Self-assembled Lipid Research Articles

Liposomes for Antibiotic Encapsulation and Delivery.

When antibiotics are administered, orally or intravenously, they pass through different organs and layers of tissue on their way to the site of infection; this can cause dilution and/or intoxication. To overcome these problems, drug delivery vehicles have been used to encapsulate and deliver antibiotics, improving their therapeutic index while minimizing their adverse effects. Liposomes are self-assembled lipid vesicles made from at least one bilayer of phospholipids with an inner aqueous compartment. Liposomes are attractive vehicles to deliver antibiotics because they can encapsulate both hydrophobic and hydrophilic antibiotics, they have low toxicity, and they can change the biodistribution of the drug. Furthermore, liposomes have been approved by regulatory agencies. However, most developmental and mechanistic research in the field has been focused on encapsulation and delivery of anticancer drugs, a class of molecules that differ significantly in chemistry from antibiotics. In this critical Review, we discuss the state of knowledge regarding the design of liposomes for encapsulation and delivery of antibiotics and offer insight into the challenges and promises of using liposomes for antibiotic delivery.

Nucleotide lipid-based hydrogel as a new biomaterial ink for biofabrication

One of the greatest challenges in the field of biofabrication remains the discovery of suitable bioinks that satisfy physicochemical and biological requirements. Despite recent advances in tissue engineering and biofabrication, progress has been limited to the development of technologies using polymer-based materials. Here, we show that a nucleotide lipid-based hydrogel resulting from the self-assembly of nucleotide lipids can be used as a bioink for soft tissue reconstruction using injection or extrusion-based systems. To the best of our knowledge, the use of a low molecular weight hydrogel as an alternative to polymeric bioinks is a novel concept in biofabrication and 3D bioprinting. Rheological studies revealed that nucleotide lipid-based hydrogels exhibit suitable mechanical properties for biofabrication and 3D bioprinting, including i) fast gelation kinetics in a cell culture medium and ii) shear moduli and thixotropy compatible with extruded oral cell survival (human gingival fibroblasts and stem cells from the apical papilla). This polymer-free soft material is a promising candidate for a new bioink design.

Enantiomers of phospholipids and cholesterol: A key to decipher lipid-lipid interplay in membrane.

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.

Selection from a pool of self-assembling lipid replicators

Replication and compartmentalization are fundamental to living systems and may have played important roles in life’s origins. Selection in compartmentalized autocatalytic systems might provide a way for evolution to occur and for life to arise from non-living systems. Herein we report selection in a system of self-reproducing lipids where a predominant species can emerge from a pool of competitors. The lipid replicators are metastable and their out-of-equilibrium population can be sustained by feeding the system with starting materials. Phase separation is crucial for selective surfactant formation as well as autocatalytic kinetics; indeed, no selection is observed when all reacting species are dissolved in the same phase. Selectivity is attributed to a kinetically controlled process where the rate of monomer formation determines which replicator building blocks are the fittest. This work reveals how kinetics of a phase-separated autocatalytic reaction may be used to control the population of out-of-equilibrium replicators in time.

Self-assembly of glycerol monooleate with the antimicrobial peptide LL-37: a molecular dynamics study.

Over the past decade, the rapid increase in the incidence of antibiotic-resistant bacteria has promoted research towards alternative therapeutics such as antimicrobial peptides (AMPs), but their biodegradability limits their application. Encapsulation into nanocarriers based on the self-assembly of surfactant-like lipids is emerging as a promising strategy for the improvement of AMPs' stability and their protection against degradation when in biological media. An in-depth understanding of the interactions between the structure-forming lipids and AMPs is required for the design of nanocarriers. This in silico study, demonstrates the self-assembly of the amphiphilic lipid glycerol monooleate (GMO) with the antimicrobial peptide LL-37 into nanocarriers on the molecular scale. Molecular dynamics (MD) simulations show the formation of direct micelles, with either one or two interacting LL-37, and vesicles in this two-component system in agreement with experimental results from small-angle X-ray scattering studies. The hydrophobic contacts between LL-37 and GMOs in water appear responsible for the formation of these nanoparticles. The results also suggest that the enhanced antimicrobial efficiency of LL-37 in these nanocarriers that was previously observed experimentally can be explained by the availability of its side chains with charged amino acids, an increase of the electrostatic interaction and a decrease of the peptide's conformational entropy upon interacting with GMO. The results of this study contribute to the fundamental understanding of lipid–AMP interactions and may guide the comprehensive design of lipid-based self-assembled nanocarriers for antimicrobial peptides.

