L α phosphatidylcholine

Proteoliposomes were prepared using the detergent-doped liposomes method^{4 (link),51 (link)}. Dry pellets of l-α-phosphatidylcholine (derived from soybean, Sigma) were dissolved in chloroform, dried in a rotary evaporator, resuspended to 20 mg/ml and sonicated in buffer containing 50 mm KPi, pH 7.5. After three freeze–thaw cycles, large unilamellar vesicles were prepared by extrusion through a polycarbonate filter with pore diameters of 400 nm. Liposomes were diluted to 4 mg/ml and destabilized beyond R_sat with Triton X-100. SEC-purified SLC26Dg in 0.2% DM was added to the liposomes at a weight ratio of 1:50 (protein/lipid) for transport assays or 1:20 (protein/lipid) for PELDOR measurements, and detergent was subsequently removed by the addition of Biobeads. For radioisotope transport assays, proteoliposomes were harvested by centrifugation for 1.5 h at 250,000 × g and resuspended in 50 mm sodium phosphate (NaPi), pH 7.5, 2 mm MgSO₄ to a lipid concentration of 20 mg/ml. After three freeze–thaw cycles, proteoliposomes were stored in liquid nitrogen until analysis. For PELDOR measurements, proteoliposomes were harvested by centrifugation for 20 min at 250,000 × g and resuspended in 50 mm KPi, pH 8.0 to a final spin concentration of 80–130 µm.

E(AH) compounds were generated using the thin-film hydration method. L-α-Phosphatidylcholine (Sigma-Aldrich), TEGO Care CG 90 surfactant (Evonik Industries AG, Germany), and AH compounds were added to 20 ml ethanol (Duksan Co.). We used a rotary evaporator (RE100-Pro, DLAB, China) to completely remove ethanol and form a lipid membrane on the flask wall. The lipid membrane was hydrated in 20 mL 5% ethanol and used as an elastic ethosome. After homogenization to produce a particle with a consistent size using a microfluidizer (M110EH, Microfluidics, USA), the unloaded components were removed using a 0.45 μM syringe filter (Advantec, Japan) and ethosomes were stirred overnight at 4°C for stabilization.

Rab18 lentiviruses (overexpression and knockdown) were produced as previously described [16]. Briefly, overexpression or knockdown plasmids with three other plasmids (pGag/Pol, pRev, pVSVG) were co-transfected into HEK293 T cells using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Cat L3000015). After 16 h, the medium was replaced with advanced DMEM containing 2% FBS, 0.01 mmol/L cholesterol (Sigma-Aldrich, Cat C8667), 0.01 mmol/L L-α-phosphatidylcholine (Sigma-Aldrich, Cat P443), 1:1000 diluted Chemically Defined Lipid (Invitrogen, Cat 11905031), and 4 mmol/L L-glutamine (Sigma-Aldrich, Cat G7513). After 48 h, supernatants containing the lentivirus were collected. Lentiviral titers were determined by tissue culture infectious dose (TCID50) in HEK293 T cells. The lentiviruses (MOI = 1) were applied to infect SUVECs. After a 12-h infection, SUVECs were cultured in fresh medium with puromycin (5 mg/mL) (Sigma-Aldrich, Cat P8833) to select stable cell lines. The empty vector CMV and random sequence vector (NTshRNA) were treated equally as controls.

To prepare stabilizer for CSP, 2.5 g L-α-phosphatidylcholine (P7443, Sigma, USA) and 3.42 g sucrose esters (Gemfont Corporation, Taiwan) were sequentially incorporated into 400 mL water. The mixed stabilizer materials were stirred at 25°C, and 40 g curcuminoid powder with curcumin, demethoxycurcumin, and bisdemethoxycurcumin (Toong Yeuan, Taiwan) were added to form a 10 % curcuminoid aqueous solution. This non-homogenously mixed solution was subjected to a high-speed homogenization pretreatment from 4,000 to 6,000g for 10 minutes using a PRO250 homogenizer (PRO Scientific, USA). Next, a nano-grade wet grinder (Netzsch-Fein mahltechnik GmbH, Germany) carried on yttria-stabilized tetragonal zirconia for circulation milling with 0.2 mm beads for 180 minutes to obtain the aqueous dispersion. The average diameter of un-nanosized curcuminoid was 5140±178 nm, and the average diameter of CSP was 59±1 nm. Finally, the nanosized CSP composed of curcumin (83.56%), demethoxycurcumin (14.13%) and bisdemethoxycurcumin (2.31%) was obtained. Right before oral administration, CSP was diluted into the drinking water at concentration 0.75 mg/mL. The vesicle control in this study contain the same stabilizer and went through the same preparation process without adding curcuminoid.

The plasmid pcDNA 3.1(−)/r‐caspase‐3 was constructed by the investigators 19. AE was purchased from Jiangxi Tiangong Technology (Jiangxi, China), and dimethylsulfoxide (DMSO), l‐α‐phosphatidylcholine, cholesterol, 3‐2,5‐diphenyl‐tetrazolium bromide (MTT), and Hochest33342 were purchased from Sigma‐Aldrich Co. (St. Louis, MO). Caspase‐3 antibodies were obtained from Cell Signaling Technology (Danvers, MA) and β‐actin antibodies and the corresponding secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RPMI 1640 medium was purchased from Hyclone (Logan, UT) and fetal bovine serum (FBS) was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). The light‐emitting diode (LED) light was purchased from Chongqingjingyu Laser Biological Company (Chongqing, China). Human gastric cancer cell line (SGC‐7901) was purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China.