pH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.

Self-assembled lipid nanoparticles (LNPs), serving as essential nanocarriers in recent COVID-19 mRNA vaccines, provide a stable and versatile platform for delivering a wide range of biological materials. Notably, LNPs with unique inverse mesostructures, such as cubosomes and hexosomes, are recognized as fusogenic nanocarriers in the drug delivery field. This study delves into the physicochemical properties, including size, lyotropic liquid crystalline mesophase, and apparent pKa of LNPs with various lipid components, consisting of two ionizable lipids (ALC-0315 and SM-102) used in commercial COVID-19 mRNA vaccines and a well-known inverse mesophase structure-forming helper lipid, phytantriol (PT). Two partial mesophase diagrams are generated for both ALC-0315/PT LNPs and SM-102/PT LNPs as a function of two factors, ionizable lipid ratio (α, 0-100 mol%) and pH condition (pH 3-11). Furthermore, the impact of different LNP stabilizers (Pluronic F127, Pluronic F108, and Tween 80) on their pH-dependent phase behavior is evaluated. The findings offer insights into the self-assembled mesostructure and ionization state of the studied LNPs with potentially enhanced endosomal escape ability. This research is relevant to developing innovative next-generation LNP systems for delivering various therapeutics.

Bottom-up cubosome synthesis without organic solvents.

HYPOTHESIS: Bottom-up synthesis of cubosomes is more energetically favourable than top-down approaches. However, bottom-up methods often rely on organic solvents such as ethanol as diluents, and lead to concurrent formation of liposomes. We propose using non-toxic diluents such as honey, glycerol and lactic acid for bottom-up cubosome synthesis. EXPERIMENTS: Cubosomes were prepared using solutions of phytantriol in a range of diluents including choline chloride-glycerol, honey, lactic acid, glycerol, and ethanol. These solutions were added dropwise to water containing the stabiliser, poloxamer 407, following an established method of cubosome synthesis. The resulting structures were characterised using small-angle X-ray scattering, DLS and cryo-TEM. FINDINGS: Cubosomes were successfully formed using a range of non-toxic diluents. This demonstrates that harmful organic solvents like ethanol are not required, and that the diluents need not be hydrotropes. Furthermore, unlike ethanol, these other diluents allowed formation of cubosomes without concurrent formation of liposomes. Given the huge potential for cubosomes in drug delivery, this new method offers a potentially useful low-cost, low-toxicity synthesis option.

Cellular uptake of self-assembled phytantriol-based hexosomes is independent of major endocytic machineries.

Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to "phase through" the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.

On the Structure and Transdermal Profile of Liquid Crystals Based on Phytantriol.

BACKGROUND: There has been a growing concern in transdermal drug technology over the past several decades. As a novel transdermal delivery system, Lyotropic liquid crystals (LLC) still face challenges such as drug loading, limited drug permeation and instability of systems. LLC system is so sensitive that a very subtle change in composition may induce a phase transformation or conversion of spatial configuration, and result in a diverse percutaneous delivery subsequently. OBJECTIVE: To find out the effects of hydrophilic and lipophilic components on the structure and transdermal properties of LLCs, hydrophilic sinomenine hydrochloride (SH) and lipophilic cinnamaldehyde (CA) was chosen as a model drug and a skin permeation enhancer, respectively, several formulations were prepared and compared. METHOD: The structure of LLC was evaluated by visual observation, Cross-polarizing light microscopy (CPLM) and Small angle X-ray diffraction (SAXS). The Franz diffusion cell was applied to investigate its skin penetration of SH across the rat skins. Fourier transform infrared spectroscopy (FTIR) was recorded to evaluate the intermolecular interaction between the LC samples and stratum corneum (SC). CONCLUSION: The results showed that a controlled transdermal process might be obtained by adjusting the ratios of different drugs or loading doses when LLCs with dual-components were applied.

Tailoring the internal structure of liquid crystalline nanoparticles responsive to fungal lipases: A potential platform for sustained drug release.

