Membrane-Inserting α-Lipid Polymers: Understanding Lipid Membrane Insertion and Effect on Membrane Fluidity

Membrane-inserting materials bearing a lipid residue at one end of their macromolecular chain, α-lipid polymers, are increasingly utilized in biological and pharmaceutical fields. Insertion of these materials into lipid membranes underlines several clinically available liposomal formulations and led to the identification of cellular targets in drug discovery. Herein, we approach this concept from the perspective of a lipid membrane to investigate the relationship between the molecular structure of the inserting α-lipid polymer and the effects that the insertion has on the membrane properties. We synthesized libraries of hydrophilic (co)-polymers comprising neutral or acidic monomers, including <i>N</i>-hydroxyethyl acrylamide (HEA), acrylic acid (AA), and 3-acrylamide propanoic acid (3-AAPA), and either of two terminal membrane-inserting moieties with different molecular structures, cholesteryl (Chol) or 1,2-dioleyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) phospholipid. We investigated the structure-function relationships combining experimental methods (laurdan generalized polarization, flow cytometry, ¹³C and wide-line ³¹P solid-state NMR, and surface plasmon resonance) in conjunction with in silico modeling. Our data indicate that insertion of α-lipid polymers increases the fluidity of a range of artificial lipid membranes as well as cell plasma membranes in Caco-2 cell culture. The extent of α-lipid polymer-membrane association, described by kinetic and thermodynamic parameters <i>K</i> _a and <i>K</i> _d, and <i>K</i> _D, respectively, depends on both (i) the nature of the membrane-inserting anchor and (ii) the length of the hydrophilic chain. Hexadecahydro-3H-cyclopenta-[<i>a</i>]-phenanthrene-structure-based cholesterol anchor shows faster and stronger membrane association than phospholipid (DOPE) ones. In addition, the shorter polymers (targeted DP = 50 as opposed to DP = 100) display a higher level of membrane association which leads to a consequent larger, from 1.3- to 2.2-fold depending on the polymer, increase of bilayer fluidity. In silico molecular modeling of Chol-(HEA)_n polymers suggests that an increase in overall membrane fluidity results from a significant disruption of the lipid organization occurring near the point of insertion of the cholesterol anchor into the membrane. This effect decreases rapidly further away from the insertion point. Our work hence shows that the insertion of α-lipid polymers has a significant effect on lipid bilayer membranes, whereby the observed increase in membrane fluidity needs to be considered when, for example, designing drug formulations or modifying biological systems where it may impact on liposomal membrane stability in biological environment or ability to retain therapeutic cargo.