Dynamic light scattering (DLS)

The size of liposomes was analyzed by dynamic light scattering technique (DLS) using a Zetasizer Nano ZSP (Malvern Instruments, Worcestershire, UK). The experiments were conducted in triplicates at 25°C. Liposomes at 0.3mM concentration were measured with and without the increasing concentrations of Pb²⁺, Cd²⁺, Co²⁺, Ni²⁺, and Mn²⁺ to assess any metal induced changes. Furthermore, it was ensured that the size of liposomes was kept at a narrow distribution with a polydispersity index (PDI) of less than 0.10 (< 10%) for each measurement.

Osmolarity of the liposomes was considered and calculated using the osmolarities of the mono- and divalent salts present in the external environment [68]. Briefly, the osmolarity of the mono- and divalent cations were determined based on the number of particles from dissolving the solution in water. The osmolarity of the divalent cation was then divided to the monovalent cation to generate a percentage change in osmolarity.