Plasmalogen-Based Liquid Crystalline Multiphase Structures Involving Docosapentaenoyl Derivatives Inspired by Biological Cubic Membranes

Abstract

Structural properties of plasmenyl-glycerophospholipids (plasmalogens) have been scarcely studied for plasmalogens with long polyunsaturated fatty acid (PUFA) chains, despite of their significance for the organization and functions of the cellular membranes. Elaboration of supramolecular assemblies involving PUFA-chain plasmalogens in nanostructured mixtures with lyotropic lipids may accelerate the development of nanomedicines for certain severe pathologies (e.g., peroxisomal disorders, cardiometabolic impairments, and neurodegenerative Alzheimer's and Parkinson's diseases). Here, we investigate the spontaneous self-assembly of bioinspired, custom-produced docosapentaenoyl (DPA) plasmenyl (ether) and ester phospholipids in aqueous environment (pH 7) by synchrotron small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). A coexistence of a liquid crystalline primitive cubic Im3m phase and an inverted hexagonal (H_II) phase is observed for the DPA-ethanolamine plasmalogen (C16:1p-22:5n6 PE) derivative. A double-diamond cubic Pn3m phase is formed in mixed assemblies of the phosphoethanolamine plasmalogen (C16:1p-22:5n6 PE) and monoolein (MO), whereas a coexistence of cubic and lamellar liquid crystalline phases is established for the DPA-plasmenyl phosphocholine (C16:1p-22:5n6 PC)/MO mixture at ambient temperature. The DPA-diacyl phosphoinositol (22:5n6-22:5n6 PI) ester lipid displays a propensity for a lamellar phase formation. Double membrane vesicles and multilamellar onion topologies with inhomogeneous distribution of interfacial curvature are formed upon incorporation of the phosphoethanolamine plasmalogen (C16:1p-22:5n6 PE) into dioleoylphosphocholine (DOPC) bilayers. Nanoparticulate formulations of plasmalogen-loaded cubosomes, hexosomes, and various multiphase cubosome- and hexosome-derived architectures and mixed type nano-objects (e.g., oil droplet-embedding vesicles or core-shell particles with soft corona) are produced with PUFA-chain phospholipids and lipophilic antioxidant-containing membrane compositions that are characterized by synchrotron SAXS and cryo-TEM imaging. The obtained multiphase nanostructures reflect the changes in the membrane curvature induced by the inclusion of DPA-based PE and PC plasmalogens, as well as DPA-PI ester derivative, and open new opportunities for exploration of these bioinspired nanoassemblies.

Keywords: SAXS; cryo-TEM; docosapentaenoyl phospholipids; hexosomes; inverted hexagonal phase; lipid cubic phase; plasmalogen-loaded cubosomes.

FIGURE 1

Schematic presentation of lamellar and non-lamellar liquid crystalline phase structures resulting from the structural polymorphism of lyotropic lipid/water mixtures: lamellar bilayer, inverted hexagonal (H_II), primitive cubic (P), bicontinuous double-diamond cubic (D), bicontinuous gyroid cubic (G), and sponge phases.

FIGURE 2

Chemical structures of custom-produced polyunsaturated fatty acid (PUFA)-ether or ester type glycerophospholipids with long tails comprised of docosapentaenoyl (DPA) chains. Plasmalogens (1-alkyl-1′-enyl,2-acyl ether phospholipids) are obtained with phosphocholine and phosphoethanolamine headgroups, namely, 1-O-1′-(Z)-hexadecenyl-2-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, C16:1p-22:5n6 PC (top), and 1-O-1′-(Z)-hexadecenyl-2-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphoethanolamine, C16:1p-22:5n6 PE (middle). As a diacyl ester lipid, 1,2-bis(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphoinositol, 22:5n6 PI, is enriched in docosapentaenoyl (DPA) moieties in order to increase the hydrophobic lipid volume with regard to the bulky phosphoinositol (PI) polar headgroup. Monoolein (bottom) is a single-chain monoglyceride used as a colipid in the studied mixed nanoassemblies.

