the particle true size. The hydrodynamic diameter, also known as the Stokes diameter, then refers to the size of a hypothetical sphere that has the same translational diffusion coefficient as the particle under analysis (Kastner and Perrie, 2016).

DLS measurement generally yields a distribution dependent on intensity, number or volume. Intensity-based measurements can be used to calculate the number and volume distributions, which are crucial for estimating the relative proportion of particles in a sample (Chu and Liu, 2000). The size distribution is determined by weighing the intensity-based distribution according to the scattered light intensity of each particle. The intensity of scattered light correlates with both the concentration of the analyte and its molecular weight. The absolute molecular weight can be ascertained by integrating light scattering measurements with concentration assessments, such as refractive index measurements. The dispersed light is unaffected by the scattering angle for molecules significantly smaller than the wavelength of the input light (Andersson et al., 2009). DLS is a rapid technique for analyzing particles of very small size, is a highly reproducible technique and with the advantage of not requiring substantial training of the operator. The limitations of DLS are related to its sensitivity to the presence of larger particles or aggregates which can cause intensity measurement distortion (Chu and Liu, 2000; Ito et al., 2004), As a result, this method is not suitable for characterizing particle size distributions that are heterogeneous or multimodal (Bell et al., 2012). Accurate dilution of the sample is also needed to minimize the ratio of signal to noise in the measurements and to prevent the occurrence of multiple light scattering. The quality of filtered medium and clean cuvettes are essential in order to prevent contamination (Kastner and Perrie, 2016).

Sikora et al. (2016) evaluated the size change of silica NPs due to the adsorption of proteins from serum which forms a “protein corona.” The authors reported an increase in particle with the adsorption of proteins at the outer particle layer (Sikora et al., 2016). However, in case of hydrophilicity and agglomeration, DLS might prove unable to accurately measure the particle size. In such cases, one can rely on other high-resolution techniques such as Differential Centrifugal Sedimentation (DCS).

Differential Centrifugal Sedimentation (DCS), also known as disk centrifugal photo-sedimentation (DCP) or analytical disc centrifugation, is a novel method for the quantification of NP size distribution as small as 2 nm, and is being used to resolve multi-modal particle distributions and to detect minute variations in particle size (Plüisch et al., 2021). The sample is introduced at the base of a centrifuge chamber, rather than at the fluid interface, enabling particles with a lower density than the fluid to ascend to the fluid’s surface. DCS was used in a study by Kruse et al. (2024), to evaluate the size distribution of ultra-small gold NPs of 1.5 nm covalently conjugated with doxorubicin and a far-red-fluorescent dye. Authors highlighted that the hydrodynamic diameter is routinely underestimated by DCS for extremely small particles due to the unknown effective density of the metal core combined with the hydrated ligand shell (Kruse et al., 2024). Ultra-small metal NPs (mean size of 2 nm), made of silver, platinum, and others, stabilized with glutathione, were characterized by DCS (Wolff et al., 2024b), allowing to confirm the NPs dispersibility in water without signs of agglomeration.

Nakajima et al. (2024) used DCS to assess the adsorptive interactions between ceramic particles and polymeric binders in slurry. Under a centrifugal force, the particles settling induce viscous resistance on the adhered binder, resulting in separation governed by the particle size and density. The authors confirmed that DCS helped in the quantitative evaluation of adsorption forces in the nano-Newton range, translating the interactions between particles and binders (Nakajima et al., 2024).

In Nanoparticle Tracking Analysis (NTA) technique, the size distribution of particles suspended in a liquid medium is assessed by analyzing their Brownian motion and light scattering (Monakhova et al., 2023). A beam of laser light passing through a prism encounters the suspended particles, which causes scattering of light. This light is focused by an optical microscope onto a camera that captures particle movement, creating a video file. This enables each particle in the frame to be tracked and its specific movement in the suspended medium to be studied (Filipe et al., 2010). It is a comparatively new special technique that can be used particularly for biological systems and molecules, such as DNA and proteins.

As in the case of DLS analysis, diffusion coefficient via NTA can be computed from the distance traveled by the particles, with the help of Stokes-Einstein equation, the distances travelled by each particle in the frame yields real-time information regarding particle size distribution. Besides, the data obtained can be used to display the number-based distribution, intensity and particle concentration (Kastner and Perrie, 2016).

