4.1.1. Particles Size (PS)

Particle size (PS) investigations serve as one of the main determining factors of biodistribution and retention of NCM in target tissues. PS is defined as the size of a hypothetical hard sphere that exhibits the same diffusion characteristics as the NCM being measured. The result is reported as mean particle size and homogeneity of size distribution. The latter parameter is expressed as the polydispersity index (PDI), which is a dimensionless parameter calculated from cumulative analysis of the DLS-measured intensity autocorrelation function [71]. A PDI value in the range <0.25 indicates a desirable homogeneity whereas a PDI value >0.5 indicates heterogeneous particle sizes [72]. While DLS provides a simple and rapid estimate of particle size, several studies suggest that DLS exhibits inherent limitations and is relatively poor at analysing multimodal particle size distributions [72,73].

By way of example, when a mixture of 20- and 100-nm nanometre particles are measured, the signal for the small particles may be lost because the signal intensity of a spherical particle of radius *r* is proportional to *r* [74]. Therefore, the scattering intensity of small particles tends to be masked by that of larger particles. Microscopy provides an accurate assessment of the size and shape of a NCM but often requires complicated sample preparation steps specific to the microscopic technique used [73], which in turn may change samples and create artefacts such as NCM agglomeration, which is particularly observed during drying prior to electron microscopy [75]. Furthermore, due to the limited throughput capability, it is difficult to obtain an accurate particle size distribution [74], and the underlying principle is that the sample status in each method is not the same.
