Cationic Bilayer Research Articles

Advances in the study of LNPs for mRNA delivery and clinical applications.

Messenger ribonucleic acid (mRNA) was discovered in 1961 as an intermediary for transferring genetic information from DNA to ribosomes for protein synthesis. The COVID-19 pandemic brought worldwide attention to mRNA vaccines. The emergency use authorization of two COVID-19 mRNA vaccines, BNT162b2 and mRNA-1273, were major achievements in the history of vaccine development. Lipid nanoparticles (LNPs), one of the most superior non-viral delivery vectors available, have made many exciting advances in clinical translation as part of the COVID-19 vaccine and therefore has the potential to accelerate the clinical translation of many gene drugs. In addition, due to these small size, biocompatibility and excellent biodegradability, LNPs can efficiently deliver nucleic acids into cells, which is particularly important for current mRNA therapeutic regimens. LNPs are composed cationic or pH-dependent ionizable lipid bilayer, polyethylene glycol (PEG), phospholipids, and cholesterol, represents an advanced system for the delivery of mRNA vaccines. Furthermore, optimization of these four components constituting the LNPs have demonstrated enhanced vaccine efficacy and diminished adverse effects. The incorporation of biodegradable lipids enhance the biocompatibility of LNPs, thereby improving its potential as an efficacious therapeutic approach for a wide range of challenging and intricate diseases, encompassing infectious diseases, liver disorders, cancer, cardiovascular diseases, cerebrovascular conditions, among others. Consequently, this review aims to furnish the scientific community with the most up-to-date information regarding mRNA vaccines and LNP delivery systems.

Mechanism of Cationic Lipid Induced DNA Condensation: Lipid-DNA Coordination and Divalent Cation Charge Fluctuations.

The condensation of nucleic acids by lipids is a widespread phenomenon in biology with crucial implications for drug delivery. However, the mechanisms of DNA assembly in lipid bilayers remain insufficiently understood due to challenges in measuring and assessing each component's contribution in the lipid-DNA-cation system. This study uses all-atom molecular dynamics simulations to investigate DNA condensation in cationic lipid bilayers. Our exhaustive exploration of the thermodynamic factors reveals unique roles for phospholipid head groups and cations. We observed that bridging cations between lipid and DNA drastically reduce charges, while mobile magnesium cations "ping-ponging" between double strands create charge fluctuations. While the first factor stabilizes the DNA-lipid complex, the latter creates attractive forces to induce the spontaneous condensation of DNAs. This novel mechanism not only sheds light on the current data regarding cationic lipid-induced DNA condensation but also provides potential design strategies for creating efficient gene delivery vectors for drug delivery.

Enveloped Viral Replica Equipped with Spike Protein Derived from SARS-CoV-2.

Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from β-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.

Dendrimer Induced Bilayer Disintegration of Hybrid Vesicles.

Physicochemical investigations on the inclusion of anionic polyamidoaminesuccinamic acid dendrimer, generation 5 (PAMAM-SA, G5) with positively charged hybrid vesicles (HCV), prepared using soylecithin, ion pair amphiphile (IPA), cholesterol and dihexadecyldimethylammonium bromide, were investigated by dynamic light scattering, transmission electron/atomic force microscopy (TEM/AFM), differential scanning calorimetry, fluorescence spectroscopy and surface pressure-time isotherm studies. Adsorption of dendrimer onto vesicle surface and subsequent bilayer disruption strongly depends on the bilayer composition and dendrimer concentration. Change in the zeta potential value with increasing dendrimer concentration suggests the dendrimer-vesicle interaction to be electrostatic in nature. AFM studies also confirm the adsorption of dendrimer as well as hole formation in the bilayer. Impact of the inclusion of dendrimer into the bilayer were further investigated through differential scanning calorimetry by monitoring the chain melting temperature and enthalpy of the chain melting processes. Dendrimer at low concentration does not alter bilayer integrity, while hole formations are noted at higher dendrimer concentration. Fluorescence anisotropy studies confirm the adsorption and subsequent bilayer disruption due to dendrimer inclusion. Dendrimer induced vesicle disintegration kinetics conclusively illustrate the transformation of cationic bilayer to monolayer and thereby exposing the role of IPA. In vitro cytotoxicity studies on PAMAM-SA, G5 and HCVs mixtures against human breast cancer cell line suggest that dendrimer-liposome aggregates (dendriosomes) exhibit substantial anticancer activities with insignificant side effects. It is expected that the dendriosomes may have application to host and deliver anticancer drug in the field of targeted drug delivery.

