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Dilute electrostatically stabilized aqueous solutions of hexa-acylated (C14) lipid A diphosphate from Escherichia coli form stable and regularly shaped colloidal crystals in a size range of approximately 50-1000 nm in width and 50-100 nm in thickness. The formation of these nanocrystals occurs over a range of volume fractions between 3.5 × 10-3 and 1.2 × 10-2 and at a low ionic strength, ~10-5. The shape of these crystals appears to be cubic or rhombohedral, and when exposed to the electron beam, these fragile nanocrystals are easily damaged. Electron diffraction patterns obtained from single particles reveal that they are orientated (001) crystals that conform to a trigonal or hexagonal unit cell (a = 3.65 ± 0.07 nm and c = 1.97 ± 0.04 nm), revealing crystal-like pore walls that exhibit structural periodicity with a spacing of 0.65 nm and are at least four times the size of the unit cell adopted by lipid A diphosphate.

It has been possible to prepare electrostatically stabilized aqueous dispersions of lipid A (diphosphate) particles of low polydispersity at low ionic strength (1-10 mM NaCl) over a range of volume fractions of 1.5 × 10-4 < < 5.75 × 10-4 (25 C). These suspensions have been characterized by transmission electron microscopy, light scattering, osmotic pressure measurements, and small-angle X-ray scattering experiments at 25 C. All four measurements yielded independent values for particle sizes, weighted-average molecular weights, number-average molecular weights, and particle surface charge. The mean values obtained are = 37.59 ± 0.75 nm, = 24.89 ± 0.88 nm, = (10.55 ± 0.78) × 106 g/mol, = (9.81 ± 0.90) × 106 g/mol, and the effective surface charge Z* = (756 ± 85). Very good experimental agreement is found for the directly measured osmotic pressure values and those determined from light scattering and small-angle X-ray scattering measurements as a function of volume fraction, , by applying liquid-state theory models. Using the particle parameters for the lipid A (diphosphate) system as determined, the scattering functions and the osmotic pressures can be compared as a function of volume fraction with no adjustable parameters. The ordering of lipid A in solution revealed a body-centered cubic (bcc) type lattice (a = 36.14 nm) at volume fractions of 3.75 × 10-4 < < 4.15 × 10-4, whereas at volume fractions of 4.15 × 10-4 < < 5.75 × 10-4 in the presence of 1.0 mM NaCl a face-centered cubic (fcc) lattice type (a = 57.25 nm) was observed. Small-angle X-ray scattering experiments also indicate the presence of long-ranged order at 1.0 mM or at 10.0 mM NaCl for lipid A dispersions of 3.75 × 10-4 < < 5.75 × 10-4.

Electrostatically stabilized aqueous dispersions of nm-sized free lipid A particles at low volume fractions (1.0×10-4 ρTEM=8.0±0.6%) yield similar values for the particle size and particle size distribution ( ρQELS=10.9±0.75 %), whereas conductivity and acid-base titrations estimate surface chemical parameters (dissociation constant, ionizable sites, and Stern capacitance). Effective particle charges were determined by fits of the integral equation calculations of the polydisperse static structure factor, S(Q), to the light scattering data. Using the particle properties as determined from these experiments, the polydisperse structure factor, S(Q), was calculated as a function of volume fraction, θ, which was found to be consistent with a S(Q) dependence on the number particle density. It can be concluded that, at low volume fractions and low ionic strength, the light scattering data are well represented by a Poisson-Boltzmann model (PBC) of fluid-like ordering of free lipid A in aqueous solution. We find that the light scattering data of this dispersion are best described by a model where only a small fraction of the ionizable phosphate groups is dissociated at neutral pH. Finally, light scattering studies of lipid A dispersions of volume fractions of 3.9×10-4

© 2012 Paradies et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mineralization of Lipid A-Phosphates in Threeand Two-Dimensional Colloidal Dispersions

A comprehensive study was performed on electrostatically stabilized aqueous dispersion of lipid A-diphosphate in the presence of bound Ca2+, Mg2+, K+, and Na+ ions at low ionic strength (0.10-10.0-mM NaCl, 25 degrees C) over a range of volume fraction of 1.0 x 10(-4)< or =phi< or =4.95 x 10(-4). These suspensions were characterized by light scattering (LS), quasielastic light scattering, small-angle x-ray scattering, transmission electron microscopy, scanning electron microscopy, conductivity measurements, and acid-base titrations. LS and electron microscopy yielded similar values for particle sizes, particle size distributions, and polydispersity. The measured static structure factor, S(Q), of lipid A-diphosphate was seen to be heavily dependent on the nature and concentration of the counterions, e.g., Ca2+ at 5.0 nM, Mg2+ at 15.0 microM, and K+ at 100.0 microM (25 degrees C). The magnitude and position of the S(Q) peaks depend not only on the divalent ion concentration (Ca2+ and Mg2+) but also on the order of addition of the counterions to the lipid A-diphosphate suspension in the presence of 0.1-microM NaCl. Significant changes in the rms radii of gyration (R2G) 1/2 of the lipid A-diphosphate particles were observed in the presence of Ca2+ (24.8+/-0.8 nm), Mg2+ (28.5+/-0.7 nm), and K+ (25.2+/-0.6 nm), whereas the Na+ salt (29.1+/-0.8 nm) has a value similar to the one found for the de-ionized lipid A-diphosphate suspensions (29.2+/-0.8 nm). Effective particle charges were determined by fits of the integral equation calculations of the polydisperse static structure factor, S(Q), to the light-scattering data and they were found to be in the range of Z*=700-750 for the lipid A-diphosphate salts under investigation. The light-scattering data indicated that only a small fraction of the ionizable surface sites (phosphate) of the lipid A-diphosphate was partly dissociated (approximately 30%). It was also discovered that a given amount of Ca2+ (1.0-5.0 nM) or K+ (100 microM) influenced the structure much more than Na+ (0.1-10.0-mM NaCl) or Mg2+ (50 microM). By comparing the heights and positions of the structure factor peaks S(Q) for lipid A-diphosphate-Na+ and lipid A-diphosphate-Ca2+, it was concluded that the structure factor does not depend simply on ionic strength but more importantly on the internal structural arrangements of the lipid A-diphosphate assembly in the presence of the bound cations. The liquidlike interactions revealed a considerable degree of ordering in solution accounting for the primary S(Q) peak and also the secondary minimum at large particle separation. The ordering of lipid A-diphosphate-Ca2+ colloidal crystals in suspension showed six to seven discrete diffraction peaks and revealed a face-centered-cubic (fcc) lattice type (a=56.3 nm) at a volume fraction of 3.2 x 10(-4)< or =phi< or =3.9 x 10(-4). The K+ salt also exhibited a fcc lattice (a=55.92 nm) at the same volume fractions, but reveals a different peak intensity distribution, as seen for the lipid A-diphosphate-Ca2+ salt. However, the Mg2+ and the Na+ salts of lipid A-diphosphate showed body-centered-cubic (bcc) lattices with a=45.50 nm and a=41.50 nm, respectively (3.2 x 10(-4)< or =phi< or =3.9 x 10(-4)), displaying the same intensity distribution with the exception of the (220) diffraction peaks, which differ in intensity for both salts of lipid A-diphosphate.