Elucidating the Polymorphism of Xanthone: A Crystallization and Characterization Study.

The aim of this work is to shed light on the polymorphism of xanthones, a class of oxygenated molecules well known for their bioactivity, including antioxidant, anticancer, and anti-inflammatory effects. Understanding the polymorphism of xanthones can enable the design of novel solid products for pharmaceutical, nutraceutical, and agrochemical applications. Prior to this work, two entries accounting for different space groups were deposited for 9-xanthone in the Cambridge Structure Database (CSD): an orthorhombic P212121 and a monoclinic P21 structure solved at room and low temperatures, respectively. However, the very high similarity between these two structures and the lack of clear differences in their physical properties (e.g., thermal behavior) suggested the possibility of the existence of only one crystal structure. In fact, the differences shown in the literature data might be related to the chosen operating parameters, as well as the instrumental resolution of the single-crystal X-ray diffraction experiments. In the work presented here, the ambiguity in the polymorphism of xanthone is investigated using thermal analysis, powder and synchrotron single-crystal XRD, and optical microscopy. Additionally, a workflow for the correct identification of twinned crystal structures, which can be applied to other polymorphic systems, is presented. Such workflow combines the collection of a large data set of high-resolution diffraction patterns using synchrotron radiation with the use of principal component analysis, a dimensionality reduction technique, for a quick and effective identification of phase transitions happening during the data collection. Crystallization experiments were designed to promote the formation of different crystal structures of xanthone that were recrystallized based on past literature and beyond.

From angular to round: in depth interfacial analysis of binary phosphatidylethanolamine mixtures in the inverse hexagonal phase.

Packing stress in the lipidic inverse hexagonal HII phase arises from the necessity of the ideally cylinder-shaped micelles to fill out the hexagonally-shaped Wigner-Seitz unit cell. Thus, hydrocarbon chains stretch towards the corners and compress in the direction of the flat side of the hexagonal unit cell. Additionally, the lipid/water interface deviates from being perfectly circular. To study this packing frustration in greater detail, we have doped 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE) with increasing molar concentrations of 1,2-palmitoyl-sn-phosphatidylethanolamine (DPPE: 0 to 15 mol%). Due to its effectively longer hydrophobic tails, DPPE tends to aggregate in the corner regions of the unit cell, and thus, increases the circularity of the lipid/water interface. From small angle X-ray diffraction (SAXD) we determined electron density maps. Using those, we analysed the size, shape and homogeneity of the lipid/water interface as well as that of the methyl trough region. At 6 and 9 mol% DPPE the nanotubular water core most closely resembles a circle; further to this, in comparison to its neighbouring concentrations, the 9 mol% DPPE sample has the smallest water core area and smallest number of lipids per circumference, best alleviating the packing stress. Finally, a three-water layer model was applied, discerning headgroup, perturbed and free water, demonstrating that the hexagonal phase is most stable in the direction of the flat faces (compression zones) and least stable towards the vertices of the unit cell (decompression zones).

Correction: Planar confined water organisation in lipid bilayer stacks of phosphatidylcholine and phosphatidylethanolamine.

Correction for 'Planar confined water organisation in lipid bilayer stacks of phosphatidylcholine and phosphatidylethanolamine' by Gerome Vancuylenberg et al., Soft Matter, 2023, https://doi.org/10.1039/D3SM00387F.

Planar confined water organisation in lipid bilayer stacks of phosphatidylcholine and phosphatidylethanolamine.

