High-resolution spectra of liposomes using MAS NMR. The case of intermediate-size vesicles.

The acquisition of high-resolution spectra of large unilacurvature and unilamellarity. Their unilamellarity is particularly advantageous for biotechnological applications, while mellar vesicles has been hampered by problems of line broadening caused by lipid and vesicle motion in the intertheir small curvature makes them better models for biological membranes than the highly curved SUVs. However, as mediate motional regime. In this paper, we introduce a method for obtaining P NMR high-resolution spectra of the vesicle size increases from that of the SUVs, the membrane tumbling becomes progressively slower (21) and the large unilamellar vesicles by slowing the vesicle dynamics into a more tractable time scale. efficiency of the orientation averaging by lateral diffusion decreases because of the low curvature of the vesicles. The Traditional models of biological membranes for NMR studies include multilamellar vesicles (MLVs) which can time scale of the molecular tumbling of the LUVs falls into the intermediate time scale regime. The tumbling of the range in size from 0.2 to 10 mm (1, 2) , small unilamellar vesicles (SUVs) with diameters of approximately 20–40 nm vesicles is too slow to average out the anisotropic interactions: the spectra have powder lineshapes distorted by partial (3) , and large unilamellar vesicles (LUVs) with diameters from 50 to 200 nm (4, 5) . The many-layered MLVs have averaging (21, 22) . Thus, the LUV spectra are not motionally averaged to high resolution like SUVs, and in addibeen widely exploited for structural and motional information (6–9) . Because of their large size, there is little averagtion, they cannot be spun to high resolution with MAS like the MLVs (23–26) . The molecular tumbling in the intermeing of the anisotropic part of the spin Hamiltonian by vesicle tumbling, making them well adapted to study by wideline diate regime diminishes the efficiency of the lineshape averaging (27, 28) . Here, we show a method to evade this ‘‘incosolid-state NMR techniques. Indeed, order parameters deduced from wideline H or P NMR spectra as well as herent averaging’’ effect by reducing the vesicles overall lineshape simulations have provided a wealth of information motion and thus moving the vesicles’ dynamics out of the on the dynamics and conformation of lipids in membranes. intermediate motional regime. For several types of investigations, such as membrane Egg phosphatidylcholine (EPC), egg phosphatidylglycpermeability, drug delivery by liposomal encapsulation erol (EPG), egg phosphtidylethanolamine (EPE), choles(10, 11) , and membrane fusion (12) , however, unilamellar terol (CHOL), and 1,2-dioleoyl-sn-glycero-3-phosphochovesicles are preferable. SUVs, generally obtained by sonicaline (DOPC) were purchased from Sigma Chemical Co. tion of MLVs, have been used as model membranes when and were used without further purification. 1,2-Dioleoyl-snthe need for unilamellarity is paramount. SUVs give rise to glycero-3-phosphate (DOPA) was synthesized in our laborahigh-resolution spectra as their fast rotational motion avertory from DOPC following the procedure of Roux et al. ages the anisotropic dipolar interaction to zero and the chem(29) . The purity of these lipids was checked by thin-layer ical shift to its isotropic value (13–20) . However, the high chromatography and by high-resolution NMR in chlorocurvature of the SUVs always raises the question of biologiform. The buffer used in the experiments was 100 mM cal relevance. N [2 -hydroxyethyl]piperazine N * [ethanesulfonic acid] Intermediate-size LUVs, which can be obtained by a rapid (Hepes) , 100 mM potassium sulfate (K2SO4) and 5 mM extrusion procedure, offer an attractive combination of low ethylendiamine tetraacetic acid (EDTA). To prepare the liposomes, the appropriate proportions of lipids were dissolved in 3 ml of chloroform and 1 ml of † To whom correspondence should be addressed. methanol to ensure homogenous mixing. The solvent was * Present address: Physics Department, Simon Fraser University, Burnaby, BC V3J 1J2 Canada slowly removed at room temperature by rotary evaporation.