Lignin as an amphiphilic polymer can undergo phase separation and form nanoparticles by aggregation driven by hydrophobic interactions. The particles prepared using solvent shifting from HL60 and HL85 demonstrate excellent colloidal stability, but interestingly smaller size compared to conventional lignin nanoparticles (LNPs) prepared under identical conditions (Figure ). The particle sizes analyzed by DLS show that the final size of particles depends on the degree of hydroxymethylation (Figure ). Considering the effect of temperature on hydroxyl content of hydroxymethylated lignin shown in this study, this trend is in line with the results of Pylypchuk et al. who found that increasing the content of aliphatic hydroxyl groups of lignin can result in smaller particle sizes. It is worth noting that as the particle size decreases the turbidity in the colloidal dispersion drops which is the result of smaller angles of light scattering (Figure ). SEM images confirm this trend. Larger particles were observed in the case of HL60NP compared to the more extensively hydroxymethylated HL85NP (Figure and). Most importantly, these hydroxymethylated particles remain reactive not only due to the hydroxymethyl groups, but also owing to the presence of free phenolic hydroxyl groups on their surfaces (Figure ). Additionally, hydroxymethylated lignins show increased solubility in the solvent mixture (acetone-water 3:1 w/w) used in the solvent shifting method, which results in higher concentrations of HLNPs in the final aqueous dispersions. In addition, the particle size of the HLNPs can be tuned by increasing the initial concentration of HL85 beyond that possible with unmodified lignin, while still achieving a good mass yield (>80%) (Figure ).