Phospholipid-based liposomes are abundantly studied in biomembrane research and used in numerous medical and biotechnological applications. Despite current extensive knowledge on membrane nanostructure and its mechanical properties under various environmental conditions, there is still a lack of understanding on interfacial lipid-water interactions. In this work, the nature of the confined water layer for L-α-phosphatidylcholine (egg-PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) in the fluid lamellar phase of multilamellar vesicles was investigated. A new model for describing three different water regions is proposed, which have been characterised using a combination of small angle X-ray scattering (SAXS) and densitometry. The three regions concern (i) 'the headgroup water', (ii) 'perturbed water' near the membrane/water interface and (iii) a core layer of 'free water' (unperturbed water). The behaviour of all three layers is discussed as a function of temperature, concerning influences of chain saturation and headgroup type. While the overall water layer and perturbed water layer thickness increase with temperature, the free water layer displays the opposite trend for PCs, and in PEs is completely absent. Furthermore, an estimate of the temperature dependent headgroup orientation is given for both, PCs and PEs. The newly presented structural data deduced from the three-water region model will be useful for future refined molecular dynamics simulations and allow a better theoretical understanding of the attractive van der Waals force between adjacent membranes.

Hyaluronic-Acid-Tagged Cubosomes Deliver Cytotoxics Specifically to CD44-Positive Cancer Cells.

Delivery of chemotherapy drugs specifically to cancer cells raises local drug doses in tumors and therefore kills more cancer cells while reducing side effects in other tissues, thereby improving oncological and quality of life outcomes. Cubosomes, liquid crystalline lipid nanoparticles, are potential vehicles for delivery of chemotherapy drugs, presenting the advantages of biocompatibility, stable encapsulation, and high drug loading of hydrophobic or hydrophilic drugs. However, active targeting of drug-loaded cubosomes to cancer cells, as opposed to passive accumulation, remains relatively underexplored. We formulated and characterized cubosomes loaded with potential cancer drug copper acetylacetonate and functionalized their surfaces using click chemistry coupling with hyaluronic acid (HA), the ligand for the cell surface receptor CD44. CD44 is overexpressed in many cancer types including breast and colorectal. HA-tagged, copper-acetylacetonate-loaded cubosomes have an average hydrodynamic diameter of 152 nm, with an internal nanostructure based on the space group Im3m. These cubosomes were efficiently taken up by two CD44-expressing cancer cell lines (MDA-MB-231 and HT29, representing breast and colon cancer) but not by two CD44-negative cell lines (MCF-7 breast cancer and HEK-293 kidney cells). HA-tagged cubosomes caused significantly more cell death than untargeted cubosomes in the CD44-positive cells, demonstrating the value of the targeting. CD44-negative cells were equally relatively resistant to both, demonstrating the specificity of the targeting. Cell death was characterized as apoptotic. Specific targeting and cell death were evident in both 2D culture and 3D spheroids. We conclude that HA-tagged, copper-acetylacetonate-loaded cubosomes show great potential as an effective therapeutic for selective targeting of CD44-expressing tumors.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Breaking Isolation to Form New Networks: pH-Triggered Changes in Connectivity inside Lipid Nanoparticles.

There is a growing demand to develop smart nanomaterials that are structure-responsive as they have the potential to offer enhanced dose, temporal and spatial control of compounds and chemical processes. The naturally occurring pH gradients found throughout the body make pH an attractive stimulus for guiding the response of a nanocarrier to specific locations or (sub)cellular compartments in the body. Here we have engineered highly sensitive lyotropic liquid crystalline nanoparticles that reversibly respond to changes in pH by altering the connectivity within their structure at physiological temperatures. At pH 7.4, the nanoparticles have an internal structure consisting of discontinuous inverse micellar "aqueous pockets" based on space group Fd3m. When the pH is ≤6, the nanoparticles change from a compartmentalized to an accessible porous internal structure based on a 2D inverse hexagonal phase (plane group p6mm). We validate the internal symmetry of the nanoparticles using small-angle X-ray scattering and cryogenic transmission electron microscopy. The high-resolution electron microscopy images obtained have allowed us for the first time to directly visualize the internal structure of the Fd3m nanoparticles and resolve the two different-sized inverse micelles that make up the structural motif within the Fd3m unit cell, which upon structural analysis reveal excellent agreement with theoretical geometrical models.

Skimmed milk structural dynamics during high hydrostatic pressure processing from in situ SAXS.

