Charlotte E. Conn

High throughput preparation and characterisation of amphiphilic nanostructured nanoparticulate drug delivery vehicles.

The preparation, characterisation and assessment of drug delivery vehicles is typically a slow and complex process. Here we present a nanostructured nanoparticle system that can be prepared and characterised in a high-throughput fashion. In particular we use phytantriol and Myverol to prepare inverse bicontinuous cubic and inverse hexagonal liquid crystalline nanoparticles loaded with 10 commonly used therapeutic agents at increasing concentration. The dispersions are prepared using automated apparatus to create different concentrations and phases using novel protocols. We are able to characterise each stabilised nanoparticle dispersion using a range of methodologies including small angle X-ray scattering, particle sizing and drug partitioning. With this information we are able to assess which drug delivery vehicle is preferred for each drug and at which concentration the drug should be loaded to ensure maximum payload and to retain particle integrity.

Nanostructured bicontinuous cubic lipid self-assembly materials as matrices for protein encapsulation

We review the use of bicontinuous cubic amphiphile self-assembly materials for protein encapsulation. The unique amphiphilic nature of bicontinuous cubic lipidic phases make them an ideal medium for the encapsulation of soluble, peripheral, and integral membrane proteins. Current and prospective applications for such systems include drug delivery, membrane protein crystallisation, biosensors and biofuel cells. We focus on the impact of the incorporated protein on the structure of the cubic phase, which has a knock-on effect on performance for many end-applications.

Soft ordered mesoporous materials from nonionic isoprenoid-type monoethanolamide amphiphiles self-assembled in water

The thermal and lyotropic liquid crystalline phase behaviour of a series of amide and monoethanolamide amphiphiles with isoprenoid-type hydrocarbon chains has been investigated. The amphiphilic nature of these molecules combined with their ability to form nanostructured self-assemblies makes them ideal candidates as delivery vehicles of bioactive molecules. For both families of molecules, increased branching in the hydrophobic chain, associated with increasing chain length, results in a decreased melting point. The melting points of the amides are significantly higher than their monoethanolamide homologues. Interestingly, both hexahydrofarnesoyl (H-farnesoyl) and phytanoyl monoethanolamide exhibit a glass transition temperature at around −72 °C to −74 °C. H-Farnesoyl and phytanoyl monoethanolamide form lyotropic liquid crystalline phases in water, whilst H-farnesoyl and phytanoyl amide form a spontaneous emulsion at the amphiphile–water interface. In particular, at room temperature H-farnesoyl and phytanoyl monoethanolamide form the Schwarz diamond (QIID) and the Schoen gyroid (QIIG) bicontinuous cubic phases which are retained down to temperatures as low as 1 °C. Furthermore, phytanoyl monoethanolamide displays a QIID, QIIG and inverse hexagonal phase (HII) at physiological temperature. Both phytanoyl and H-farnesoyl monoethanolamide form mesoporous cubic phases at room temperature that are easily dispersed into cubosomes. The robust nature of the nanostructured phase formation of these two monoethanolamides over a wide range of temperatures makes them ideal candidates for a variety of applications.

Thermotropic and lyotropic liquid crystalline phases of Guerbet branched-chain -D-glucosides

The effect of chain branching on glycolipid thermotropic and lyotropic phases was investigated for a series of synthetic β-D-glucosides derived from Guerbet alcohols, whose total hydrocarbon chain length ranged from C8 to C24. The compounds, which can be viewed as isosteric mimics for glycoglycerolipids, were synthesised in high purity and their liquid crystalline phases were studied using optical polarising microscopy (OPM), and small-angle X-ray diffraction. When dry, the shortest compound (total C8) exhibits a monotropic Lα phase while longer ones (C16 and C20) adopt inverse hexagonal HII phases. The C24 compound forms an ordered lamellar phase at room temperature, but exhibits a metastable HII phase upon cooling. Curiously the intermediate chain length homologue (C12) adopts an isotropic inverse micellar (L2) phase in the dry state over the range of temperatures studied. Upon hydration, the C8 compound dissolves, and the C12 compound forms a fluid lamellar Lα phase. The C16 Guerbet glucoside (i.e. β-Glc-C10C6) exhibits an inverse bicontinuous cubic phase of space group Ia3d in excess water, never previously observed in branched-chain lipids, and very seldom observed in excess water. The C20 compound remains in the HII phase upon hydrating, with the lattice parameter swelling substantially.