A supramolecular system mimicking the infection process of an enveloped virus through membrane fusion

Membrane fusion is an essential step for the entry of enveloped viruses, such as human immunodeficiency virus and influenza virus, into the host cell, often triggered by the binding of membrane proteins on the viral envelope to host cell membrane. Recently, external stimuli was shown to trigger membrane fusion in an artificial system. Direct observation of artificial membrane fusion using a giant unilamellar vesicle (GUV), which is similar in size to a cell, is useful as a biological model system. However, there are no model systems for studying membrane fusion of enveloped viruses with host cells. Here, we report a supramolecular model system for viral entry into a GUV or cell through membrane fusion. The system was constructed by complexing a cationic lipid bilayer on an anionic artificial viral capsid, self-assembled from viral β-annulus peptides. We demonstrate that the cationic enveloped artificial viral capsid electrostatically interacts with the anionic GUV or cell, and the capsid enters the GUV or cell through membrane fusion. The model system established in this study will be important for analyzing membrane fusion during infection of a natural virus.

Aspirin-Induced Ordering and Faster Dynamics of a Cationic Bilayer for Drug Encapsulation.

The lipid dynamics and phase play decisive roles in drug encapsulation and delivery to the intracellular target. Thus, understanding the dynamic and structural alterations of membranes induced by drugs is essential for targeted delivery. To this end, united-atom molecular dynamics simulations of a model bilayer, dioctadecyldimethylammonium bromide (DODAB), are performed in the absence and presence of the usual nonsteroidal anti-inflammatory drug (NSAID), aspirin, at 298, 310, and 345 K. At 298 and 310 K, the bilayers are in the interdigitated two-dimensional square phases, which become rugged in the presence of aspirin, as evident from height fluctuations. At 345 K, the bilayer is in the fluid phase in both the absence and presence of aspirin. Aspirin is preferentially located near the oppositely charged headgroup and creates void space, which leads to an increase in the interdigitation and order parameters. Although the center of mass of lipids experiences structural arrest, they reach the diffusive regime faster and have higher lateral diffusion constants in the presence of aspirin. Results are found to be consistent with recent quasi-elastic neutron scattering studies that reveal that aspirin acts as a plasticizer and enhances lateral diffusion of lipids in both ordered and fluid phases. Different relaxation time scales of the bonds along the alkyl tails of DODAB due to the multitude of lipid motions become faster upon the addition of aspirin. Our results show that aspirin insertion is most favorable at physiological temperature. Thus, the ordered, more stable, and faster DODAB bilayer can be a potential drug carrier for the protected encapsulation of aspirin, followed by targeted and controlled drug release with antibacterial activity in the future.

Influence of l-Dopa adsorption time and concentration on Au nanorods aggregation for SERS quantitative analysis

Levodopa (l-Dopa) is an important neurotransmitter from the amino acid family and is also associated with the treatment of Parkinson's disease. The obtention of the l-Dopa SERS signal in colloid is a challenge due to the zwitterionic charge promoting repulsion from nanoparticle surface and leading to poor or not active SERS signal. In this work, SERS signal of l-Dopa was successfully obtained using Au nanorods (AuNRs) covered with a cationic lipid bilayer and using the 785 nm laser line. The UV–Vis extinction, dynamic light scattering, and zeta potential results indicated an influence of l-Dopa adsorption time and l-Dopa concentration on AuNRs aggregation. The SERS signal followed this time dependence, being the characteristic l-Dopa band observed only after 30 min. The l-Dopa quantitative analysis using the standard addition method presented a linear SERS signal at 439 cm−1 with a limit of detection of 6.7 × 10−7 mol L−1 for peak intensity, and 7.9 × 10−7 mol L−1 for band integrated area.

