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

2. 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

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

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

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

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

7. 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

8. Interaction of 1μM B12I122- under +100 mV constant potential
Current (pA)
Time (s)
Current (pA)
Time (s)

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

10. Interaction of 1μM B12I122- under -100 mV constant potential
Current (pA)
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)
Time (s)

11. 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

12. Interaction of 0.25 μM B12I122- -100mV potential applied in cycles
At lower concentrations Very clear gating
Interaction of 0.25 μM B12I mV 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 B12I mV 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)

13. 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

14. +cluster
+detergent
10 mM HEPES 0.15 M NaCl

15. 10 mM HEPES 0.15 M NaCl
At 1 M KCl: no leakage!

16. 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

17. 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

18. Thank you for your attention!

20. Interaction of 0.1 mM BSH in 0.15 M KCl at -70mV constant potential
Time (s)
-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