Disulphide polymerizeable phosphatidylcholines : characterization of membrane physical properties and investigations of in vivo behavior

by Handel, Tracy Marie

Abstract (Summary)
In the past ten years, polymerizeable amphiphiles have been recognized as an important class of synthetic phospholipids due to their ability to modify the mechanical and chemical stability of membranes. The motivation behind studies of polymerizeable phospholipids is based on the potential importance of stable membranes in a wide variety of applications including reactivity control, encapsulation technologies and drug delivery. However, compared to nonpolymerizeable lipids, there is a relative paucity of information correlating membrane physical properties with the structure of the polymeric lipid. The present investigation involves the synthesis of a series of disulfide polymerizeable phosphatidylcholines, the characterization of the physical properties of membranes formed from these lipids, and an investigation of the biodistributions, vascular clearance rates and degradation rates of these liposomes in vivo. The structures of the lipids under investigation are analogous to saturated phosphayitdylcholines but with a thiol either alpha to the carbonyl of the acyl chain ([alpha]-THIOL) or at the chain terminus ([omega]-THIOL). It is found that the presence and position of the polymerizeable moiety drastically alters the physical characteristics of the membranes. A variety of physical techniques have been utilized to understand both the bulk properties of the lipids such as morphology and permeability, as well as the molecular details of the lipid conformation and dynamics. On the basis of such studies including Raman, FT-IR and DSC, it appears that the presence of the polymerizeable group at the interfacial region ([alpha]-THIOLS) causes a reduction in the lipid packing and an increase in chain disorder compared to nonpolymerizeable analogs. For the monomeric form of the [alpha]-THIOLS, the decreased interlipid interaction may be ascribed to the presence of an additional hydrophilic pendant group at the interface that interferes with tight crystalline packing, most likely by a combination of steric and hydration effects. In contrast to the general expectations for polymerized versus nonpolymerized phospholipids, upon polymerization, the membrane disorder is augmented even further. We believe this to be a consequence of the conformational restrictions of polymerization which inhibit the ability of the polymeric lipids to adopt a highly ordered and uniform packing state. Instead it is suspected that the reduced conformational freedom of polymerized lipids promotes the formation of surface defects between unlinked polymer chains. As a morphological consequence, the polymeric [alpha]-THIOLS tend to form smaller and largely unilamellar vesicles on dispersion in water. Furthermore because of the disorder in the hydrocarbon chain region, they are quite permeable to entrapped solutes and when administered in vivo, are cleared rapidly from the circulatory system. The latter effect is likely the result of facilitated absorption of opsonizing proteins into the disordered membrane surface which subsequently accelerates vascular clearance and uptake by the cells of the reticuloendothelial system (RES). On the basis [superscript 31]P NMR relaxation measurements, it was observed that the motion of the headgroups in polymeric [alpha]-THIOLS was reduced relative to nonpolymerizeable analogs. Furthermore a reduced chemical shift anisotropy of polymeric [alpha]-THIOLS in the liquid-crystalline state relative to nonpolymerizeable lipids was observed and suggests an alteration in the average orientation of the headgroup for the polymer. Because the headgroup of phosphatidylcholines is zwitterionic, changes in the average orientation can have marked effects on the electrostatic properties of the membrane surface, which in turn can affect membrane morphology and interactions with cell-surfaces and proteins. For [omega]-THIOLS, a distinctly different behavior was observed compared to the [alpha]-THIOLS. For the monomers and especially the polymers, results from vibrational spectroscopy and DSC suggest that the membrane conformational order and rigidity is increased relative to nonpolymerizeable phosphatidylcholine analogues. However, in contrast to the [alpha]-THIOLS that form normal self-sealed liposomal structures in both the monomeric and polymeric state, polymerization induces the transformation of [omega]-THIOLS into bilayer fragments lacking an internal aqueous compartment. The most likely explanation for this was derived from [superscript 13]C NMR relaxation experiments, which indicated that the mobility at the bilayer midplane of polymeric [omega]-THIOLS is as restricted as the interfacial region. This contrasts to nonpolymerizeable phospholipids and [alpha]-THIOLS, which have the bilayer interior as the most fluid portion of the membrane. The rigidity at the midplane may prohibit the ability of the polymeric [omega]-THIOLS to form curved or continuous multilamellar sheets or to respond to transient defects in the membrane without fragmentation. An interesting and unexpected result concerning the [omega]-THIOLS was retention of a phase transition after polymerization. For most polymeric lipids that have the polymerizeable moiety at the chain terminus, polymerization has resulted in the disappearance of the transition due to crosslinking of the hydrocarbon chains. The presence of the transition in polymerized [omega]-THIOLS may be evidence for the fact that polymerization results in a predominance of intra- rather than inter-leaflet coupled chains.
Bibliographical Information:

Advisor:John D. Baldeschwieler

School:California Institute of Technology

School Location:USA - California

Source Type:Master's Thesis



Date of Publication:01/18/1989

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