Antigen/Adjuvant-Displaying Enveloped Viral Replica as a Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidate.

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled β-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

Transient Coatings from Nanoparticles Achieving Broad-Spectrum and High Antimicrobial Performance.

Cationic and hydrophilic coatings based on casting and drying water dispersions of two different nanoparticles (NPs) onto glass are here described and evaluated for antimicrobial activity. Discoid cationic bilayer fragments (BF) surrounded by carboxy-methylcellulose (CMC) and poly (diallyl dimethyl ammonium) chloride (PDDA) NPs and spherical gramicidin D (Gr) NPs dispersed in water solution were cast onto glass coverslips and dried, forming a coating quantitatively evaluated against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. From plating and colony forming units (CFU) counting, all strains interacting for 1 h with the coatings lost viability from 105 to 106, to zero CFU, at two sets of Gr and PDDA doses: 4.6 and 25 μg, respectively, or, 0.94 and 5 μg, respectively. Combinations produced broad spectrum, antimicrobial coatings; PDDA electrostatically attached to the microbes damaging cell walls, allowing Gr NPs interaction with the cell membrane. This concerted action promoted optimal activity at low Gr and PDDA doses. Further washing and drying of the deposited dried coatings showed that they were washed out so that antimicrobial activity was no longer present on the glass surface. Significant applications in biomedical materials can be foreseen for these transient coatings.

Anomalous Diffusion of Polyelectrolyte Segments on Supported Charged Lipid Bilayers.

This work provides mesoscale models for the anomalous diffusion of a polymer chain on a heterogeneous surface with rearranging randomly distributed adsorption sites. Both the "bead-spring" model and oxDNA model were simulated on supported lipid bilayer membranes with various molar fractions of charged lipids, using Brownian dynamics method. Our simulation results demonstrate that "bead-spring" chains exhibit sub-diffusion on charged lipid bilayers which agrees with previous experimental observations for short-time dynamics of DNA segments on membranes. In addition, the non-Gaussian diffusive behaviors of DNA segments have not been observed in our simulations. However, a simulated 17 base pairs double stranded DNA, using oxDNA model, performs normal diffusion on supported cationic lipid bilayers. Due to the number of positively charged lipids attracted by short DNA is small, the energy landscape that the short DNA experiences during diffusion is not as heterogeneous as that experienced by long DNA chains, which results in normal diffusion rather than sub-diffusion for short DNA.

Thermotropic Behavior and Structural Organization of C24:1 Sulfatide Dispersions and Its Mixtures with Cationic Bilayers.

C24:1 sulfatide (SF) is an endogenous activator of type II NKT cells. The thermotropic behavior and structure of SF dispersions and its mixtures (4.8-16.6 mol %) with cationic dioctadecyldimethylammonium bromide (DODAB) bilayers were investigated by differential scanning calorimetry and electron paramagnetic resonance spectroscopy. The non-interdigitated lamellar structures formed by pure SF display broad thermal events around 27.5 °C when heated and cooled. These events disappear upon mixing with DODAB, showing complete lipid miscibility. SF decreases the DODAB gel-phase packing, with a consequent decrease in phase-transition temperatures and cooperativity upon heating. In contrast, SF increases the rigidity of the DODAB fluid phase, resulting in a smaller decrease in transition temperatures upon cooling. The hysteresis between heating and cooling decreased as the SF molar fraction increased. These effects on DODAB are similar to the ones described for other glycolipids, such as αGalCer and βGlcCer. This might be due to the orientation of the rigid and planar amide bond that connects their sphingoid bases and acyl chains, which result in a V-shaped conformation of the glycolipid molecules. The current results may be important to plan and develop new immunotherapeutic tools based on SF.