FIGURE 3

(A) Synchrotron small-angle X-ray scattering (SAXS) patterns of a hydrated bulk-phase plasmenyl-phospholipid [DPA-ethanolamine plasmalogen (C16:1p-22:5n6 PE)] at a lipid/water ratio of 50/50 (wt%/wt%) and aqueous phosphate buffer environment (1.10^{− 2} M) of pH 7 with added 2,6-di-tert-butyl-4-methylphenol (BHT). Pattern (1) corresponds to a freshly prepared plasmalogen/water sample examined by synchrotron SAXS. Plots (2)–(4) display the progressive vanishing of the cubic phase peaks upon multiple SAXS recordings with the same sample. (B) Indexing of the Bragg diffraction peaks resolved in the SAXS pattern (1) from (A). In a linear scale, the two sets of Bragg peaks index: (i) a primitive cubic phase of the Im3m space group of symmetry [purple bars corresponding to the (110), (200), (211), (220), (310), (222), (321), (400), and (411) cubic lattice reflections for peak positions spaced in the ratio √2:√4:√6:√8:√10:√12:√14:√16:√18] and (ii) an inverted hexagonal (H_II) phase [green bars corresponding to the (10), (11), and (20) reflections at peak positions spaced in the ratio 1:√3:√4]. Temperature is 22°C.

FIGURE 4

Synchrotron small-angle X-ray scattering (SAXS) patterns of bulk-phase mixtures of monoolein (MO) with incorporated 15 mol.% docosapentaenoyl (DPA, 22:5n6)-modified plasmalogen-phosphoethanolamine C16:1p-22:5n6 PE (red plot), plasmalogen phosphocholine C16:1p-22:5n6 PC (blue plot), or DPA-diacyl phosphoinositol 22:5n6-22:5n6 PI ester lipid (green plot). The red, blue, and green plots correspond to 15/85 (mol/mol) ratio between the polyunsaturated fatty acid (PUFA) phospholipids and monoolein (MO). The orange plot presents the SAXS pattern for the pure MO/buffer system. The lipid/water ratio is 40/60 (wt/wt). Aqueous phase: 1.10^{− 2} M phosphate buffer containing 2,6-di-tert-butyl-4-methylphenol (BHT). Temperature is 22°C.

FIGURE 5

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B,C) cryogenic transmission electron microscopy (cryo-TEM) images of a self-assembled nanoparticulate plasmalogen-phosphocholine (C16:1p-22:5n6 PC)/monoolein (MO)/vitamin E/coenzyme Q₁₀/VPGS-PEG₁₀₀₀ system with a plasmalogen-PC/MO molar ratio of 15/85 (mol/mol) and added vitamin E (10 mol.%) and coenzyme Q₁₀ (1 mol.%). Aqueous phase: 1.10^{− 2} M phosphate buffer containing 2,6-di-tert-butyl-4-methylphenol (BHT). Dispersion content: 5 wt% lipid/95 wt% aqueous buffer. The set of Bragg peaks in (A) indexes an inner cubic structure of the double-diamond Pn3m cubic lattice space group. The bars indicate the (110), (111), (200), (211), (220), (221), (310), and (311) reflections, for which the peak positions are spaced in the ratio √2:√3:√4:√6:√8:√9:√10:√11 … The topologies of the cubosome particles, stabilized by VPGS-PEG₁₀₀₀ (6 mol.%), and coexisting vesicular membranes are presented in (B,C).

FIGURE 6

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B–F) cryogenic transmission electron microscopy (cryo-TEM) images of a nanoparticulate plasmalogen-phosphocholine (C16:1p-22:5n6 PC)/vitamin E/VPGS-PEG₁₀₀₀ system obtained by self-assembly and dispersion in an excess aqueous phosphate buffer (1.10^{− 2} M) phase containing 2,6-di-tert-butyl-4-methylphenol (BHT). Dispersion content: 5 wt% lipid phase/95 wt% aqueous phase.

FIGURE 7

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B–F) cryogenic transmission electron microscopy (cryo-TEM) images of a dispersed plasmalogen-phosphoethanolamine (C16:1p-22:5n6 PE)/dioleoylphosphocholine (DOPC)/coenzyme Q₁₀/VPGS-PEG₁₀₀₀ system at a docosapentaenoyl (DPA)-plasmalogen PE/monoolein (MO) molar ratio of 15/85 (mol/mol) and containing a coenzyme Q₁₀ (1 mol.%) and VPGS-PEG₁₀₀₀ (6 mol.%). Aqueous phase: 1.10^{− 2} M phosphate buffer with dissolved 2,6-di-tert-butyl-4-methylphenol (BHT). Dispersion content: 5 wt% lipid phase/95 wt% aqueous buffer phase.