Understanding the changes in milk at a nanostructural level during high-pressure (HP) treatment can provide new insights to improve the safety and functionality of dairy products. In this study, modifications of milk nanostructure during HP were studied in situ by small-angle X-ray scattering (SAXS). Skimmed milk was pressurized to 200 or 400 MPa at 25, 40 or 60 °C and held for 5 or 10 min, and the effect of single- and double-HP treatment was also investigated. In most cases, the SAXS patterns of skimmed milk are well fitted with a three-population model: a low-q micellar feature reflecting the overall micelle size (~0.002 Å-1), a small casein cluster contribution at intermediate-q (around 0.01 Å-1) and a high-q (0.08-0.1 Å-1) population of milk protein inhomogeneities. However, at 60 °C a scattering feature of colloidal calcium phosphate (CCP) which is normally only seen with neutron scattering, was observed at 0.035 Å-1. By varying the pressure, temperature, holding and depressurization times, as well as performing cycled pressure treatment, we followed the dynamic structural changes in the skimmed milk protein structure at different length scales, which depending on the processing conditions, were irreversible or reversible within the timescales investigated. Pressure and temperature of the HP process have major effects, not only on size of casein micelles, but also on "protein inhomogeneities" within their internal structure. Under HP, increasing processing time at 200 MPa induced re-association of the micelles, however, the changes in the internal structure were more pressure-dependent than time dependent.

Coupling Phase Behavior of Fatty Acid Containing Membranes to Membrane Bio-Mechanics.

Biological membranes constantly modulate their fluidity for proper functioning of the cell. Modulation of membrane properties via regulation of fatty acid composition has gained a renewed interest owing to its relevance in endocytosis, endoplasmic reticulum membrane homeostasis, and adaptation mechanisms in the deep sea. Endowed with significant degrees of freedom, the presence of free fatty acids can alter the curvature of membranes which in turn can alter the response of curvature sensing proteins, thus defining adaptive ways to reconfigure membranes. Most significantly, recent experiments demonstrated that polyunsaturated lipids facilitate membrane bending and fission by endocytic proteins - the first step in the biogenesis of synaptic vesicles. Despite the vital roles of fatty acids, a systematic study relating the interactions between fatty acids and membrane and the consequent effect on the bio-mechanics of membranes under the influence of fatty acids has been sparse. Of specific interest is the vast disparity in the properties of cis and trans fatty acids, that only differ in the orientation of the double bond and yet have entirely unique and opposing chemical properties. Here we demonstrate a combined X-ray diffraction and membrane fluctuation analysis method to couple the structural properties to the biophysical properties of fatty acid-laden membranes to address current gaps in our understanding. By systematically doping pure dioleoyl phosphatidylcholine (DOPC) membranes with cis fatty acid and trans fatty acid we demonstrate that the presence of fatty acids doesn't always fluidize the membrane. Rather, an intricate balance between the curvature, molecular interactions, as well as the amount of specific fatty acid dictates the fluidity of membranes. Lower concentrations are dominated by the nature of interactions between the phospholipid and the fatty acids. Trans fatty acid increases the rigidity while decreasing the area per lipid similar to the properties depicted by the addition of saturated fatty acids to lipidic membranes. Cis fatty acid however displays the accepted view of having a fluidizing effect at small concentrations. At higher concentrations curvature frustration dominates, leading to increased rigidity irrespective of the type of fatty acid. These results are consistent with theoretical predictions as detailed in the manuscript.

Influence of a pH-sensitive polymer on the structure of monoolein cubosomes.

Cubosomes consist in submicron size particles of lipid bicontinuous cubic phases stabilized by surfactant polymers. They provide an appealing road towards the practical use of lipid cubic phases for pharmaceutical and cosmetic applications, and efforts are currently being made to control the encapsulation and release properties of these colloidal objects. We overcome in this work the lack of sensitivity of monoolein cubosomes to pH conditions by using a pH sensitive polymer designed to strongly interact with the lipid structure at low pH. Our cryo-transmission electron microscope (cryo-TEM) and small-angle X-ray scattering (SAXS) results show that in the presence of the polymer the cubic phase structure is preserved at neutral pH, albeit with a larger cell size. At pH 5.5, in the presence of the polymer, the nanostructure of the cubosome particles is significantly altered, providing a pathway to design pH-responsive cubosomes for applications in drug delivery.