High-Throughput Production and Structural Characterization of Libraries of Self-Assembly Lipidic Cubic Phase Materials

A protocol is presented for the high-throughput (HT) production of lyotropic liquid crystalline phases from libraries of lipids and lipid mixtures using standard liquid dispensing robotics, implementing methods that circumvent the problems traditionally associated with handling the highly viscous cubic phase. In addition, the ability to structurally characterize lipidic phases and assess functionality for membrane proteins contained within cubic phases, in a HT manner, is demonstrated. The techniques are combined and exemplified using the application of membrane protein crystallization within lipidic cubic phases.

Enhanced uptake of an integral membrane protein, the dopamine D2L receptor, by cubic nanostructured lipid nanoparticles doped with Ni(II) chelated EDTA amphiphiles

Intrinsic difficulties in characterizing the structure of combined membrane protein–lyotropic liquid crystalline lipidic cubic systems have hampered the development of techniques such as membrane protein (MP) crystallization, which remain largely empirical with consequently low success rates. Here we have incorporated an integral membrane protein and important neurological drug target, the dopamine D2L receptor, within nanostructured nanoparticles of lipidic cubic phase, known as Cubosomes. We show that MPs are incorporated within Cubosomes and that they exert a structural effect which is qualitatively similar to that seen in bulk cubic phase for some systems, exemplifying the potential of Cubosomes to characterize MP incorporation. In addition we have shown, for Cubosomes doped with Ni(II) chelated EDTA amphiphiles, that the strong affinity interaction between the bio-engineered histidine(His)-tag on the protein and the Ni(II) chelated EDTA headgroup of the doped amphiphile leads to enhanced interaction between the membrane protein and the nanostructured cubic nanoparticle. This indicates that protein loading within a cubic phase can be increased as required either to facilitate crystal growth within cubic mesophases or for drug loading. In addition it exemplifies the potential of Cubosome nanostructured nanoparticles to be targeted to specific sites in the body.

High-throughput analysis of the structural evolution of the monoolein cubic phase in situ under crystallogenesis conditions

We have tracked the structural evolution of the monoolein bicontinuous cubic phase under in meso crystallogenesis conditions. Significantly, all measurements have been carried out in situ within a crystallisation plate reproducing the exact conditions during a crystallisation trial. The structure of the MO cubic phase, doped with a small concentration of amyloid-beta peptide, was measured 1 day, 5 days, 7 days and 21 days after addition of PACT crystallisation screen, which systematically varies cation, anion, pH and polyethylene glycol (PEG). The components of the screen had a significant impact on the structure of the cubic phase. We have rationalised the structural variation with respect to the effect of the individual screen components. Specifically addition of higher Mw PEG effected a transition from a diamond cubic phase (QIID) to a gyroid cubic phase (QIIG) across the majority of the plate and the QIIG phase became more prevalent with time. The effect of individual salts was correlated with their position in the Hofmeister series. Changes in pH and buffer system had a more minor effect on mesophase structure. We have discussed the implications of the observed structural changes with respect to the putative mechanism of protein crystal growth within a lipidic cubic phase.

Investigation of the Effect of Sugar Stereochemistry on Biologically Relevant Lyotropic Phases from Branched-Chain Synthetic Glycolipids by Small-Angle X-ray Scattering

