Lipid and Polymer-Lipid Systems

The explosion of knowledge in the biological and biochemical sciences would not have been possible without the techniques of the physical sciences; however, the theoretical framework of physical chemistry has been largely irrelevant to that progress. The situation poses a philosophical dilemma, the physical and biological sciences remaining disjoint. A case is made that the puzzle will be resolved if it be recognised that the present framework is deeply flawed. Emerging theories in physical chemistry that correct present inadequacies lead to the hope that the barriers will disappear.

General aspects of association phenomena in polymer and surfactant solutions, with emphasis on mixed solutions, are outlined and the role of fluorescence as a key approach to unravel molecular mechanisms is reviewed. Some illustrations of how fluorescence spectroscopy has been successfully used to study important issues, such as molecular association, microstructure, and molecular dynamics, in these systems are considered. These achievements provide an important contribution to the understanding and control of macroscopic properties, biological functions, and industrial applications of these systems.

We present a method to extract from pulsed-field-gradient spin-echo NMR measurements, the droplet size distributions in emulsions and the self-diffusion coefficient of the dispersed phase. The method is applied to butter and water/extra-virgin olive oil emulsions. The droplet size distributions are in agreement with optical microscopy analysis. The butter size distribution is a log—normal form and the effect of the salt on the diffusion coefficient of the aqueous phase is evaluated in the case of the salted butter.

The existence of a uniform phase at the alveolar surface is demonstrated by polarizing microscopy and cryogenic transmission electron microscopy (cryo-TEM). Rabbit lungs were opened and the alveolar surface film was directly deposited on optical microscope coverslips and on electron microscope grids. In the polarizing microscope, the alveolar surface material deposited exhibits a uniform birefringence, which shows that the alveolar surface is lined by a coherent liquid-crystalline phase; a surface phase. Cryo-TEM, with reduced risks of artifacts compared to conventional electron microscopy, shows the existence of a mechanically stable and continuous surface structure of regularly spaced bilayers oriented along the grid surface over large distances (microns). The periodicity observed by cryo-TEM indicates that the surface phase has the same inner organization as the ultrastructure known as tubular myelin (TM). Two alternative structures of TM are considered: the traditional crossed bilayer structure and a bilayer structure free from intersections according to the so-called crossed layers of parallels (CLP) minimal surface. Evidence presently available favors the CLP minimal surface structure alternative. The implication of the existence of a surface phase on the barrier function of the alveolar surface zone is finally considered.

The performance of two principally different kinds of intended stabilizers is reviewed. Especially any influence of the stabilizers on the liposome properties, such as structure, permeability and surface potential, is discussed. Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers have not been shown to function satisfactorily as stabilizers in combination with phospholipids. The incorporation of triblock copolymers of different segment length leads to structural breakdown of the liposome structure even at low concentrations. In addition, incorporation of the copolymers results in extensive leakage of encapsulated material. Neither have there been any reports of unambiguous proof of efficient steric repulsion due to the coating by copolymers. In contrast poly(ethylene glycol) lipids [PEG(2000)-phosphatidyl ethanol-amine] efficiently provide a steric barrier to the liposomes. This is, however, only true if the surface concentration is kept below a rupture limit. An important additional effect is that the permeability of encapsulated hydrophilic cargo is reduced in the presence of PEG lipids. The most common PEG lipids contain a carbamate linkage that introduces a negative surface potential at the liposome surface. However, at medium ionic strength similar to physiological conditions the surface potential is small and does not contribute to any important extent to colloidal stabilization.

The extent of acyl migration and hydrolysis phenomena in glycerol monooleate/water based systems and how they are affected by acetic acid and oleic acid has been examined. To facilitate analysis and interpretation of the data, samples with a low water content, 2–5 wt%, which form a liquid reverse micellar, L₂, phase, were chosen. Acyl migration and hydrolysis were monitored versus time by ¹³C NMR analysis. In addition, the acid value to monitor hydrolysis has been also determined. The formation of small amounts of glycerol-2-monooleate as well as hydrolysis products was observed. These phenomena depend on the water content and storage conditions. The implications of these findings for the observed phase behaviour of the glycerol monooleate/water system are discussed.

The equilibrium self-diffusion of water contained within the porous structure of three different types of starch, viz. corn, wheat and potato starch was investigated. The samples were exposed to humid air until they reached a certain water content, the value of which was determined from the ¹H NMR free induction decays. The water self-diffusion coefficient was determined by means of a pulsed-field-gradient stimulated-echo experiment. This experiment is susceptible to cross-relaxation effects, i.e. the transfer of longitudinal magnetization during the experiment; therefore, the cross-relaxation process was first quantified by means of Goldman— Shen-type experiments. The water self-diffusion could then be rationalized in terms of a distribution of diffusion coefficients, with an average value much reduced compared to bulk water. No dependence on the diffusion time in the range from 70 to 340 ms was observed, indicating that there are no impermeable barriers for the diffusion of water on a length scale of microns.