The smallest particle size that can be estimated by this technique depends on the intensity of the scattered light. The intensity of the scattered light is affected by factors, such as particle size, laser power wavelength, angle and refractive index of the particle and suspending medium, and particle shape. Refractive index of the particles being analyzed determines the lower limit detected. The upper limit, on the other hand, is affected by Brownian movement of the particles which tends to decrease with increase in particle size. NTA measurements are based on the assessment of hydrodynamic diameter of each particle under study. Thus, this avoids errors due to the presence of large particles or their aggregates, being NTA a complementary technique to DLS. NTA usually analyses the motion of individual NPs rather than the sum of their motion. The technique allows higher resolution of sample containing mixed population of particles and analysis of fluorescent particles (Braeckmans et al., 2010). NTA also allows to investigate the size distribution of NPs of diameters in the range of 10–1,000 nm (Filipe et al., 2010). In comparison to DLS, NTA is found to be very accurate for size evaluation of mono- and polydisperse NP populations. NTA, however, requires well-trained personnel for precise measurement. User systematic error in selection of field of view cannot be ruled out. Comparing the time of experiment, NTA usually takes longer time to be carried out than DLS. In addition, the measurement chamber has to be cleaned manually after each measurement (Kastner and Perrie, 2016).

Gross et al. (2016) investigated the particle size, as well as the concentration of variable sized NPs in polymeric suspensions and protein samples. The authors confirmed the very accurate and reproducible particle size measurements and concentration in monodisperse systems, under well-defined and constant parameter conditions (Gross et al., 2016).

Hubert et al. (2020) showed that the NTA technique can potentially be used to characterize submicron-sized protein aggregates and particles in a range of lyophilized and in-solution pharmaceutical products. The authors also showed that the average size of standard 200 nm NPs was within 10% of the expected particle size (Hubert et al., 2020).

Among various imaging techniques available for NPs size analysis, microscopic techniques, such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM), represent the most important ones.

X-Ray Diffraction (XRD) represents one of the primary techniques to describe the structural properties and crystallinity of NPs. This method provides a rough estimation regarding particle size, using Debye Scherrer formula (Khan et al., 2017; Kumar and Rao, 2019), besides allowing the identification of single and multiple NPs (Emery et al., 2016). However, for NPs with sizes lower than 100 of atoms, the proper measurement and acquisition of structural attributes may pose problems. Besides, the diffractograms can be influenced by NPs with amorphous properties and variable atom-atom bonds (Ingham, 2015).

X-ray diffraction is known to vary according to the atoms composing the crystal lattice and their arrangement. When a sample is exposed to a beam of X-ray and undergoes diffraction, the distances between the planes of atoms can be measured employing the Bragg’s Law (Equation 2): n λ = 2 d   sin ⁡ θ (2) where n is an integer in the order of the diffracted beam, λ represents the wavelength of the incident beam of X-ray, d denotes the distance between adjacent planes of atoms (the d-spacings), and θ is the angle formed by incidence of the X-ray beam. The d-spacings can also be calculated as the geometry of the XRD instrument is specifically designed to facilitate this measurement. The sample’s particular set of d-spacings is what allows the X-ray scan to provide a precise “fingerprint” of the material. XRD can also estimate crystallite size using Scherrer’s equation, making it a key tool in nanoparticle characterization. The interpretation of the scan entails comparison with the standard references patterns, on the basis of which the materials are identified (Pandian, 2014).

Statistically representative data are among the benefits of XRD. However, the diffractogram obtained for NPs with size less than 3 nm may be too broad. Moreover, the samples meant for XRD analysis need to be in powder form, which is usually obtained by drying the colloidal dispersion of NPs (Mourdikoudis et al., 2018), and this treatment can modify the polymorphism of the particles under study.

Upadhyay et al. (2016) checked the size of magnetized Fe₃O₄ particles by means of XRD with the Scherer formula using the broadening of the most intense peak. The size of Fe₃O₄ particles ranged from 9 nm to 14 nm when the synthesis temperature increased from room temperature to 100°C. The size increased from 27 nm to 53 nm with increasing the reaction period from 10 min to 2 h. The lattice parameter for the analyzed samples ranged from 0.8397 to 0.8414 nm (Upadhyay et al., 2016).

Nath et al. (2020) published a study on XRD peak broadening analysis by different models. Cadmium selenide (CdSe) NPs were prepared by first reducing selenium powder into sodium hydrogen selenide (NaHSe) with sodium borohydride (Nath et al., 2020). To this selenium precursor, a mixture of distilled water, 3-mercaptoacetic acid and cadmium chloride was added to finally obtain CdSe NPs. XRD was used for the analysis of their structural attributes and crystallinity. XRD peak broadening analysis was carried out to estimate the elastic properties of the CdSe NPs. Various properties studied with the help of Scherrer plot were stress, intrinsic strain and energy density. Models, such as Williamson–Hall plot, Halder-Wagner Method and Size-Strain Plot were employed, in addition to particle size determination. The values of average particle size of CdSe NPs determined by TEM and SEM analysis were found to be 36.23 nm and 36.2 nm, respectively. The authors confirmed that the values obtained from Halder-Wagner Method and Size Strain Plot for the particle size of CdSe NPs were close to those obtained from TEM, SEM and AFM analysis (Nath et al., 2020).