Polypeptide Bilayer Assembly-Mediated Gene Delivery Enhances Chemotherapy in Cancer Cells.

Developing gene vectors with high transfection efficiency and low cytotoxicity to humans is crucial to improve gene therapy outcomes. This study set out to investigate the use of cationic polypeptide bilayer assemblies formed by coil-sheet poly(l-lysine)-block-poly(l-benzyl-cysteine) (PLL-b-PBLC) as gene vectors that present improved transfection efficiency, endosomal escape, and biocompatibility compared to PLL. The formation of the polyplexes was triggered by hydrogen bonding, hydrophobic interactions, and electrostatic association between the cationic PLL segments and the negatively charged plasmid encoding p53, resulting in self-assembled polypeptide chains. Transfection efficiency of these polyplexes increased with increments of PLL-to-PBLC block ratios, with PLL15-b-PBLC5 bilayers exhibiting the best in vitro transfection efficiency among all, suggesting that PLL-b-PBLC bilayer assemblies are efficient in the protection and stabilization of genes. The polypeptide bilayer gene vector reversed the cisplatin sensitivity of p53-null cancer cells by increasing apoptotic signaling. Consistent with in vitro results, mouse xenograft studies revealed that PLL15-b-PBLC5/plasmid encoding p53 therapy significantly suppressed tumor growth and enhanced low-dose cisplatin treatment, while extending survival of tumor-bearing mice and avoiding significant body weight loss. This study presents a feasible gene therapy that, combined with low-dose chemotherapeutic drugs, may treat genetically resistant cancers while reducing side effects in clinical patients.

Characterization and Differential Cytotoxicity of Gramicidin Nanoparticles Combined with Cationic Polymer or Lipid Bilayer.

Gramicidin (Gr) nanoparticles (NPs) and poly (diallyl dimethyl ammonium) chloride (PDDA) water dispersions were characterized and evaluated against Gram-positive and Gram-negative bacteria and fungus. Dynamic light scattering for sizing, zeta potential analysis, polydispersity, and colloidal stability over time characterized Gr NPs/PDDA dispersions, and plating and colony-forming units counting determined their microbicidal activity. Cell viabilities of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in the presence of the combinations were reduced by 6, 7, and 7 logs, respectively, at 10 μM Gr/10 μg·mL−1 PDDA, 0.5 μM Gr/0. 5μg·mL−1 PDDA, and 0.5 μM Gr/0.5 μg·mL−1 PDDA, respectively. In comparison to individual Gr doses, the combinations reduced doses by half (S. aureus) and a quarter (C. albicans); in comparison to individual PDDA doses, the combinations reduced doses by 6 times (P. aeruginosa) and 10 times (C. albicans). Gr in supported or free cationic lipid bilayers reduced Gr activity against S. aureus due to reduced Gr access to the pathogen. Facile Gr NPs/PDDA disassembly favored access of each agent to the pathogen: PDDA suctioned the pathogen cell wall facilitating Gr insertion in the pathogen cell membrane. Gr NPs/PDDA differential cytotoxicity suggested the possibility of novel systemic uses for the combination.

Embedding a membrane protein into an enveloped artificial viral replica.

Natural enveloped viruses, in which nucleocapsids are covered with lipid bilayers, contain membrane proteins on the outer surface that are involved in diverse functions, such as adhesion and infection of host cells. Previously, we constructed an enveloped artificial viral capsid through the complexation of cationic lipid bilayers onto an anionic artificial viral capsid self-assembled from β-annulus peptides. Here we demonstrate the embedding of the membrane protein Connexin-43 (Cx43), on the enveloped artificial viral capsid using a cell-free expression system. The expression of Cx43 in the presence of the enveloped artificial viral capsid was confirmed by western blot analysis. The embedding of Cx43 on the envelope was evaluated by detection via the anti-Cx43 antibody, using fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM). Interestingly, many spherical structures connected to each other were observed in TEM images of the Cx43-embedded enveloped viral replica. In addition, it was shown that fluorescent dyes could be selectively transported from Cx43-embedded enveloped viral replicas into Cx43-expressing HepG2 cells. This study provides a proof of concept for the creation of multimolecular crowding complexes, that is, an enveloped artificial viral replica embedded with membrane proteins.

