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

Abstract

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 N-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-sn-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 K _a and K _d, and K _D, respectively, depends on both (i) the nature of the membrane-inserting anchor and (ii) the length of the hydrophilic chain. Hexadecahydro-3H-cyclopenta-[a]-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.

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Effect of membrane-inserting polymers on fluidity of cell plasma membrane, as assessed by laurdan assay. (a) Top: chemical structure of cholesterol (Chol)-, propanoic acid-2-[[(butylthio)­thioxomethyl]­thio]-carbonate (PABTC)- and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) phospholipid-containing homo- and random copolymers utilized in this work. PABTC-containing polymers were used here as non-membrane-inserting control materials. Bottom: Laurdan membrane fluidity assay: Caco-2 cells were incubated for 30 min with 0.1 μM Chol-, DOPE-, and non-membrane-inserting PABTC-terminated control polymers. Low molecular weight RAFT agents (Chol, PABTC, and DOPE, structures in Chart S1), were also tested (right panel). Polymer name codes indicate both the relative proportion of the different repeating units and the total number of repeating units (n, also indicated as DP, that is, the degree of polymerization of the tested polymers). For example, (HEA_0.9-AA_0.1)_n indicates a polymer that has a total number of repeating units of “n”, 90% of which come from N-(2-hydroxyethyl)­acrylamide (HEA) and 10% from acrylic acid (AA). DP (n) of each polymer is reported under each bar column. Results are expressed as delta generalized polarization (ΔGP), which is here the difference between the cells incubated with polymers and that of untreated cells. A positive value represents an increase in membrane rigidity, while a negative value represents an increase in membrane fluidity, compared with untreated cells. (b) Chemical structure and abbreviations for monomers utilized to synthesize the materials used in this study. (c) Membrane fluidity kinetic assay (assessed by laurdan assay on Caco-2 cells, at variable polymer concentration, incubation time: 60 min). Statistical significance of the results was assessed through a one-way ANOVA test, comparing the fluidity values obtained with the values measured for untreated cells (** P < 0.01, *** P < 0.001, n = 2).

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α-Lipid polymers insert into both the cell plasma membrane and the artificial lipid bilayers. (a) Insertion of cholesterol- and DOPE-terminated (HEA)_n polymers in the plasma membrane of Caco-2 cells depends on polymer chain length and nature of polymer chain-end. Caco-2 cells were incubated for 20 min with 0.1 μM polymer solutions and analyzed by imaging flow cytometry (FACS). Polymers are fluorescently tagged at the ω-end with O-alkyl pyranine F = MalPyr; λ_ex = 377 nm, λ_em = 420 nm (Scheme S3). Scale bars: 10 μm. (b) ¹³C solid-state magic angle spinning (MAS) NMR (ssNMR) confirms binding of cholesterol- or DOPE-terminated (HEA)_n homopolymers to immobilized DMPC:cholesterol model lipid bilayer (DMPC:cholesterol 1:1 mol:mol), comparing cross-polarization (CP) and direct excitation. Solid lines are spectra performed with cross-polarization, and dotted lines are spectra performed with direct excitation. Light blue panels highlight characteristic resonances for the HOCH₂ and CH₂NH groups of the (HEA)_n polymers. The spectra intensities are normalized to the signal at 70.5 ppm, corresponding to the glycerol CH resonance of DMPC.

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(a) Effect of polymer chain length and end-group on polymer binding to immobilized model lipid bilayer (DOPC:DSPC:cholesterol 2:1:1 molar ratio), as quantified by surface plasmon resonance (SPR). Left. The lipid bilayer was immobilized on an SPR L1 sensor chip, and the dissociation constant K _D of cholesterol- or DOPA-terminated poly­(2-hydroxyethyl acrylamide) (HEA)_n polymers was calculated. Higher K _D values are indicative of greater dissociation, hence weaker binding, of cholesterol- or DOPA-terminated (HEA)_n materials from the immobilized lipid bilayer. (b) Effect of cholesterol- or DOPA-terminated (HEA)_n materials on bilayer fluidity on model DMPC:cholesterol 1:1 membrane, as assessed by solid-state wide-line ³¹P NMR analysis. Polymers were tested at a membrane lipid:polymer 10:1 mol:mol ratio. The isotropic peak around 0 ppm in DOPE-(HEA)₄₇ indicates the presence of nonbilayer structures, suggesting polymer-induced partial membrane shedding.

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(a) Coarse-graining scheme for (HEA)_n polymers and details of polymer backbone dihedral distributions inferred from atomistic simulations. (b) Analysis of 300 ns CG simulations of atactic (HEA)₅₈ in water. Top left: RMSD from initial structure; top right: radius of gyration; bottom left: distance matrix for initial structure; bottom right: mean distance matrix. (c) Deuterium order parameters calculated along lipid chains within radial shells: (a) within 2 nm, (b) between 2 and 5 nm, and (c) beyond 5 nm of the embedded cholesterol group. Data for control simulations (embedded cholesterol, no polymer) are also shown. (d) Snapshots from the 500 ns atomistic simulation of Chol-(HEA)₅₈ in a DPPC bilayer. Blue: HEA, red: cholesterol: green: linker; gray: lipid with head groups shown space-filling and tails (left panel only) as rods.

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Aggregation simulations for Chol-(HEA)₅₈ and Chol-(HEA)₁₂₀ (blue) on a DPPC membrane surface (gray) (top view).