Synthetic branched-chain glycolipids are suitable as model systems in understanding biological cell membranes, particularly because certain natural lipids possess chain branching. Herein, four branched-chain glycopyranosides, namely, 2-hexyl-decyl-α-D-glucopyranoside (α-Glc-OC10C6), 2-hexyl-decyl-β-D-glucopyranoside (β-Glc-OC10C6), 2-hexyl-decyl-α-D-galactopyranoside (α-Gal-OC10C6), and 2-hexyl-decyl-β-D-galactopyranoside (β-Gal-OC10C6), with a total alkyl chain length of 16 carbon atoms have been synthesized, and their phase behavior has been studied. The partial binary phase diagrams of these nonionic surfactants in water were investigated by optical polarizing microscopy (OPM) and small-angle X-ray scattering (SAXS). The introduction of chain branching in the hydrocarbon chain region is shown to result in the formation of inverse structures such as inverse hexagonal and inverse bicontinuous cubic phases. A comparison of the four compounds showed that they exhibited different polymorphism, especially in the thermotropic state, as a result of contributions from anomeric and epimeric effects according to their stereochemistry. The neat α-Glc-OC10C6 compound exhibited a lamellar (Lα) phase whereas dry α-Gal-OC10C6 formed an inverse bicontinuous cubic Ia3d (QII(G)) phase. Both β-anomers of glucoside and galactoside adopted the inverse hexagonal phase (HII) in the dry state. Generally, in the presence of water, all four glycolipids formed inverse bicontinuous cubic Ia3d (QII(G)) and Pn3m (QII(D)) phases over wide temperature and concentration ranges. The formation of inverse nonlamellar phases by these Guerbet branched-chain glycosides confirms their potential as materials for novel biotechnological applications such as drug delivery and crystallization of membrane proteins.

Lamellar crystalline self-assembly behaviour and solid lipid nanoparticles of a palmityl prodrug analogue of Capecitabine--a chemotherapy agent.

An amphiphile prodrug, 5-deoxy-5-fluoro-N(4)-(palmityloxycarbonyl) cytidine or 5-deoxy-5-fluoro-N(4)-(hexadecanaloxycarbonyl) cytidine (5-FCPal), consisting of the same head group as the commercially available chemotherapeutic agent Capecitabine, linked to a palmityl hydrocarbon chain via a carbamate bond is reported. Thermal analysis of this prodrug indicates that it melts at ∼115 °C followed quickly by degradation beginning at ∼120 °C. The neat solid 5-FCPal amphiphile acquires a lamellar crystalline arrangement with a d-spacing of 28.6±0.3 Å, indicating interdigitation of the hydrocarbon chains. Under aqueous conditions, solid 5-FCPal is non-swelling and no lyotropic liquid crystalline phase formation is observed. In order to assess the in vitro toxicity and in vivo efficacy in colloidal form, solid lipid nanoparticles (SLNs) with an average size of ∼700 nm were produced via high pressure homogenization. The in vitro toxicity of the 5-FCPal SLNs against several different cancer and normal cell types was assessed over a 48 h period, and IC(50) values were comparable to those observed for Capecitabine. The in vivo efficacy of the 5-FCPal SLNs was then assessed against the highly aggressive mouse 4T1 breast cancer model. To do so, the prodrug SLNs were administered orally at 3 different dosages (0.1, 0.25, 0.5 mmol/mouse/day) and compared to Capecitabine delivered at the same dosages. After 21 days of receiving the treatments, the 0.5 mmol dose of 5-FCPal exhibited the smallest average tumour volume. Since 5-FCPal is activated in a similar manner to Capecitabine via a 3 step enzymatic pathway with the final step occurring preferentially at the tumour site, formulation of the prodrug into SLNs combines the advantage of selective, localized activation with the sustained release properties of nanostructured amphiphile self-assembly and multiple payload materials thereby potentially creating a more effective anticancer agent.

Effect of lipid architecture on cubic phase susceptibility to crystallisation screens

The proposed mechanism for in meso crystallisation depends, at least initially, on retention of the underlying cubic phase. However, a crystallisation trial requires screening across a wide range of crystallisation conditions, containing polymers, salts, buffers and at varying pH, all of which are known to drive structural changes in lipid phases. We have previously shown that the lipid monoolein (MO) is relatively robust to the components of the PACT crystallization screen. Here we extend our research to determine the susceptibility of the 3-D ordered cubic phase formed by four different lipids; monoolein, phytantriol, phytanoyl monoethanolamide and H-farnesoyl monoethanolamide, to two different crystallisation screens (the PACT and PEG-ion screens) in situ, within a 96-well crystallisation plate. Addition of screen is shown to result in rich and varied phase behaviour with the transformation to 1-D ordered lamellar, 2-D ordered hexagonal and disordered micellar phases in many wells. We have rationalized the structural changes for each lipid by a consideration of the osmotic stress exerted by the PEG components, and the position of various anions and cations present in the Hofmeister series. The nanostructure of the cubic phase is shown to be the most important parameter affecting the susceptibility of the cubic phase structure to the components of the screen. In particular, a reduction in lipid bilayer thickness and water channel diameter increases the susceptibility.