Curcumin

All solvents and reagents were reagent grade; H₂O was distilled and deionised. Tanshinone IIA was purchased from Sigma (St. Louis, MO) and was dissolved in ethanol (100%). The two turmeric preparations contained the curcuminoids curcumin, desmethoxycurcumin, and bis-demethoxycurcumin as follows: (1) Naturex #140500 (Avignon, France); 95% curcuminoids (high-performance liquid chromatography, Supplementary Fig. S1D), (2) curcumin powder 95%, 89.88% curcuminoids and volatile oils of turmeric rhizome (Dolcas Biotech LLC; PCCA, Houston, TX). Both were dissolved in dimethylsulfoxide (DMSO) (Sigma; St. Louis, MO), prior to addition to cell cultures. Resveratrol (99.0%; Jiaherb) and EGCG (95.3%; Taiyo Green Power Co., Ltd) were obtained from PCCA (Houston, TX), and were dissolved in ethanol. All solutions were sonicated for 10 min. The source of the compounds for the gene expression studies was as follows: curcumin (≥98% curcuminoid content) (CAS number 458-37-7) (Thermo Fisher Scientific, NJ, USA; prepared by Acros Organics for Thermo Fisher; stored at room temperature under nitrogen), (−)-epicatechin gallate, and Resveratrol (Thermo Fisher).

Exosomes were loaded with different molecules. The hydrophobic drug Curcumin (Acros Organics™, Thermo-Fisher Scientific) was loaded into exosomes (0.5% loading w/w) by incubation for 10 min in the dark. Curcumin–exosomes were then isolated by ultracentrifugation at 120,000×g for 1 h at 15 °C in an SW32 Ti rotor (Optima TL Ultracentrifuge, Beckman Coulter) and resuspended in MilliQ water. Exosomes were also loaded with nucleic acids (random siRNA, DNA-CH or miRNA145) by electroporation as previously described [42 (link)]. Briefly, exosome pellet was resuspended in PBS 0.1X and gently mixed with the appropriated µl of siRNA, DNA-CH or miRNA (same loading than for EMNs) in a final volume of 400 μl into 0.4 cm electroporation cuvettes. Exosomes were then electroporated using a Gene Pulser II Electroporator (Bio-Rad), at 300 V and 25 μF of capacitance. Lastly, exosomes were incubated in ice for 30 min to allow the exosome membrane to be fully restored. To get rid of free nucleic acids, exosomes were diluted with cold PBS and isolated again by ultracentrifugation at 120,000×g for 90 min at 15 °C in 70.1 Ti rotor (Optima TL Ultracentrifuge).

Human Aβ_1-42 was purchased from Peptide Institute Inc. (Osaka, Japan), curcumin (Figure 1) from Thermo Fisher Scientific K.K. (Waltham, MA, USA), and GT863 (purity: 99.1%) (Figure 1) from Green Tech Co., Ltd. (Kyoto, Japan). Nicardipine was obtained from Merck KGaA, NNC 55-0396 from Cayman Chemical Company (Ann Arbor, MI, USA), and PD173212 from Abcam PLC. (Cambridge, UK). curcumin and GT863 were dissolved in DMSO, with a final concentration of 0.1%. The other chemical reagents used were commercially available special-grade products.