Supplementary Material for: Palmitic Acid Curcumin Ester Facilitates Protection of Neuroblastoma against Oligomeric Aβ<sub>40</sub> Insult

<b><i>Background/Aims:</i></b> The generation of reactive oxygen species (ROS) caused by amyloid-β (Aβ) is considered to be one of mechanisms underlying the development of Alzheimer’s disease. Curcumin can attenuate Aβ-induced neurotoxicity through ROS scavenging, but the protective effect of intracellular curcumin on neurocyte membranes against extracellular Aβ may be compromised. To address this issue, we synthesized a palmitic acid curcumin ester (P-curcumin) which can be cultivated on the cell membrane and investigated the neuroprotective effect of P-curcumin and its interaction with Aβ. <b><i>Methods:</i></b> P-curcumin was prepared through chemical synthesis. Its structure was determined via nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). An MTT assay was used to assess Aβ cytotoxicity and the protective effect of P-curcumin on SH-SY5Y cells. The effect of P-curcumin on Aβ-induced ROS production <i>in vitro</i> and <i>in vivo</i> were assessed based on changes in dichlorofluorescein (DCF) fluorescence. A spectrophotometric method was employed to detect lipid peroxidation. To mimic the interaction of P-curcumin on cell membranes with Aβ, liposomes were prepared by thin film method. Finally, the interactions between free P-curcumin and P-curcumin cultivated on liposomes and Aβ were determined via spectrophotometry. <b><i>Results:</i></b> A novel derivative, palmitic acid curcumin ester was prepared and characterized. This curcumin, cultivated on the membranes of neurocytes, may prevent Aβ-mediated ROS production and may inhibit the direct interaction between Aβ and the cellular membrane. Furthermore, P-curcumin could scavenge Aβ-mediated ROS as curcumin <i>in vitro</i> and <i>in vivo</i>, and had the potential to prevent lipid peroxidation. Morphological analyses showed that P-curcumin was better than curcumin at protecting cell shape. To examine P-curcumin’s ability to attenuate direct interaction between Aβ and cell membranes, the binding affinity of Aβ to curcumin and P-curcumin was determined. The association constants for free P-curcumin and curcumin were 7.66 × 10<sup>4</sup> M<sup>-1</sup> and 7.61 × 10<sup>5</sup> M<sup>-1</sup>, respectively. In the liposome-trapped state, the association constants were 3.71 × 10<sup>5</sup> M<sup>-1</sup> for P-curcumin and 1.44× 10<sup>6</sup> M<sup>-1</sup> for curcumin. With this data, the thermodynamic constants of P-curcumin association with soluble Aβ <i>(ΔH, ΔS</i>, and Δ<i>G</i>) were also determined. <b><i>Conclusion:</i></b> Cultivated curcumin weakened the direct interaction between Aβ and cell membranes and showed greater neuroprotective effects against Aβ insult than free curcumin.