The hydrophobic effect, a ubiquitous process in biology, is a primary thermodynamic driver of amphiphilic self-assembly. It leads to the formation of unique morphologies including two highly important classes of lamellar and micellar mesophases. The interactions between these two types of structures and their involved components have garnered significant interest because of their importance in key biochemical technologies related to the isolation, purification, and reconstitution of membrane proteins. This work investigates the structural organization of mixtures of the lamellar-forming phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and two zwitterionic micelle-forming surfactants, being n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12 or DDAPS) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-Lyso-PC), when assembled by water vapor hydration with X-ray diffraction measurements, brightfield optical microscopy, wide-field fluorescence microscopy, and atomic force microscopy. The results reveal that multilamellar mesophases of these mixtures can be assembled across a wide range of POPC to surfactant (POPC:surfactant) concentration ratios, including ratios far surpassing the classical detergent-saturation limit of POPC bilayers without significant morphological disruptions to the lamellar motif. The mixed mesophases generally decreased in lamellar spacing (D) and headgroup-to-headgroup distance (D_hh) with a higher concentration of the doped surfactant, but trends in water layer thickness (D_w) between each bilayer in the stack are highly variable. Further structural characteristics including mesophase topography, bilayer thickness, and lamellar rupture force were revealed by atomic force microscopy (AFM), exhibiting homogeneous multilamellar stacks with no significant physical differences with changes in the surfactant concentration within the mesophases. Taken together, the outcomes present the assembly of unanticipated and highly unique mixed mesophases with varied structural trends from the involved surfactant and lipidic components. Modulations in their structural properties can be attributed to the surfactant's chemical specificity in relation to POPC, such as the headgroup hydration and the hydrophobic chain tail mismatch. Taken together, our results illustrate how specific chemical complexities of surfactant-lipid interactions can alter the morphologies of mixed mesophases and thereby alter the kinetic pathways by which surfactants dissolve lipid mesophases in bulk aqueous solutions.

Atomic force microscopy (AFM) in conjunction with microfluidic delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. While AFM is well-known for its spatial precision in imaging and 2D nanolithography, the development of AFM-based nanotechnology into 3D nanoprinting requires overcoming the technical challenges of controlling material delivery and interlayer registry. This work demonstrates the concept of 3D nanoprinting of amphiphilic molecules such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Various formulations of POPC solutions were tested to achieve point, line, and layer-by-layer material delivery. The produced structures include nanometer-thick disks, long linear spherical caps, stacking grids, and organizational chiral architectures. The POPC molecules formed stacking bilayers in these constructions, as revealed by high-resolution structural characterizations. The 3D printing reached nanometer spatial precision over a range of 0.5 mm. The outcomes reveal the promising potential of our designed technology and methodology in the production of 3D structures from nanometer to continuum, opening opportunities in biomaterial sciences and engineering, such as in the production of 3D nanodevices, chiral nanosensors, and scaffolds for tissue engineering and regeneration.

Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.

The constructs of lipid molecules follow self-assembly, driven by intermolecular interactions, forming stacking of lipid bilayer films. Achieving designed geometry at nano- to micro-levels with packing deviating from the near-equilibrium structure is difficult to achieve due to the strong tendency of lipid molecules to self-assemble. Using ultrasmall (<fL) droplets containing designed molecules, our prior work has demonstrated that molecular assembly, in principle, is governed mainly by transient inter-molecular interactions under their dynamic spatial confinement, i.e., tri-phase boundaries during drying. As a result, the assemblies can deviate, sometimes significantly, from the near-equilibrium structures of self-assembly. The present work applies the approach and concept to lipid molecules using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Taking advantage of the high spatial precision and the minute size of the delivery probe in our combined atomic force microscopy and microfluidic delivery, the transient shape of each liquid droplet is regulated. In doing so, the final geometry of the POPC assemblies has been regulated to the designed geometry with nanometer precision. The results extend the concept of controlled assembly of molecules to amphiphilic systems. The outcomes exhibit high potential in lipid-based biomaterial science and biodevice engineering.

Knowledge of AFM tip and lipid bilayer stack interactions is essential for designing and developing nano- to mesoscale lipid constructs. In this study, a model was developed to reveal the complex molecular mechanism governing tip–lipid interactions. This complexity in the interaction forces during penetration leading to a disturbance of lipid bilayers is explored using coarse-grained MARTINI molecular dynamics simulations for a multibilayer 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) lipid system and an AFM tip apex. Force–distance profiles acquired during AFM tip penetration displayed a sawtooth pattern with a periodicity consistent with the bilayer thickness. This computation provides mechanistic insights into this process: a layer-by-layer penetration with distinct features in force–distance traces to specific molecular events such as climbing, adhesion, and compression. The outcomes benefit development of engineered lipid-based materials used in drug and vaccine delivery and fabrication of lipid-based nanodevices and biosensors.