RESUMO
OBJECTIVES: To investigate the impact of freezing in -80°C on the structure of isolated low density lipoproteins (LDLs), using nanotechnology, such as Atomic Force Microscopy (AFM). DESIGN AND METHODS: Blood EDTA plasma was obtained from healthy subject and used immediately to isolate LDL by sequential ultracentrifugation at 10°C in 55,000 rpm for 3h, using a Beckmann XL-90 ultracentrifuge (75Ti rotor), in the presence of KBr in PBS. LDLs were then diluted with PBS until final concentrations of 5 and 15 mg LDL/dl. After initial observation, samples were frozen in -80°C for two weeks and observed again after thawing. Experiments were performed in triplicate on two smooth and clean substrates of different hydrophobicity, glass (HOPG) and Si (c-Si). Statistical significance was set at 0.05. RESULTS: Macroscopically, LDL particles formed aggregations in a dendroid layout. There were no differences between images taken from both substrates (HOPG and c-Si). Frozen samples presented significantly smaller LDL particles, than fresh ones. In specific, mean diameter of LDL particle in the fresh LDL sample was 19.77 nm, ranging from 13.34 to 28.76 nm. The frozen LDL sample had a mean diameter of 5.2 nm, ranging from 2.0 to 8.0 nm, which was significantly different from the unfrozen. CONCLUSIONS: Atomic Force Microscopy showed that freezing of LDL causes alterations in their size.