Lipid-based lyotropic liquid crystalline phase transitions as a novel assay platform using birefringence as the visual signal output.

Amphiphilic lipids can often form various lipid-based lyotropic liquid crystalline phases when they are exposed to aqueous environments, which includes the inverse hexagonal (H2), inverse cubic (V2), inverse discontinuous cubic (I2) and lamellar (Lα) phases. Their different interactions with crossed-polarised light make some phases appear bright and some dark, which offers great potential in developing a novel universal assay platform using birefringence as the visual signal output. Here, we have developed a novel strategy for constructing an assay platform using lyotropic liquid crystalline phases as the signal transducer and using changes in their birefringence upon exposure to lipase as the signal output. The hydrolysis of the ester group of glyceryl monooleate (GMO) by lipase induced a phase transition from cubic phase to hexagonal phase and consequently "turned on" the birefringence as the signal output. The change in the intensity of the birefringence depended on both the exposure time and concentration of lipase. The streptavidin-biotin affinity was utilised to demonstrate the potential of the birefringence assay platform, using free biotin as the model analyte, biotinylated lipase and streptavidin-coated magnetic beads in the competitive format. A semi-quantitative assay with a detection limit of 5 μg mL-1 to free biotin as the model analyte was achieved in the visual birefringence mode. This study demonstrated a proof-of-concept lyotropic assay platform with birefringence as the visual signal output that could be deployed as an electronics- and colour-free diagnostic device for a wide range of applications, for example to indicate the presence of toxins in water.

Preparation of Nanostructured Lipid Drug Delivery Particles Using Microfluidic Mixing.

Cubosomes are highly ordered self-assembled lipid particles analogous to liposomes, but with internal liquid crystalline structure. They are receiving interest as stimuli responsive delivery particles, but their preparation typically requires high energy approaches such as sonication which is not favourable in many applications. Here we investigated the impact of microfluidic preparation on particle size distribution and internal structure of cubosomes prepared from two different lipid systems, phytantriol and glyceryl monooleate (GMO). The impact of relative flow rates of the aqueous and organic streams, the total flow rate and temperature were investigated in a commercial microfluidic system. The particle size distribution and structure were measured using dynamic light scattering and small angle X-ray scattering respectively. Phytantriol based particles were robust to different processing conditions, while cubosomes formed using GMO were more sensitive to composition both locally and globally, which reflects their preparation using other techniques. Thus, in summary microfluidics represents a reproducible and versatile method to prepare complex lipid particle dispersions such as cubosomes.

Hybrid Plasmonic Photoreactors as Visible Light-Mediated Bactericides.

Photocatalytic compounds and complexes, such as tris(bipyridine)ruthenium(II), [Ru(bpy)3]2+, have recently attracted attention as light-mediated bactericides that can help to address the need for new antibacterial strategies. We demonstrate in this work that the bactericidal efficacy of [Ru(bpy)3]2+ and the control of its antibacterial function can be significantly enhanced through combination with a plasmonic nanoantenna. We report strong, visible light-controlled bacterial inactivation with a nanocomposite design that incorporates [Ru(bpy)3]2+ as a photocatalyst and a Ag nanoparticle (NP) core as a light-concentrating nanoantenna into a plasmonic hybrid photoreactor. The hybrid photoreactor platform is facilitated by a self-assembled lipid membrane that encapsulates the Ag NP and binds the photocatalyst. The lipid membrane renders the nanocomposite biocompatible in the absence of resonant illumination. Upon illumination, the plasmon-enhanced photoexcitation of the metal-to-ligand charge-transfer band of [Ru(bpy)3]2+ prepares the reactive excited state of the complex that oxidizes the nanocomposite membrane and increases its permeability. The photooxidation induces the release of [Ru(bpy)3]2+, Ag+, and peroxidized lipids into the ambient medium, where they interact synergistically to inactivate bacteria. We measured a 7 order of magnitude decrease in Gram-positive Arthrobacter sp. and a 4 order of magnitude decrease in Gram-negative Escherichia coli colony forming units with the photoreactor bactericides after visible light illumination for 1 h. In both cases, the photoreactor exceeds the bactericidal standard of a log reduction value of 3 and surpasses the antibacterial effect of free Ag NPs or [Ru(bpy)3]2+ by >4 orders of magnitude. We also implement the inactivation of a bacterial thin film in a proof-of-concept study.