The interaction between DNA and oppositely charged surfactants has been investigated by several techniques, like fluorescence microscopy, electron microscopy, phase diagram determination, and ellipsometry. The phase behaviour is more strongly associative than that in previously studied systems. A precipitate is formed for very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles and that the addition of anionic surfactant can release free DNA back into solution from a compact globular complex between DNA and cationic surfactant. Finally, we investigated DNA interactions with polycations, chitosans with different chain lengths, by fluorescence microscopy, in vivo transfections assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

We report an investigation of the aggregation and molecular recognition properties of phospholi-pid derivatives containing a nucleosidic unit on the phospholipid polar head. Long chain 5′-(1,2-dioleoyl-sn-glycero(3)phospho)-adenosine, 5′-(1,2-dioleoyl-sn-glycero(3)phospho)-uridine, 5′-(1,2-dioleoyl-sn-glycero(3)phospho)-cytidine, and 5′-(1,2-dioleoyl-sn-glycero(3)phospho)-ethenoadenosine phospholipid derivatives form vesicles, while short-chain 5′-(1,2-dioctanoyl-sn-glycero(3)phospho)-adenosine and 5′-(1,2-dioctyl-sn-glycero(3)phospho)-uridine form ellipsoidal micelles. We demonstrated that the arrangement of these novel surfactants in supramolecular devices, driven by hydrophobic interactions, provides the cooperative effect necessary to promote recognition between the complementary bases, though bases are exposed to an aqueous environment. In particular, despite the presence of the aqueous environment, molecular recognition occurs between the phospholiponu-cleoside complementary bases via stacking and hydrogen bonding in a way that strictly resembles the Watson-Crick recognition pattern of DNA and RNA. Generally, hydrogen-bond-mediated interactions are not effective in aqueous systems, since water forms strong hydrogen-bond interactions with host molecules. These findings highlight the importance of phospholipid nucleosidic derivatives as model devices for molecular recognition in biological systems and for biotechnological applications.

The quaternary system comprising sodium dodecyl sulfate/ water/pentanol/dodecane has been studied at high oil content and at two different water-to-surfactant ratios. Of particular interest is a highly flow birefringent, isotropic fluid phase, which occupies an island in the pseudo-ternary phase diagrams between the L₂ phase and the L₃ phase and which is in close proximity to the dilute lamellar phase. In order to investigate its microstructure, the self-diffusion coefficient of the solvent was measured in this phase using Fourier transform pulsed-field-gradient spin-echo NMR spectroscopy, and its value was found to be significantly lower than those in the L₃ phase of the same system. Comparative conductivity measurements were also performed, and they showed that the conducting material in the phase forms an infinitely connected network. In conclusion, it seems reasonable to describe the phase microstructure as a connected perforated bilayer or possibly a connected three-dimensional network of reverse cylinders. The conductivity of the L_x phase decreases strongly upon stirring. Taking into account the flow birefringence, this is interpreted as a shear-induced phase transition to a lamellar phase.

The phase behavior and aggregate structure of the didodecyldimethylammonium bromide (DDAB)-sodium taurodeoxycholate—water mixed surfactant system have been investigated in the dilute (above 95 wt% water) cationic-rich area, at 25 °C. A combination of techniques has been used, including polarizing and video-enhanced light microscopy, cryogenic transmission electron microscopy and water self-diffusion NMR. Between the dilute lamellar phase (~4-30 wt% DDAB) and the very narrow isotropic solution (below 0.5 wt% DDAB), the lamellar dispersions formed by DDAB contain different types of vesicle structures (vesicles, microtubules and multilamellar structures). On addition of bile salt to the DDAB dispersions, strong electrostatic headgroup interactions and geometric packing effects (owing to the unusual molecular structure of the anionic surfactant) are present. The vesicle aggregates, however, are able to incorporate an amount of bile salt up to roughly 20 mol%. Further addition induces a macroscopic phase separation, with the formation of a strong white dispersion. The light and electron micrographs show that the vesicles are spherical and integral, undergoing an increase in size when the bile salt concentration is varied between 0 and 10 mol%. At higher bile salt concentration, smaller vesicles, tubular structures and submicron-sized dispersion droplets are observed. Water self-diffusion NMR measurements give further information regarding vesicle size and poly-dispersity.

Glyceryl monooleate (GMO) and other unsaturated long-chain monoglycerides show peculiar phase behaviour characterised by the presence of one or more cubic liquid-crystalline phases. In the present study, hydrolysis of cubic lipid-water phases formed by GMO were determined. Human pancreatic carboxyl ester lipase (CEL), colipase-dependent lipase with colipase, human duodenal contents and rat pancreas homogenate were added and the release of free oleic acid from GMO was determined. It was found that pancreatic enzymes hydrolyse GMO cubic lipid-water phases in vitro. In the presence of concentrations of sodium taurocholate, sodium taourodeoxycholate or sodium glycocholate, above 3 mM, the hydrolysis by pancreatic enzymes was stimulated. The optimal pH for the hydrolysis of GMO by CEL was 7.5–8.5, by colipase-dependent lipase 6–9 with a peak at 6.5 and by rat pancreas homogenate 6–8.5 with a peak at 7.3. The hydrolysis of GMO with human duodenal content was slow but increased with time. These results indicated that pancreatic enzymes hydrolyse GMO in cubic lipid-water phases. The hydrolysis is effected by pH and the concentration of bile salts.