FIGURE 8

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B–F) cryogenic transmission electron microscopy (cryo-TEM) images of a nanoparticulate plasmalogen-phosphoethanolamine (C16:1p-22:5n6 PE)/monoolein (MO)/VPGS-PEG₁₀₀₀ system with a docosapentaenoyl (DPA)-plasmalogen PE/MO molar ratio of 20/80 (mol/mol) and dispersed by VPGS-PEG₁₀₀₀ (6 mol.%). Aqueous phase: 1.10^{− 2} M phosphate buffer with dissolved 2,6-di-tert-butyl-4-methylphenol (BHT). Dispersion content: 5 wt% lipid phase/95 wt% aqueous phase.

FIGURE 9

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B–F) cryogenic transmission electron microscopy (cryo-TEM) images of a self-assembled nanoparticulate plasmalogen-phosphoethanolamine (C16:1p-22:5n6 PE)/monoolein (MO)/vitamin E/coenzyme Q₁₀/VPGS-PEG₁₀₀₀ system with a plasmalogen-PE/MO molar ratio of 20/80 (mol/mol) and added vitamin E (5 mol.%) and coenzyme Q₁₀ (1 mol.%). Aqueous phase: 1.10^{− 2} M phosphate buffer containing 2,6-di-tert-butyl-4-methylphenol (BHT). Dispersion content: 5 wt% lipid phase/95 wt% aqueous phase.

FIGURE 10

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B–F) cryogenic transmission electron microscopy (cryo-TEM) images of a self-assembled nanoparticulate plasmalogen-phosphoethanolamine (C16:1p-22:5n6 PE)/monoolein (MO)/vitamin E/coenzyme Q₁₀/VPGS-PEG₁₀₀₀ system with a plasmalogen-PE/MO molar ratio of 15/85 (mol/mol) and added vitamin E (10 mol.%) and coenzyme Q₁₀ (1 mol.%). Aqueous phase: 1.10^{− 2} M phosphate buffer containing 2,6-di-tert-butyl-4-methylphenol (BHT). The Bragg peak positions spaced in the ratio 1:√3:√4 correspond to (10), (11), and (20) reflections of an inverted hexagonal (H_II) phase inner organization of the lipid nanoparticles (hexosomes). Dispersion content: 5 wt% lipid phase/95 wt% aqueous phase. The topologies of the hexosome nanoparticles surrounded with soft coronas and stabilized by VPGS-PEG₁₀₀₀ (6 mol.%) and fragments of a dense inverted hexagonal (H_II) phase are presented in (B–F).

FIGURE 11

(A) Synchrotron small-angle X-ray scattering (SAXS) pattern and (B–F) cryogenic transmission electron microscopy (cryo-TEM) images of a self-assembled nanoparticulate docosapentaenoyl (DPA)-diacyl phosphoinositol (22:5n6-22:5n6 PI)/monoolein (MO)/vitamin E/coenzyme Q₁₀/VPGS-PEG₁₀₀₀ system with a DPA-diacyl PI/MO molar ratio of 15/85 (mol/mol) and added vitamin E (10 mol.%) and coenzyme Q₁₀ (1 mol.%). The PEGylated amphiphile VPGS-PEG₁₀₀₀ is included at 6 mol.%. Aqueous phase: 1.10^{− 2} M phosphate buffer containing 2,6-di-tert-butyl-4-methylphenol (BHT). Dispersion content: 5 wt% lipid/95 wt% aqueous buffer.

FIGURE 12

Summary of the different nanoscale topology types of docosapentaenoyl (DPA) plasmalogen- and ester-based liquid crystalline multiphase structures generated by spontaneous assembly in excess aqueous medium. Examples of individual cubosome and hexosome particles are given together with multiphase nanoparticles and intermediate structures formed as a result of inhomogeneous curvature distribution in the studied polycomponent lipid mixtures.