Lipid lateral segregation driven by diacyl cyclodextrin interactions at the membrane surface

Abstract

Cyclodextrins are hydrophilic molecular cages with a hydrophobic interior allowing the inclusion of water-insoluble drugs. Amphiphilic cyclodextrins obtained by appending a hydrophobic anchor were designed to improve the cell targeting of the drug-containing cavities through their liposome transportation in the organism. After insertion in model membranes, they were found to induce a lateral phase separation into a pure lipid phase and a fluid cyclodextrin-rich phase (L(CD)) with reduced acyl chain order parameters, as observed with a derivative containing a cholesterol anchor (M. Roux, R. Auzely-Velty, F. Djedaïni-Pilard, and B. Perly. 2002. Biophysical Journal, 8:813-822). We present another class of amphiphilic cyclodextrins obtained by grafting aspartic acid esterified by two lauryl chains on the oligosaccharide core via a succinyl spacer. The obtained dilauryl-beta-cyclodextrin (betaDLC) was inserted in chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) membranes and studied by deuterium NMR ((2)H-NMR). A laterally segregated mixed phase was found to sequester three times more lipids than the cholesteryl derivative (approximately 4-5 lipids per monomer of betaDLC), and a quasipure L(CD) phase could be obtained with a 20% molar concentration of betaDLC. When cooled below the main fluid-to-gel transition of DMPC-d54 the betaDLC-rich phase stays fluid, coexisting with pure lipid in the gel state, and exhibits a sharp transition to a gel phase with frozen DMPC acyl chains at 12.5 degrees C. No lateral phase separation was observed with partially or fully methylated betaDLC, confirming that the stability of the segregated L(CD) phase was governed through hydrogen-bond-mediated intermolecular interactions between cyclodextrin headgroups at the membrane surface. As opposed to native betaDLC, the methylated derivatives were found to strongly increase the orientational order of DMPC acyl chains as the temperature reaches the membrane fluid-to-gel transition. The results are discussed in relation to the "anomalous swelling" of saturated phosphatidylcholine multilamellar membranes known to occur in the vicinity of the main fluid-to-gel transition.

SCHEME 1

Chemical structures of the βDLC derivatives.

FIGURE 1

²H-NMR powder (a) and de-Pake-ed spectra (b–h) of DMPC-d54 membranes recorded at 21°C, without (a, b) and with (c–h) 2.5%, 5%, 7.5%, 10%, 20%, and 30% βDLC expressed in molar %. The bold digits on the pure DMPC spectrum (b) point to the quadrupolar splittings attributed to the methyl deuterons (1), the vicinal C13 methylene deuterons of the sn-1 (2), and sn-2 (3) chains and those of the plateau region (4). The components (I) and (II) described in the text are indicated on spectrum (e). The de-Pake-ed spectra were scaled with normalization factors obtained by area normalization of their related FT spectra (a).

FIGURE 2

²H-NMR de-Pake-ed spectra obtained from DMPC-d54 membranes containing 7.5% (a, b) and 20% (c, d) molar of βDLC. The stacked plots (b, d) show the spectra recorded at 37°C and 30°C and from 25 to −12°C (1°C step, going from the upper traces to the lower traces). The traces (a) and (c) show the spectra obtained at 37°C. The components (I) and (II) described in the text are indicated on spectrum (a). The de-Pake-ed spectra were scaled with normalization factors obtained by area normalization of their related FT spectra. Spectrum e: DMPC-d54 membranes containing 20% molar of βDLC at 15°C. Spectrum f: pure DMPC-d54 membranes at 30°C.

FIGURE 3

²H-NMR de-Pake-ed methyl resonances of DMPC-d54 membranes recorded at 0°C (a, b) and −12°C (c–i), without (a, c) and with (b, d–i) 2.5%, 5%, 7.5%, 10%, 20%, and 30% βDLC expressed in molar %.

FIGURE 4

First moment M1 of the ²H-NMR powder spectra of DMPC-d54 membranes as a function of temperature (°C): (•) pure and with (○) 2.5%, (×) 5%, (*) 7.5%, (♦) 10%, (▪) 20%, and (□) 30% (molar %) of βDLC.

FIGURE 5

²H-NMR de-Pake-ed spectra of DMPC-d54 membranes recorded at 20°C, either pure (a) or with a 10% molar concentration of βDLC (b), DimβDLC (c), TrimβDLC (d), or cholesterol (e).

FIGURE 6

Temperature dependence of DMPC-d54 acyl chain order parameters S_CD of the plateau region and sn-1 C13 methylene resonances measured from the deuterium de-Pake-ed spectra (top) and of the average order parameters 〈S_CD〉 of the myristoyl chains calculated from the first moment M1 of the powder spectra (bottom). The membranes were either pure (○) or contained 10% molar concentration of βDLC (▪), TrimβDLC (•), or cholesterol (□). With the exception of the βDLC data (▪), all spectral moments M1 were measured from deuterium NMR spectra of DMPC-d54 membranes in the fluid state, containing no component which could be attributed to gel state lipids, i.e., no signal around ±61 kHz. Average order parameters 〈S_CD〉 of βDLC-containing membranes below Tc, calculated from a fluid powder pattern obtained after subtraction of a small gel component with intensities around ±61 kHz (<10% of the whole signal) is also shown (*). The inset in the top panel shows the TrimβDLC concentration dependence of the methylene plateau deuteron order parameters at 20°C (•).