Inherently PET/CT Dual Modality Imaging Lipid Nanocapsules for Early Detection of Orthotopic Lung Tumors.

Accurate diagnosis of cancer at an early stage is the key to reduce cancer mortality and improve survival. PET imaging has high sensitivity but low spatial resolution, while CT imaging has good spatial location information. Therefore, the combination of PET and CT imaging can provide complementary advantages to achieve accurate early diagnosis of tumors. However, currently developed PET or CT imaging agents have only a single function. Here, we designed and constructed a self-assembled lipid nanocapsule encapsulated with iodixanol and labeled with self-chelated 64Cu for precise PET/CT imaging of tiny lung tumor. The lipid nanocapsule self-assembled in water using LPPC-Ce6, a conjugate of chlorin e6 (Ce6) and lysophosphatidylcholine (LPPC), to form a bilayer vesicular structure. 64Cu was embedded in the center of the tetrapyrrole ring of Ce6 by natural capture ability for Cu2+ ions. GLT21.T, the aptamer targeting lung cancer, was conjugated to the surface of the lipid nanocapsules. Iodixanol was loaded into the cavity of the lipid nanocapsule (64Cu@LCI-apt). In the nanostructure, the loading of iodixanol was sufficiently high, and the specific activity could be flexibly adjusted according to imaging requirements. The prepared 64Cu@LCI-apt achieves excellent radiolabeling efficiency, stability and effective targeting of lung tumor. In an early orthotopic lung cancer model, 64Cu@LCI-apt demonstrated the capabilities of sensitive PET imaging and enhanced contrast CT imaging to enable efficient high-quality PTE/CT imaging of tiny orthotopic lung tumor with a diameter of 500 μm. 64Cu@LCI-apt has great potential for early, sensitive, and accurate diagnosis of tumors through dual-mode PET/CT imaging.

Biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment

The gut microbiota represents a huge community of microorganisms that play essential roles in immune modulation and homeostasis maintenance. Microbiota transplantation is an important approach to prevent and treat disease as it can inhibit pathogen colonization and positively modulate bacterial composition. However, the development of oral bacterial therapeutics has been restricted by low bioavailability and limited retention in the gastrointestinal tract. Here, we report a simple yet highly efficient method to coat gut microbes via biointerfacial supramolecular self-assembly. Coating can be performed within 15 min by simply vortexing with biocompatible lipids. Bacteria coated with an extra self-assembled lipid membrane exhibit significantly improved survival against environmental assaults and almost unchanged viability and bioactivity. We demonstrate their enhanced efficacies in oral delivery and treatment using two murine models of colitis. We suggest that biointerfacial supramolecular self-assembly may provide a unique platform to generate advanced bacterial therapeutics for the treatment of various diseases.

Stabilization of self-assembled lipids in exfoliated organo-nanosheets

Layered materials represent interesting 2D confinement matrix for both the preservation of organic compounds and were suggested as carriers for biomolecules. An organoclay was prepared with the use of a clay mineral modified by a cationic surfactant and used as a confinement matrix for assemblies formed by 1-monoolein lipid. Monoolein self assembles in water in various lyotropic and thermotropic liquid crystalline phases which were associated to organo-nanosheets resulting from a prior delamination procedure of the organoclay. The lamellar organization confined within the nanosheets displays a different phase sequence than the bulk state with its existence on wide range of temperature.

Structural Transformation in Vesicles upon Hydrolysis of Phosphatidylethanolamine and Phosphatidylcholine with Phospholipase C.