Binding affinity and conformation of a conjugated AS1411 aptamer at a cationic lipid bilayer interface.

Aptamers have been widely used in the detection, diagnosis, and treatment of cancer. Owing to their special binding affinity toward cancer-related biomarkers, aptamers can be used for targeted drug delivery or bio-sensing/bio-imaging in various scenarios. The interfacial properties of aptamers play important roles in controlling the surface charge, recognition efficiency, and binding affinity of drug-delivering lipid-based carriers. In this research, the interfacial behaviors, such as surface orientation, molecular conformation, and adsorption kinetics of conjugated AS1411 molecules at different cationic lipid bilayer interfaces were investigated by sum frequency generation vibrational spectroscopy (SFG-VS) in situ and in real-time. It is shown that the conjugated AS1411 molecules at the DMTAP bilayer interface show a higher binding affinity but with slower binding kinetics compared to the DMDAP bilayer interface. The analysis results also reveal that the thymine residues of cholesteryl conjugated AS1411 molecules show higher conformational ordering compared to the thymine residues of the alkyl chain conjugated AS1411 molecules. These understandings provide unique molecular insight into the aptamer-lipid membrane interactions, which may help researchers to improve the efficiency and safety of aptamer-related drug delivery systems.

Structural properties of cationic surfactant-fatty alcohol bilayers: insights from dissipative particle dynamics.

Bilayers, self-assembled by cationic surfactants and fatty alcohols in water, are the basic units of lamellar gel networks - creamy formulations extensively used in cosmetics and pharmaceutics. Mesoscopic modelling and study of the bilayers formed by single- or double-tail cationic surfactants (CTAC or DHDAC), and fatty alcohols (FAs) in the lamellar fluid and gel phases were employed. Fatty alcohols with alkyl tail equal to or greater than the surfactant alkyl tail, i.e., C16FA or C18FA and C22FA, were considered. A model formulation was explored with the FA concentration greater than that of the surfactant and the structure of the fluid and gel bilayers in tensionless state characterised via the density profiles across the bilayers, orientational order parameters of the surfactant and FA chains, intrinsic analysis of the bilayer interfaces, and bending rigidity. The intrinsic analysis allows identification and quantification of the coexistence of the interdigitated and non-interdigitated phases present within the gel bilayers. The FA chains were found to conform the primary scaffolding of the bilayers while the surfactant chains tessellate bilayer monolayers from their water-hydrophobic interface. Further, the overlap of the FA chains from the apposed monolayers of the fluid bilayers rises with increasing FA length. Finally, the prevalence of the non-interdigitated phase over the interdigitated phase within the gel bilayers becomes enhanced upon the FA length increase with a preference of the surfactant chains to reside in the non-interdigitated phase rather than the interdigitated phase.

Dynamics and local environment of an aromatic counterion bound to di-chain cationic surfactant bilayers studied by avoided level crossing muon spin resonance: evidence for counterion condensation.

