Ordering of lipid A-monophosphate clusters in aqueous solutions
Authors: Faunce Chester A. , Reichelt Hendrik , Quitschau Peter , Paradies Henrich H. 
Published: September 2007

In this investigation, a study of the self-assembly of electrostatically stabilized aqueous dispersions of nanometric lipid A-monophosphate clusters from Escherichia coli was carried out in three different volume-fraction regimes. The experimental techniques used in the investigation were osmotic pressure, static and quasielastic light scattering, scanning electron microscopy and transmission electron microscopy, and small-angle x-ray scattering. Experiments were carried out at low ionic strength (I=0.1–5.0mM NaCl) at 25°C. At volume fractions between 1.5×10−4⩽ϕ⩽5.4×10−4, the lipid A-monophosphate clusters had an average rms hydrodynamic diameter of d¯=7.5nm, and a weighted-average molecular weight of (1.78±0.23)×105gmol−1. Quasielastic light scattering (LS) experiments yield similar values for the particle size and particle size distribution compared to electron microscopy, small-angle x-ray scattering, and LS experiments. When the volume fraction was increased to a higher regime 5.4×10−4⩽ϕ⩽9.50×10−4, much larger clusters of lipid A monophosphate formed. The clusters detected in this volume-fraction range were assembled from between 8 and 52 of the d¯=7.5nm clusters and the assemblies are densely packed in such a way that colloidal crystals composed of the monodisperse microspheres are in physical contact with their nearest neighbors. Clusters that formed in volume fractions between 10.0×10−4⩽ϕ⩽40.0×10−4 revealed a weighted-average molecular weight of (10.15±0.17)×106gmol−1 and a hydrodynamic diameter of ∼d¯=70.6nm. The crossover volume fraction between the small and the large clusters appeared at ϕcr=5.05×10−4. In the intermediate volume-fraction range, the scattered intensity I(Q) vs Q curves (light and x rays) showed asymptotic behavior. From the asymptotic curves, the scattered intensity, the relationship between the average mass and radius, and the fractal dimension df were determined. The df value, which was evaluated from the expression I(Q)∝RGdf, was found to be 1.67±0.03, a value that was virtually independent of the ionic strength (0.1–5.0mM NaCl) at 25°C. Even at a very low ionic strength (I=0.10mM NaCl), lipid A monophosphate formed a number of differently shaped clusters. Electron microscope images showed that two types of self-assembled clusters existed at the lowest volume-fraction range studied and also dominated the images taken at the higher volume-fraction regimes. One type of cluster showed a cubic morphology and a size variation of 50–100nm, while another type took on the appearance of a quadratic cylinder, with dimensions of 50×150nm2. The other clusters appeared in various shapes: dimers, trimers, and distorted tetramers, which were quite different from the ones previously observed for lipid A diphosphate. Small-angle x-ray diffraction experiments on lipid A-monophosphate clusters suspended in water, containing 5mM NaCl (25°C), indicated the existence of long-range order of d¯=7.5nm. At low polydispersity, two distinct types of lipid A-monophosphate colloidal clusters were able to form at low polydispersity and were subsequently identified using light scattering, small-angle x-ray scattering, and selected-area electron diffraction. From an analysis of experimental results obtained from these clusters, distinct peaks could be assigned to a body-centered cubic (bcc) lattice, with a=49.5±1.8nm. The solution structure found for lipid A diphosphate at volume fractions of 3.75×10−4⩽ϕ⩽4.15×10−4 also exhibited a (bcc)-type lattice; however, a=36.1nm [C. A. Faunceet al. J. Phys. Chem. 107, 2214 (2003)]. Using the particle and cluster properties determined from small-angle x-ray scattering, light scattering, and osmotic-pressure measurements as a function of volume fraction, good agreement was found between the directly measured osmotic-pressure values and those calculated from scattering experiments.