Figure 2

(A) Partition of compounds in an U-tube experiment. Concentration of biguanide on the trans-side of the U-tube at 48 h and 72 h at 25 °C, after addition of 250 µM of biguanide on the cis-side. (B) Variation in the internal pH of HPTS-containing EYPC liposomes. Intravesicular solution: 1 mM HPTS, 10 mM HEPES, and 100 mM NaCl, adjusted to pH = 7.4, and extravesicular solution: 10 mM HEPES and 100 mM NaCl, adjusted to pH = 7.4. Biguanidiums were injected after 50 s at 5 mM (50 mol% relative to the 10 mM EYPC concentration), a NaOH pulse was induced at 300 s, and the liposomes were lysed with Triton-X at 550 s. Each curve is the average of three independent measurements. (C) Fluorescence of safranin O in the EYPC liposomes. Biguanidium salts were injected at 5 mM (50 mol% relative to the 10 mM EYPC concentration) at 50 s. Each curve is the average of three independent measurements. (D) Mitochondrial penetration of compound 1b and metformin. Mitochondrial isolation was performed according to the previously reported protocol²¹. For each experiment, an anti-HA IP was performed on KP4 cells expressing pMXs-3XHA-EGFP-OMP25 that were treated for 3 h with 15 μM metformin, 15 μM compound 1b or vehicle. This shows mitochondrial penetration and accumulation of the drugs at 15 μM concentration after 3 h. As positive controls were used methanol solutions of metformin and 1b at 15 μM, as negative control methanol and untreated mitochondria as vehicle.