Similar to XRD technique, small-angle X-ray scattering (SAXS) method allows elastic scattering at a specific angle in a solid; however, the SAXS detector covers small angle of scattering (Modrow, 2005). Typically, this technique is used to measure the size, size distribution and shape of NPs.

Wang et al. (2008) utilized SAXS to assess the structural changes in platinum NPs as a function of temperature. Wide-angle X-ray scattering (WAXS) allows the diffraction maxima at larger angles because the distance between the detector and the NPs sample is smaller than in SAXS (Wang et al., 2008).

Navarro Oliva et al. (2024) used both SAXS and WAXS in the size characterization of nanocomposites based on Fe₃O₄ and polyvinylidene fluoride (PVDF) of 6, 9, and 14 nm. SAXS and WAXS allowed to distinguish α-phase into the β-phase of PVDF (Navarro Oliva et al., 2024). The scattering patterns yielded information regarding the size and structure of the NP aggregates, the lamellar structure of the PVDF (Navarro Oliva et al., 2024), and besides informing the type of α, β, γ, and δ polymorphs (Concha et al., 2024).

X-Ray Absorption Spectroscopy (XAS) ascertains the X-ray absorption of the sample as a function of energy. Every element has an exquisite set of characteristic absorption edges which correspond to different binding energies of its electrons, enabling element selectivity by XAS. This technique encompasses X-ray absorption near edge structure (XANES or NEXAFS) and extended X-ray absorption fine structure (EXAFS) methods (Mourdikoudis et al., 2018). It provides information regarding coordination number, bond distances and disorders of the neighboring atoms. As it does not need any long-range order, it can be widely employed for a variety of materials with some modifications in mechanical and optical design, entire XAS spectrum can be recorded with millisecond resolution. Further, it is one of the most appropriate tools to observe structural modification during a reaction process and the change in the oxidation state of metal cations in situ. Despite these advantages, one major challenge of all X-ray based techniques is that they yield ordinary information. Therefore, XAS can be complimentary to other techniques, such as XRD, UV-visible spectroscopy, Fourier Transformed Infra-Red (FTIR), Raman and mass spectroscopy, for broader view of the NPs and more consolidated results (Nayak et al., 2018). Wu et al. (2002) (Wu et al., 2002) employed XAS for detailed structural determination of titanium oxide NPs. de Oliveira et al. (2020) coupled XAS with X-ray diffraction and used these techniques to explore the bottom-up mechano-synthesis of gold NPs in the presence of different reducing agents. The authors demonstrated that these techniques provide new directions for mechanochemical research of advanced electronic substances (de Oliveira et al., 2020).

Ultraviolet visible (UV-Vis) spectroscopy is another low-cost characterization technique often employed for investigation of nanoscale materials (Carpentieri and Domenici, 2024). It is capable of handling a great number of NPs and the sample preparation is relatively easy (Tomoda et al., 2020). In this technique, intensity of light reflected from a sample is measured and compared to the intensity of reflected light from the reference standard (Tomaszewska et al., 2013). NPs possess optical characteristics, and their shape, concentration, size, state of agglomeration and refractive index are other features which make UV-Vis spectroscopy a vital technique to carry out for identification, characterization and study of NPs stability (Mourdikoudis et al., 2018). Sensitivity, simplicity and selectivity of this technique, together with the short duration of measurement and no requirement of calibration, are some merits of UV-Vis spectroscopy over other size determination techniques (Tomaszewska et al., 2013). This technique is also a non-destructive robust tool for quick recognition of the different types of NPs. Silver, copper and gold NPs exhibit typical UV-visible extinction spectra in the visible region. The absorbance (350–400 nm) can also be employed to determine the gold colloid concentration (Mourdikoudis et al., 2018).

