# Characterization of Nanoparticle Batch-To-Batch Variability
## 3 Results
### Highlights of Variations in Physicochemical Properties between Different Batches per NM  
#### 3.2.1 Silica NMs  
As shown in Figure 1F the St\(\ddot{\mathrm{o}}\)ber synthesized silica NM batches SiINP03, 05, 06, 07, 09 and 10 only slightly induced DCF oxidation at 400 \(\upmu\)g/mL (around 2-fold over control). However, batch 08 was more reactive (5.9-fold over control) and batch 04 was extremely reactive (24.2-fold over control). Further analysis of the batches by ICP-MS and ICP-OES was performed in order to identify any potential contamination that might explain the unusual behaviour of batches SiINP04 and SiINP08. Figure 1D shows the mass concentration of alkali and alkaline earth metals detected in the different batches. Figure 1: St\(\ddot{\mathrm{o}}\)ber synthesized silica SiINP03–10 (**A**) Production parameters for the different St\(\ddot{\mathrm{o}}\)ber synthesized silica nanoparticles; (**B**) Zeta potential representing the surface charge in -mV at pH 8.4 \(\pm\) 0.57; (**C**) Representative transmission electron microscopy (TEM) images (**D**) Selected impurities present in the different St\(\ddot{\mathrm{o}}\)ber synthesized SiO\({}_{2}\) NM batches. The mass concentrations of elements above detection limit are shown. The data represent the mean of two assays; (**E**) Particle diameters \(\mathrm{d_{P}}\) in nm: Z-Average of \(\mathrm{d_{P}}\) determined in aqueous suspension using dynamic light scattering (DLS) (black bars) and modal value \(\mathrm{x_{M}}\) of the number size distribution in the aerosol phase measured by mobility spectrometry (SMPS) (red bars); (**F**) Potential to induce ROS. The test NMs were delivered as suspensions of 5 mg/mL in water. The graph shows the Dichlorofluorescein (DCF) fluorescence induced by the different batches at 400 \(\upmu\)g/mL relative to a sample without NMs (control). 0.3 \(\upmu\)M H\({}_{2}\)O\({}_{2}\) was used as a positive control. Additional tested was the commercial material SiINP02. The data represent mean values of two independent experiments with three replicates \(\pm\) s.e.m. The [PERSON] synthesized SilNP04 batch, which showed a significantly increased ROS induction compared to control (Figure 1F) also had one other noticeable characteristic: It is the only batch with a detectable amount of magnesium (Figure 1D). Different Mg concentrations in cerium dioxide NMs have previously been shown to have a modest influence on the ROS production by [PERSON] et al. [37], therefore this might explain the different behaviour of SilNP04 in terms of its ROS production. For SilNP08 no conspicuous properties were detected which could explain the increased DCF fluorescence. As shown in Figure 2F, the flame synthesized SilNP012 to SilNP016 moderately induced DCF oxidation (around 3-fold over control), which was comparable to the reactivity of commercial SiO\({}_{2}\) (Aerosil(r)200, Evonik, Essen, Germany). Only batch 17 was more reactive (6.2-fold over control). Interestingly, none of the metals determined in batch SilNP017 stood out in terms of their concentration, meaning that the source of the high ROS production potential of this batch is not related to metal contaminants. Data from literature showed no potential of amorphous SiO\({}_{2}\) NMs, either flame or Stober synthesized, to generate ROS in a cell-free system, while quartz particles did. The tests were performed using electron paramagnetic resonance (EPR) spectroscopy [38] but also by the DCF assay [39]. However, in a very recent study flame or Stober synthesized NMs were tested for their potential to induce ROS, again by EPR analysis [7]. Both NMs induced ROS, however, flame synthesized NMs were much more efficient presumably due to the presence of siloxane rings at the surface. Whether the discrepancy of these different studies is due to differences in synthesis conditions needs to be addressed in the future. Additionally, whether SilNP017 had additional siloxane rings relative to the other batches and the cause of this, could be a topic for future research.