Spontaneous charged lipid transfer between lipid vesicles.

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

Mechanical Characterization of Ultralow Interfacial Tension Oil-in-Water Droplets by Thermal Capillary Wave Analysis in a Microfluidic Device.

Measurements of the ultralow interfacial tension and surfactant film bending rigidity for micron-sized heptane droplets in bis(2-ethylhexyl) sodium sulfosuccinate-NaCl aqueous solutions were performed in a microfluidic device through the analysis of thermally driven droplet interface fluctuations. The Fourier spectrum of the stochastic droplet interface displacement was measured through bright-field video microscopy and a contour analysis technique. The droplet interfacial tension, together with the surfactant film bending rigidity, was obtained by fitting the experimental results to the prediction of a capillary wave model. Compared to existing methods for ultralow interfacial tension measurements, this contactless, nondestructive, all-optical approach has several advantages, such as fast measurement, easy implementation, cost-effectiveness, reduced amount of liquids, and integration into lab-on-a-chip devices.

Electrostatic swelling of bicontinuous cubic lipid phases.

Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by (a) increasing the bilayer stiffness by adding cholesterol and (b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.

X-ray diffraction of lipid model membranes.

In this chapter the use of X-ray diffraction to study the structure of lyotropic phases and lipid model membranes is described. Determination of the phase symmetry and lattice parameters from small-angle X-ray scattering (SAXS), and of the nature of the hydrocarbon chain packing from wide-angle X-ray scattering (WAXS), are discussed. Methods by which the sign of the interfacial curvature of non-lamellar phases may be determined are then presented. Finally, the calculation of electron density profiles from the intensities of the observed Bragg peaks is described, for the lamellar phase and for the inverse hexagonal phase.

Solid state NMR of lipid model membranes.

In this chapter we describe the use of solid state nuclear magnetic spectroscopy to study the structure of lyotropic phases and lipid model membranes and show its ability to probe, site specifically, at a sub-Ångstrom resolution. Here, we demonstrate the immense versatility of the technique and its ability to provide information on the different liquid crystalline phases present. A multinuclear for example (31)P, (1)H, and (13)C approach is able to elucidate both the structure and dynamics over a wide variety of timescales. This coupled with a non-perturbing label (2)H is able to provide information such as the order parameters for a wide variety of different liquid phases.

Self-assembly and applications of ultraconcentrated nanoparticle solutions.

We demonstrate a highly efficient method for concentrating, purifying and separating gold nanoparticles. The method relies on localized density gradients that can be formed at an aqueous | organic phase interface. We show that this method is able to concentrate aqueous gold nanoparticles to the point where confinement leads to variable interparticle separations. Furthermore, the physical properties of the resulting solution are drastically altered when compared to water. For example, densities higher than 4.5 g/cm(3) could be generated without nanoparticle aggregation. As far as we are aware, this is one of the highest reported densities of an aqueous solution at room temperature. Finally, the compositions of the solutions generated are highly dependent on parameters such as particle size and background analyte making this technique highly advantageous for the separation of multimodal NP populations and chemical purification, with 99.5% and >99.9% efficiency, respectively.

A 3-D hexagonal inverse micellar lyotropic phase.

Lipids that are found in cell membranes form a variety of self-assembled phases in the presence of water. Many of these structures are liquid-crystalline with structural motifs mirrored in cells and organelles and can be exploited in the delivery of drugs and genes. We report the discovery of a lyotropic liquid crystalline phase based on a 3-D hexagonal close-packed arrangement of inverse micelles, of space group P6(3)/mmc. This is the first new inverse lyotropic liquid-crystalline phase to be reported for two decades and is the only known lyotropic phase whose structure consists of a close packing of identical inverse micelles.