Avoided level crossing muon spin resonance (ALC-μSR) has been used to study the reorientational dynamics of muon-spin-labelled 2,4,6-trimethylbenzoate (246TMB-) counterions and their interaction with DODMAC (dioctadecyldimethylammonium chloride) bilayers in the Lα and Lβ liquid crystalline states. The muoniated radical anion formed by the addition of muonium to the secondary carbons of the aromatic ring of 246TMB- is used as a local spin probe. The muon and methylene proton hyperfine parameters and the electron spin relaxation rate (λe) of the muoniated spin probe were determined as a function of temperature by modelling the ALC-μSR spectra with Monte Carlo numerical simulations. The observation of a Δ1 resonance indicates that 246TMB- is undergoing anisotropic motion and doesn't reside in the aqueous layer in either the Lα and Lβ phases. The lack of an abrupt change in the hyperfine parameters or λe when the system goes from the Lβ to the Lα lamellar liquid crystalline phases suggests that 246TMB- is located at the oil-water interface rather than within the bilayer. The hyperfine parameters indicate that 246TMB- is undergoing large amplitude reorientational motion about a preferred orientation resulting from the bilayer's electric field. The interaction between 246TMB- and the bilayer decreases and the amplitude of the wobbling-in-a-cone motion increases with increasing temperature. The temperature dependence of the electron spin relaxation rate indicates the barrier to reorientation is 41.7 kJ mol-1.

Effect of DODAB Nano-Sized Cationic Bilayer Fragments against Leishmania amazonensis.

The dioctadecyldimethylammonium bromide (DODAB) is a double-chained cationic lipid with potent bactericide and fungistatic activities; however, its toxicity on protozoan parasites is still unknown. Here, we show the antileishmanial activity of DODAB nano-sized cationic bilayer fragments on stationary-phase promastigotes and amastigotes of Leishmania amazonensis, the causative agent of cutaneous leishmaniasis. Upon treatment with DODAB, we analyzed the parasite surface zeta-potential, parasite viability, cellular structural modifications, and intracellular proliferation. The DODAB cytotoxic effect was dose-dependent, with a median effective concentration (EC50) of 25 µM for both life-cycle stages, comparable to the reported data for bacteria and fungi. The treatment with DODAB changed the membrane zeta-potential from negative to positive, compromised the parasite’s morphology, affected the cell size regulation, caused a loss of intracellular organelles, and probably dysregulated the plasma membrane permeability without membrane disruption. Moreover, the parasites that survived after treatment induced small parasitophorous vacuoles and failed to proliferate inside macrophages. In conclusion, DODAB displayed antileishmanial activity, and it remains to be elucidated how DODAB acts on the protozoan membrane. Understanding this mechanism can provide insights into the development of new parasite-control strategies.

Binding of warfarin differently affects the thermal behavior and chain packing of anionic, zwitterionic and cationic lipid membranes

Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·104 and 2.82·104 M−1, respectively) and lower for DOTAP (0.46·104 M−1). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types.

Supramolecular organization of α-galactosylceramide in pure dispersions and in cationic DODAB bilayers

α-galactosylceramide (α-GalCer; KRN7000) strongly stimulates NKT cells. The structures of α-GalCer assemblies and of cationic DODAB bilayers containing α-GalCer were investigated by differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy. Assemblies of α-GalCer have a very tightly packed gel phase, causing spin labels to cluster and display spin exchange interactions. An endothermic phase transition is observed by DSC, leading to a fluid phase. This phase transition peak disappears upon mixing with DODAB, showing that up to 9 mol% α-GalCer is miscible with the cationic lipid. ESR spectra show that α-GalCer decreases DODAB gel phase packing, resulting in a decrease of gel-fluid transition temperature and cooperativity in DSC thermograms of mixed bilayers. In contrast, α-GalCer increases the rigidity of the fluid phase. These effects are probably due to the conformation of the rigid amide bond that connects the phytosphingosine base of α-GalCer to its long and saturated acyl chain. Possibly, α-GalCer adopts a V-shaped conformation because of the perpendicular orientation of the amide bond towards the axes of the hydrocarbon chains. Apparently, the effect of the amide bond configuration is a key structural feature for the interaction between ceramide-based glycolipids and DODAB molecules, since we have previously reported a similar decrease of gel phase packing and increase in fluid phase rigidity for DODAB bilayers containing C24:1β-glucosylceramide. Since the structure of delivery systems is critical to the biological activity of α-GalCer, this work certainly contributes to the planning and development of novel immunotherapeutic tools.