Dodecaborate clusters forms stable pores in lipid membranes Melinda Bartok, Lucia Lozano White, Mengxi Wang, Dennis Ledwon and Detlef Gabel 16th ICNCT, 14-19 June 2014, Helsinki, Finland

Why to use Boron clusters? TEM of tissue from glioma patients after infusion with BSH prior to surgery BSH stained with polyclonal primary Ab and secondary red-fluorescent Ab, Hoechst stained cell nucleus

Halogenated boron clusters B12H122- Zeta potential DSC Cryo-TEM Leakage Toxicity on cells BLM 7.2 Å B12I122- 11.0 Å

Zeta potential of 0.5 mM DPPC, DSPC and DPhPC liposomes with dodecaiodo dodecaborate Binding const.: DPhPC 0.0047 mM DPPC 0.0087 mM DSPC 0.017 mM

Cryo-TEM images of 5mM DPPC liposomes alone and with 50mM B12I122- 200 nm

Black lipid membrane technique Teflon film Bilayer Solvent annulus Electrode Cis Trans Salt solution Monolayer

Membrane potential is responsible for pore formation – 50 mV +100 mV ΔV=150 mV – 100 mV ΔV= – 50 mV 1. Many pores No pores – 50 mV +100 mV ΔV=50 mV – 100 mV ΔV= – 150 mV 2. Many pores No pores – 50 mV +100 mV ΔV=100 mV – 100 mV ΔV= – 100 mV 3. Few pores

Interaction of 1μM B12I122- under +100 mV constant potential Current (pA) 150 190 230 270 340 380 420 Time (s) Current (pA) 450 490 530 570 640 680 720 Time (s)

Maximum size of the created pores 5 – 7.30 min const. potential of +100 mV Current (pA) Pore size: 25 Å Current (pA)

Interaction of 1μM B12I122- under -100 mV constant potential Current (pA) 150 190 230 270 340 380 420 Time (s) We also saw that it is concentration dependent-needing concentrations of 1 μM to see pores and sometimes 0,5 μM for transient pores Current (pA) 450 490 530 570 Time (s)

Maximum size of the created pores Current (pA) 7.30 – 10min const. potential of -100 mV Pore size: 45  Å Off-scale pore, we can see how it tries to close, only to open further and then completely close in just 1 milisecond Current (pA) The event goes off scale, but membrane remains stable

Interaction of 0.25 μM B12I122- -100mV potential applied in cycles At lower concentrations Very clear gating Interaction of 0.25 μM B12I122- -100mV potential applied in cycles -85pA = 6 Å pore +50pA= 5 Å pore -20pA = 3 Å pore 0 mV - 100 mV 0 mV - 100 mV 0 mV - 100 mV Current (pA) 23 24 25 26 27 Time (min) Interaction of 0.25 μM B12I122- -100mV constant potential -400pA = 13 Å pore -700pA= 17 Å pore -450pA = 13 Å pore -250pA = 10 Å pore -350pA = 12 Å pore RIGHT SIDE CYCLES (Attempt 2) // RIGHT SIDE CONSTANT (Attempt 2) Current (pA) 13 14 15 16 17 Time (min)

Interaction of B12I122- on decane containing membrane under different potentials 5000 pA 31.5 Å pore -5000 pA 20 25 30 35 40 minutes 1 µM 2 µM 3 µM -100 mV -100 mV -20 mV

+cluster +detergent 10 mM HEPES 0.15 M NaCl

10 mM HEPES 0.15 M NaCl At 1 M KCl: no leakage!

Cooperative pore formation These clusters stick together 5 clusters care 50 A of length, which is the same size as the DPhPC bilayer (5nm = 50 A) K+ ion sorrounded by 4 water molecules 7- 8 clusters stick together to span the bilayer 5 -6 clusters across to create a big enough pore Leaving a 10 Å diameter pore

CONCLUSIONS Holes need potential, concentration and time Higher concentrations: big holes with short life span Lower concentrations: smaller holes with long life span Average size of holes is around 5-10 Å Holes as big as 45 Å in diameter Closing mechanism much quicker than opening Holes in solvent-containing membranes persist for hour(s) In liposomes, 1 M KCl prevents leakage

Thank you for your attention!

Interaction of 0.1 mM BSH in 0.15 M KCl at -70mV constant potential Time (s) 0 20 40 60 80 100 -200 -400 -600 -800 -1000 -1200 Current (pA) Interaction of 0.1 mM BSH in 0.15 M KCl at -70mV constant potential BSH 0.1 mM 150 mM KCl