Interaction of N,N,N-trialkylammonioundecahydro-closo-dodecaborates with dipalmitoyl phosphatidylcholine liposomes (Appendix II)

The results of the zeta potential measurements indicate that the compounds bind to the liposomal surface, as drastic changes of the surface potential occur upon addition of the

Discussion

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compounds. The binding to the hydrophobic part of the membrane becomes energetically more favorable with increasing chain length, shown by the dissociation constants of the ABs.

From the compound/lipid concentrations ratio of BuAB and HxAB, it appears that a single lipid head group does not represent one binding site for a cluster molecule, but rather that each binding site comprises several lipid molecules. In case of BuAB and HxAB, and perhaps also PrAB, it is fair to say that the alkylated substance behaves like a conventional surfactant and are hence able to interact with the lipophilic core of the membrane.

(Paternostre et al., 1995) In contrast, MeAB and EtAB show no surfactant-like behavior.

The isotherms obtained from plotting zeta potential versus the root of compound concentrations indicate that the binding of further molecules is influenced by the already bound molecules through electrostatic repulsion forces and consequently the association affinity is reduced.

Within the Poisson-Boltzmann theory the surface potential is proportional to the surface charge density at low concentration (Debye-Hückel approximation). This range can be described by a binding constant. The calculated dissociation constants of the substances from the surface potential reflect the previous qualitative observations.

The limiting zeta potential of around -100 mV measured for HxAB and calculated from fitted curves for the other ABs is a rarely negative potential for liposomes. Liposomes incubated with BSH (Awad et al., 2009) do not exhibit such negative potentials and the incorporation of negatively charged arsonolipids (Fatourus et al., 2005) or dodecaborate cluster lipids (Justus et al., 2007) leads only to potentials of -50 mV and -67 mV, respectively. Only liposomes consisting of pure phosphytidylserine exhibit zeta potentials around -100 mV. A more detailed molecular simulation of interactions between the ABs and liposomes is required to explain the low potentials. It might be speculated that the water layer around the liposome surface is affected so that the slipping plane, where the zeta potential is detected, is shifted closer to the liposomal surface and consequently the shielding of the negative charges is decreased.

In the case of MeAB, the complete loss of the pre-transition temperature in the DSC profile indicates that it interacts with the choline head groups of DPPC but no interference with the lipid tails occurs. In contrast, HxAB acts more as a detergent and causes a complete disappearance of the peak representing the gel-to-liquid crystalline phase transition. It seems that it penetrates and incorporates into the liposomal membrane similar to TRITON-100 and perfluorinated drugs (PFOS) so that the anionic part is most likely located at the lipid-water interface whereas the alkyl chains interfere with the palmitoyl chains of DPPC which cause significant disruption of the packing of the lipid tails of DPPC. (Goi et al., 1986; Lehmler and Bummer, 2004; Lehmler et al., 2006)

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On the basis of the existing data the following hypothesis for the compound binding to the liposomal surface is proposed: All ABs interact with their doubly negatively charged dodecaborate cluster with the positively charged choline head groups, and the positively charged ammonium group is probably in contact with the deeper-laying negatively charged phosphate. ABs with short alkyl chains only exhibit interactions in the head group region which are probably electrostatic in nature. Additional effects affecting the water layer cannot be excluded.

As the length of the alkyl chains increases, additional interferences with the lipid tails are involved. Thus the interactions are electrostatic and additionally lipophilic.

From this hypothesis is not evident how deep the ABs protrude into the lipid bilayer and hence the binding model shown in Fig. 35 is only qualitative in its nature.

Figure 35: Binding model for ABs to the liposomal surface. The lipids and the ABs are presented in the ball-and-stick model and additionally the van der Waals radii for all atoms are shown.

The compound/lipid ratio used in cryo-TEM induces drastic changes in the morphology. Thus the influence of MeAB and EtAB leads to large bilayer sheets which are stable after heating up to 37°C and storage for one week. The other ABs differ as they are able to form micelles by themselves. This fact suggests that they share important properties with conventional ionic micelle-forming surfactants which are well known to insert into liposomal membranes, and may, at sub-solubilizing concentrations, induce a range of structural transformations of the liposomes. The insertion of surfactants into the outer lipid leaflet of the membrane frequently also leads to a process in which small liposomes are budded off from the original liposome. (Heerklotz, 2008) Thus the transformations occurring after addition of PrAB and BuAB are not surprising.

In the case of HxAB the formed concentric multilayered liposomes with a rather homogeneous spacing of 13 nm are more difficult to understand. So far these onion ring-like structures are only known for pegylated liposomes, liposomes incubated with DNA (Clement

Discussion

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et al., 2005; Letrou-Bonneval et al., 2008) or for miniemulsion polymerization with lanthanide complexes (Ramírez et al., 2006). For HxAB, it can only be speculated that a compound layer around the liposome prevents a closer approach of the membranes.

MeAB initiates a fusion process at smaller concentrations compared to BSH. (Awad et al., 2009) Therefore it seems that the change of the SH-group to the N(CH3)3-group makes the substances more effective in view of lipid mixing. It should be noted, however, that both compounds have different net charges and that the fusion mediated by BSH was tested on DMPC liposomes which have a lower phase transition temperature as DPPC liposomes.

Thus DMPC liposomes are in the fluid phase during fusion whereas DPPC liposomes remain in the gel phase.

The fusion process was not indeed tested for EtAB. The bilayers in cryo-TEM are, however, obviously the product of fusion and consequently this process is highly probable also for this compound.

The leakage induced by ABs is probably not caused by fusion based on the fact that the time scale of both processes is different. In addition the fusion process depends on the lipid concentration; the concentration is quite high in the lipid mixing experiment, but quite low in the leakage experiment. In addition we have shown before that leakage from DPPC liposomes occurs also when fusion processes are prevented by the incorporation of PEG chains into the liposomal membrane. (Gabel et al., 2007) The complex leakage kinetics, which is not first order, might rather indicate the formation of transient defects or holes. The peptide melittin, which is known to form pores, shows similar leakage kinetics. In addition the pore formation depends on the peptide concentration; an increasing concentration leads to faster leakage. (Schwarz et al., 1992) The same tendency is observed for all ABs.

The data of this study indicate that interactions of ABs with other membranes (e.g., cell membranes) must be assumed. The different toxic modes of actions are feasible: the binding of ABs to the cell membrane will result in a higher membrane potential followed by a change of cell behavior. In addition the ability of the ABs to induce leakage by pore formation might affect the permeability of the cell membrane and consequently influence the cell function. A complete disruption of the cell membrane cannot be excluded in the case of BuAB and HxAB with their detergent-like behavior.

5.3 Dodecaborate cluster lipids with variable head groups for boron neutron