This study provides insights into dynamic nanostructural changes in phospholipid systems during hydrolysis with phospholipase C, the fate of the hydrolysis products, and the kinetics of lipolysis. The effect of lipid restructuring of the vesicle was investigated using small-angle X-ray scattering and cryogenic scanning electron microscopy. The rate and extent of phospholipid hydrolysis were quantified using nuclear magnetic resonance. Hydrolysis of two phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), results in the cleavage of the molecular headgroup, causing two strikingly different changes in lipid self-assembly. The diacylglycerol product of PC escapes the lipid bilayer, whereas the diacylglycerol product adopts a different configuration within the lipid bilayer of the PE vesicles. These results are then discussed concerning the change of the lipid configuration upon the lipid membrane and its potential implications in vivo, which is of significant importance for the detailed understanding of the fate of lipidic particles and the rational design of enzyme-responsive lipid-based drug delivery systems.

Artificial High Density Lipoprotein Nanoparticles in Cardiovascular Research.

Lipoproteins are endogenous nanoparticles which are the major transporter of fats and cholesterol in the human body. They play a key role in the regulatory mechanisms of cardiovascular events. Lipoproteins can be modified and manipulated to act as drug delivery systems or nanocarriers for contrast agents. In particular, high density lipoproteins (HDL), which are the smallest class of lipoproteins, can be synthetically engineered either as nascent HDL nanodiscs or spherical HDL nanoparticles. Reconstituted HDL (rHDL) particles are formed by self-assembly of various lipids and apolipoprotein AI (apo-AI). A variety of substances including drugs, nucleic acids, signal emitting molecules, or dyes can be loaded, making them efficient nanocarriers for therapeutic applications or medical diagnostics. This review provides an overview about synthesis techniques, physicochemical properties of rHDL nanoparticles, and structural determinants for rHDL function. We discuss recent developments utilizing either apo-AI or apo-AI mimetic peptides for the design of pharmaceutical rHDL formulations. Advantages, limitations, challenges, and prospects for clinical translation are evaluated with a special focus on promising strategies for the treatment and diagnosis of atherosclerosis and cardiovascular diseases.

Amphiphilic Lipids: Nature‐Inspired Design and Application of Lipidic Lyotropic Liquid Crystals (Adv. Mater. 35/2019)

In article number 1900818, Raffaele Mezzenga, Laurent Sagalowicz, and co-workers review the thermodynamics and physics underlying the self-assembly of amphiphilic lipids. The link to Nature's deployment of these fascinating lyotropic liquid-crystalline phases and the harnessing of their properties to enable a range of technological, biomedical and food applications are reviewed.

DNA Origami as Scaffolds for Self-Assembly of Lipids and Proteins.

Since first being reported in 2006, the DNA origami approach has attracted increasing attention due to programmable shapes, structural stability, biocompatibility, and fantastic addressability. Herein, we provide an account of recent developments of DNA origami as scaffolds for templating the selfassembly of distinct biocomponents, essentially proteins and lipids, into a diverse spectrum of integrated supramolecular architectures. First, the historical development of the DNA origami concept is briefly reviewed. Next, various applications of DNA origami constructs in controllable directed assembly of soluble proteins are discussed. The manipulation and self-assembly of lipid membranes and membrane proteins by using DNA origami as scaffolds are also addressed. Furthermore, recent progress in applying DNA origami in cryoelectron microscopy analysis is discussed. These advances collectively emphasize that the DNA origami approach is a highly versatile, fast evolving tool that may be integrated with lipids and proteins in a way that meets future challenges in molecular biology and nanomedicine.

Evidence of Increased Hydrophobicity and Dynamics inside the Tail Region of Glycolipid Self-Assemblies Using 2-n-Alkyl-Pyrene Derivatives to Probe Different Locations.