Lipases are key components in the mechanisms underlying the persistence and virulence of infections by fungi, and thus also promising triggers for bioresponsive lipid-based liquid crystalline nanoparticles. We here propose a platform in which only a minor component of the formulation is susceptible to cleavage by lipase and where hydrolysis triggers a controlled phase transition within the nanoparticles that can potentially allow for an extended drug release. The responsive formulations were composed of phytantriol, which was included as a non-cleavable major component and polysorbate 80, which serves both as nanoparticle stabilizer and potential lipase target. To monitor the structural changes resulting from lipase activity with sufficient time resolution, we used synchrotron small angle x-ray scattering. Comparing the effect of the two different lipases used in this work, lipase B from Candida Antarctica, (CALB) and lipase from Rhizomucor miehei (RMML), only CALB induced phase transition from bicontinuous reverse cubic to reverse hexagonal phase within the particles. This phase transition can be attributed to an increasing amount of oleic acid formed on cleavage of the polysorbate 80. However, when also a small amount of a cationic surfactant was included in the formulation, RMML could trigger the corresponding phase transition as well. The difference in activity between the two lipases can tentatively be explained by a difference in their interaction with the nanoparticle surface. Thus, a bioresponsive system for treating fungal infections, with a tunable selectivity for different types of lipases, could be obtained by tuning the composition of the nanoparticle formulation.

Impact of preparation method and variables on the internal structure, morphology, and presence of liposomes in phytantriol-Pluronic(®) F127 cubosomes.

The formation of significant proportions of liposomes during the preparation of dispersed cubic phase particles presents a problem in trying to understanding cubosome behavior with a view to use in applications such as drug delivery. In this study, the variables impacting on liposome formation during cubosome production were interrogated. Bottom-up (BU) and top-down (TD) approaches were employed to prepare submicron sized liquid crystalline dispersions (cubosomes) of phytantriol in water with varying amounts of Pluronic(®)F127 (F127) as a stabilizer. In the BU approach, ethanol was used as a hydrotrope and was later removed using a rotary evaporator, whereas in the TD approach the bulk liquid gel was dispersed using ultrasonication. We aimed at finding the optimum ratio of phytantriol-to-F127 resulting in stable, liposome-free dispersions, whether this depends on the preparation method and the resulting morphology of the particles. The average particle size and zeta potential of the samples were measured using dynamic light scattering (DLS). Cryogenic transmission electron microscopy (Cryo-TEM) images showed a substantial number of liposomes in addition to cubosomes in the dispersion containing 4-1 (w/w) phytantriol-to-F127 prepared by the BU approach compared to very low liposome content with the TD approach. The effects of the amount of F127 in both approaches, amount of ethanol on the BU method and temperature on the TD method were investigated using small-angle X-ray scattering (SAXS). The cubosomes displayed cubic double-diamond (Pn3m) internal structure with a lattice parameter of approximately 6nm. In summary using the TD approach, with 4:1 phytantriol:F127 provided stable cubosome dispersion with minimal liposome co-existence.

Stabilising cubosomes with Tween 80 as a step towards targeting lipid nanocarriers to the blood-brain barrier.

Coating nanoparticles with the surfactant Tween 80 have been previously shown to enhance drug delivery across the blood-brain barrier (BBB). The aim of this study was to investigate whether Tween 80 could be used to stabilise phytantriol-based cubosomes thereby enabling potential application in delivering macromolecular therapeutics to the brain. Cubosome particles with their large internal and external surface area by virtue of their nanostructure are ideal for delivery of macromolecules. Phase behaviour studies were conducted using a combination of optical microscopy and small-angle X-ray scattering (SAXS) and the addition of Tween 80 to mixtures of phytantriol and water resulted in a rich array of lyotropic mesophases. In particular, a large cubic phase region and a two-phase region of readily dispersed cubosomes is observed. Cubosomes with different concentrations of Tween 80 and phytantriol as the liquid crystal forming lipid were prepared using the solvent precursor method and their physical properties were investigated. A combination of dynamic light scattering, cryogenic electron tomography and SAXS shows formation of well-defined cubosomes with a narrow size distribution and the Im3m cubic structure. Collectively, the results confirm that Tween 80 can effectively stabilize phytantriol cubosomes, opening the possibility for future application in drug delivery across the BBB. Moreover, well-defined, homogenous cubosome formulations prepared using the mild solvent precursor dilution method has significant implications for large-scale production of cubosomes, which currently is a major barrier to the application of cubosomes in the clinic.