Boron Cluster Anions Dissolve En Masse in Lipids Causing Membrane Expansion and Thinning

Abstract

Boron clusters are applied in medicinal chemistry because of their high stability in biological environments and intrinsic ability to capture neutrons. However, their intermolecular interactions with lipid membranes, which are critical for their cellular delivery and biocompatibility, have not been comprehensively investigated. In this study, we combine different experimental methods - Langmuir monolayer isotherms at the air-water interface, calorimetry (DSC, ITC), and scattering techniques (DLS, SAXS) - with MD simulations to evaluate the impact of closo-dodecaborate clusters on model membranes of different lipid composition. The cluster anions interact strongly with zwitterionic membranes (POPC and DPPC) via the chaotropic effect and cause pronounced expansions of lipid monolayers. The resulting lipid membranes contain up to 33 mol % and up to 52 weight % of boron cluster anions even at low aqueous cluster concentrations (1 mM). They show high (μM) affinity to the hydrophilic-hydrophobic interface, affecting the structuring of the lipid chains, and therefore triggering a sequence of characteristic effects: (i) an expansion of the surface area per lipid, (ii) an increase in membrane fluidity, and (iii) a reduction of bilayer thickness. These results aid the design of boron cluster derivatives as auxiliaries in drug design as well as transmembrane carriers and help rationalize potential toxicity effects.

Keywords: Chaotropic effect; Cluster anions; Monolayers; Noncovalent interactions; Superchaotropic ions.

Figure 1

a) Chemical structures (top, =BH, =BCl, =BBr, and =BI) and space‐filling molecular models (bottom) of dodecaborate clusters with increasing cross‐sectional area (calculated from their maximal van‐der‐Waals radii).[ ¹² , ¹³ , ¹⁴ ] b) Chemical structures of the phospholipids selected to evaluate cluster‐lipid interactions. POPC and DPPC both have the same zwitterionic head, but POPC possesses an unsaturated hydrocarbon chain. DPPG has the same saturated alkyl chains as DPPC but differs in its negatively charged head group.

Figure 2

Cluster‐membrane interactions of different phospholipid monolayers on subphases containing chaotropic anions (1.0 mM). a) Left: π‐A isotherms for POPC monolayers on a subphase with different anions. Right: Mean area per molecule at a constant surface pressure of 20 mN/m in dependence on increasing chaotropicity (from left to right). b) Left: π‐A isotherms for DPPC monolayers on a subphase with different boron clusters. Right: LE‐LC transition surface pressure in dependence on increasing chaotropicity (from left to right). c) Left: π‐A isotherms for DPPG monolayers on a subphase with different boron cluster solutions. Right: LE‐LC transition surface pressure as a function of increasing chaotropicity (from left to right).

Figure 3

Top: ζ‐Potential measurements of a) POPC, b) DPPC, and c) DPPG liposomes (250 μM phospholipids in 10 mM Tris buffer, pH 7.4) as a function of boron cluster concentration after incubation with the clusters for 2 hours at 25 °C. Bottom: Microcalorimetric titrations of POPC liposomes with d) B₁₂Cl₁₂ ²⁻, e) B₁₂Br₁₂ ²⁻, and f) B₁₂I₁₂ ²⁻. Raw ITC data (upper panels, “direct” titrations) for sequential injections of the liposomal suspensions to the boron cluster solutions, and apparent reaction heats obtained from the integration of the microcalorimetric traces (lower panels). All experiments were conducted in 10 mM Tris buffer, pH 7,4. Lipid/cluster concentrations in mM: d) 2.6/0.023; e) 2.5/0.038; f) 3/0.05.

Figure 4

MD simulation snapshots (at 500 ns, 30 °C) of a neat POPC lipid bilayer and b) in the presence of B₁₂I₁₂ ²⁻; polar heads, dark grey; hydrophobic tails, light grey; B₁₂I₁₂ ²⁻, purple; water and sodium counter‐cations are not shown for clarity. c) POPC chemical structure (top) and simulated S _CD values (bottom) vs carbon segment number for the acyl chains of POPC alone (black line) and in the presence of clusters. The left panel represents the sn‐palmitoyl chain and the right panel the sn‐oleoyl chain.

Figure 5

Electron density profile (top) of POPC alone (20 mM, black line) and in the presence of B₁₂I₁₂ ²⁻ (4 mM: pink line, 10 mM: dark pink, 20 mM: purple) and (bottom) illustration of the presumed cluster position in the membrane.

Figure 6

Effects of anions on lipid membranes in dependence on their chaotropicity and the required concentrations to produce significant effects.

Figure 7

Proposed mechanism of boron cluster insertion. a) Unperturbed lipid bilayer. b) Cluster insertion pushes apart the polar heads, subsequently the hydrocarbon chains reorient, stimulating c) membrane fluidity and thinning. Note that b) is a hypothetical state, only drawn for visualization as a stepwise process, affecting (i) expansion and (ii) thinning.