UV-Vis spectroscopy was used to confirm the synthesis of silver NPs from fungal isolates (Santos et al., 2022) and from hydroalcoholic extract of leaves of Schinus terebinthifolius Raddi (de Oliveira et al., 2021). Haiss et al. (2007) (Haiss et al., 2007) published an interesting study on the use of UV-Vis spectra to assess the concentration and size of gold NPs, varying between 5 and 100 nm. Hussain et al. (2020) (Hussain et al., 2020) used UV–Vis spectroscopy to characterize gold NPs in varying ethanol-water volumetric ratios in the solvent mixture. The maximum surface plasmon resonance (SPR) absorption wavelength depends on the size and shape of NPs. The maximum wavelength value of the prepared gold NPs exhibited a shift, meaning that the size of NPs increased with the increase of ethanol-water volumetric ratio. The maximum wavelength of gold NPs produced in a solvent mixture with 20% and 50% volumetric percentage of ethanol was found to appear in the shorter absorption wavelength regions (514 nm and 520 nm), which suggested that gold NPs synthesized with lower ethanol percentage were smaller in size. Also, the maximum wavelength of NPs, prepared with ethanol-water volume percentage of 80%, displayed a shift towards higher wavelength regions. This implies that larger sized particles were formed, while a broad trend of the plots indicates unevenly shaped gold NPs (Hussain et al., 2020).

Nuclear Magnetic Resonance (NMR) Spectroscopy is a useful analytical method for structural and quantitative characterization of nanomaterials. NMR was used to study interaction between ligand and surface of magnetic NPs (Wolff et al., 2024a). This technique helps in the routine investigation of molecular scale NPs, in both solid and solution phases. Particularly, it is useful for analysis of the production and final concentration of metallic NPs (Mourdikoudis et al., 2018). NMR also provides a more convenient and advanced way of surface chemistry evaluations and size determination of NPs. All NMR reports have inherent low sensitivity of Zeeman splitting (Marbella and Millstone, 2015). Guo and Yarger (2018) employed NMR spectroscopy for thoroughly studying characterization of gold NPs for surface chemistry investigation, size determination and structural evaluations. The sizes of gold NPs assessed by NMR agreed with TEM outcomes (Guo and Yarger, 2018).

Other useful techniques for NP size measurement include ferromagnetic resonance (FMR), dynamic light scattering (DLS) or photon correlation spectroscopy (PCS), and matrix assisted laser desorption/ionization (MALDI). Inductively coupled plasma mass spectroscopy (ICP-MS) is a powerful elemental analysis technique used to determine the composition and concentration of nanoparticles. While it does not directly measure nanoparticle size, single-particle ICP-MS (spICP-MS) can estimate size distributions by analyzing individual nanoparticle ionization events.

Table 1 lists examples of size measurement techniques applied to NPs for different size ranges and purposes.

The merits of in vitro toxicity assessment include relatively lower cost and fewer ethical concerns, when compared to the in vivo screening assays. In vitro cytotoxicity is mainly assessed through necrosis, apoptosis, proliferation, oxidative stress and DNA damage assays (Lama et al., 2020; Tirumala et al., 2021). Evaluation of in vitro toxicity using various cell lines has been used and reported in a number of publications [e.g., (Silva et al., 2019a; Silva et al., 2019b]. Cell viability and lethality are the two most common characteristics assessed for in vitro measurement of toxicity for different types of NPs (Silva et al., 2019a; Silva et al., 2019b; Mao et al., 2022).

An increased interest in investigating the effects of NPs size on targeting cellular uptake has been observed over the years (Augustine et al., 2020; Galindo-Camacho et al., 2023). Numerous research groups have conducted in vitro studies involving variable cell types and different NPs, and assessed the impact of size and surface charge on their uptake into the cells and on drug targeting.

In vivo toxicity evaluation brings NPs a step closer to its clinical relevance. Precision in the control of physicochemical characteristics, such as size, surface charge, shape and stability of NPs, is of utmost importance in in vivo studies since these properties govern their residence time in the blood stream and also their site specific targeting properties (Kumar et al., 2017; Sabourian et al., 2020; Yagublu et al., 2022). In vivo toxicological evaluation is generally performed using appropriate animal models. The choice of an adequate experimental model for estimating toxicity (in vitro and in vivo) is of vital significance. In vitro models (cell lines) easily help to analyze toxic effects of nanoparticulate matter on individual components of cells and tissues, but in vivo evaluation makes it possible to assess the NPs toxicity for individual organ and to the whole organism (Ajdary et al., 2018; Gnach et al., 2015). The assessment techniques for in vivo toxicity comprise studies regarding biodistribution, clearance, hematology, histopathology and serum chemistry.

The most commonly employed animal models, for in vivo studies, include mice and rats, due to their explicit genome, which makes them most appropriate for studying the histopathological changes and functions of various organs (such as hepatic, hematological, renal, nervous and immune systems). Besides these, rabbits, zebra fish, Drosophila melanogaster and Caenorhabdtis alegans also find use in toxicological investigations (Yang et al., 2017; Severino et al., 2023).