New designer biofluorophores are being increasingly used in the investigation of complex cellular processes. In this study, we utilized new derivatives of pyrene (Py), i.e., 2-n-alkyl-pyrenes (Py-C4 and Py-C8), in order to probe different regions inside the hydrophobic tail of n-dodecyl β-d-maltoside (βMal-C12) in two different phases (cubic ↔ lamellar). Although the sensitivity to the local environment is reduced compared to that of Py, attaching C4 and C8 at the 2-position of Py can provide a possible means to probe the local hydrophobicity in different parts of the tail region. The absence of excimer fluorescence and the ratio of the vibronic fluorescence peak intensities (I1/I3) in a lipid environment indicate the existence of Py as monomers in the hydrophobic region, similar to hydrophobic solvation, yet close to the headgroup region. When Py is replaced by Py-C4 and Py-C8, there is a small increase in hydrophobicity (reduction in I1/I3) as the Py moiety is pulled deeper inside the tail region of both cubic and lamellar phases. The larger space of the tail region in the lamellar phase is reflected as more local hydrophobicity measured by the probes which can penetrate deep inside, whereas the curved structure of the cubic phase limits the available space for the probes. Three fluorescence lifetime components were measured in lipid, indicating the heterogeneous nature of the hydrophobic region. In the lamellar phase, a large reduction in the average lifetime value, led by the long decay component, was measured for Py-C4 (reduction by 25%) and Py-C8 (45%) compared to that of the parent Py. This observation suggests the presence of a mechanism of interaction more collisional than static between the Py moiety and the tail region of the bilayer unit due to the ample space provided by the lamellar phase as the probe is buried deeper inside the hydrophobic region. A much smaller effect was observed in the cubic phase and was correlated with the tight environment around the probes, which stems from the increased curvature of the cubic phase. The current results provide a deeper understanding of the hydrophobic region during phase transition of lipid self-assembly which is important for better control during the process of membrane-protein crystallization.

Heteronuclear NMR spectroscopy of proteins encapsulated in cubic phase lipids.

Lipidic cubic phases, which form spontaneously via the self-assembly of certain lipids in an aqueous environment, are highly prospective nanomaterials with applications in membrane protein X-ray crystallography and drug delivery. Here we report 1H-15N heteronuclear single/multiple quantum coherence (HSQC, HMQC) spectra of 15N-enriched proteins encapsulated in inverse bicontinuous lipidic cubic phases obtained on a standard commercial high resolution NMR spectrometer at ambient temperature. 15N-enriched proteins encapsulated in this lipidic cubic phase show: (i) no significant changes in tertiary structure, (ii) significantly reduced solvent chemical exchange of backbone amides, which potentially provides a novel concept for quantifying residue-specific hydration; and (iii) improved spectral sensitivity achieved with band-selective excitation short-transient (BEST) spectroscopy, which is attributed to the presence of an abundant source of 1H nuclear spins originating from the lipid component of the cubic phase.

Toward Cluster Materials with Ordered Structures via Self‐Assembly of Heterocluster Janus Molecules

Cluster materials have attracted much attention because of their unique chemical and physical properties, hitherto unseen in bulk materials. Inspired by the lipid self-assembly principle, a series of heterocluster Janus molecules (HCJMs) with atomic precision have been rationally designed and synthesized by connecting different clusters via covalent bonds for the construction of nanomaterials and nano-objects. Due to their amphiphilicity, HCJMs self-assemble into cluster-containing nanomaterials or nano-objects with versatile ordered structures beyond those observed in conventional crystals. Their hybrid composition and nanoscale size are also greatly advantageous in the study of their fine structure by electron microscopy techniques, and enable their formation mechanisms to be unraveled. Finally, the influence of the characteristics of the HCJMs on the structure and properties of the self-assembled nano-objects are explored comprehensively. This synthesis strategy will promote further development of cluster materials with advanced functions via rational molecular design toward the construction of hierarchical nanostructures via molecular self-assembly.

Self-Assembly of Aldehyde Lipids in Water

Lipid based self-assembled structures has been applied for drug delivery systems. It is well known that lipid self-assembled structures depend on the chemical structures of lipids, however the self-assembled structures of oxidized lipids with asymmetrical structures remain unclear. In this study, 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (9-al) was used to investigate its self-assembly structure. The molecular dynamics (MD) simulation was performed with the system consisting of 128 lipids and 8,960 simple point charge (SPC) water molecules. Initially, all lipids and water molecules were randomly generated in the simulation box, then molecular trajectories were integrated and collected over microsecond times. The results showed that the equilibrium self-assembled structures of 9-al lipid preferred to form a micelle structure. We analyzed the lipid’s angle distribution and the lipid’s chain length to determine the lipid geometry. The results are in agreement with the previous MD simulation (Boonnoy, P.; et al. J. Phys. Chem. Lett. 2015, 6, 4884 – 4888) that showed the deformation of aldehyde lipid bilayer into micelle structure due to their shorter and highly mobile of aldehyde functional group in oxidized chain.

Design of Sealable Custom-Shaped Cell Mimicries Based on Self-Assembled Monolayers on CYTOP Polymer.

In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.