In vivo assessment studies may be focused on changes in the tissue structure (Jans and Huo, 2012), apoptosis (Mallidi et al., 2009; Sharma et al., 2009), inflammation and infiltration in major organs (spleen, kidney, brain, lungs and heart), but may also be aimed to investigate certain organ systems, which tend to concentrate NPs on account of their structures specificity (Yang et al., 2017). Elucidating the biodistribution of NPs in various organs may prove beneficial to guide the optimization of NPs, in all respects including their size. After animal sacrifice, the main organs or tissue are removed, commonly to explore distribution of NPs, but also to assess morphological and histological changes and signs of toxicity. The ultra-structural modification and effect of the NPs on tissue can be clearly revealed employing electron microscopy analyses. Functional injury can be detected with the help of diverse biomarkers and sophisticated high-tech instruments (Yang et al., 2017; Joseph et al., 2023). Furthermore, NPs can be detected in live or sacrificed animal models through radio labelling (Dai et al., 2021).

Gold NPs were found to cause apoptosis and acute inflammation in liver, accumulation in various organs, as well as penetration potential in sperm head and tail regions (Cho et al., 2009; Wiwanitkit et al., 2009). A study was carried out with the aim of checking the in vivo toxicity of zinc NPs at concentration of 14–20 μg/mL for 12 h in order to ascertain the impact of treatment on DNA damage, cell viability, apoptosis and ROS production (Sharma et al., 2012).

Silver NPs biodistribution and toxicity were investigated using CD-1 mice by intravenous injection of 10 mg/kg of silver NPs with different particle size, i.e. 10, 40, and 100 nm, possessing different shell coatings. Each type of synthesized NPs caused toxicity to tissues, but larger sized particles were less toxic, apparently because they are less prone to penetration (Recordati et al., 2015).

Jimeno-Romero et al. (2016) evaluated the biodistribution, bioaccumulation and biological effects of disodium laureth sulfosuccinate (DSLS) stabilized titanium dioxide (TiO₂) NPs in marine mussels (Mytilus galloprovincallis). This in vivo study used 0.1, 1, and 10 mg titanium/L concentration, either in bulk or as TiO₂ NPs (sized 60 and 180 nm). A considerable accumulation of NPs was noted in mussels dosed with TiO₂ NPs, localized in lysosomes, endosomes and residual bodies of digestive cells, as well as in the lumen of digestive tubules. The NPs measuring 60 nm were internalized to a larger extent in lysosomes of digestive cells as compared to 180 nm ones. This was confirmed by quantitative analysis of black silver deposits following autometallography (Jimeno-Romero et al., 2016). The larger sized particles were found in the lumen of gut where they were present as large aggregates. This resulted in notably decreased stability of lysosomal membrane to both the types of NPs, though greater in extent upon exposure to those with 60 nm diameter. The membrane stability was also affected by exposure to TiO₂ in bulk and DSLS-stabilized NPs. Therefore, it was concluded that membrane stability was dependent on the size of the NPs, and also on the biological effects caused by DSLS and TiO₂ (Jimeno-Romero et al., 2016).

Balmuri et al. (2017), Balmuri et al. (2017) synthesized two different types of cadmium oxide NPs, by calcinations of Cd(OH)₂ without any organic molecule (CdO-1) and with cadmium-citrate coordination polymer (CdO-2). The authors compared the toxicity of the two types of cadmium oxide NPs, on zebra fish showing those obtained by calcination with CdO-2 reduced toxicity, which was revealed by lower levels of oxidative stress, rescue of hepatic carboxylexterases and diminished metallothionein activity. These results were also supported by histopathological examination. The study articulated the toxic effects of cadmium oxide NPs in aquatic animals and also illustrated that the toxicity could be significantly reduced by carbon coverage (Balmuri et al., 2017).

Kang et al. (2020) designed infrared fluorophore conjugated polyethylene glycols (PEGs) in different sizes, and investigated their size-dependent enhanced permeability and retention (EPR) effect, and checked their pharmacokinetics, biodistribution and renal clearance in tumor-bearing mice. The targeting efficiency of PEGylated particles was confirmed by measuring tumor-to-background ratio. While the smaller sized variants of PEGs (≤20 kDa, 12 nm) displayed marked tumor targeting with either very low or non-specific uptakes, the larger sized variants (>20 kDa, 13 nm) were found to accumulate significantly in major organs, such as liver, lungs and pancreas (Kang et al., 2020).