Comparison of bulk and microfluidic methods to monitor the phase behaviour of nanoparticles during digestion of lipid-based drug formulations using in situ X-ray scattering.

The performance of orally administered lipid-based drug formulations is crucially dependent on digestion, and understanding the colloidal structures formed during digestion is necessary for rational formulation design. Previous studies using the established bulk pH-stat approach (Hong et al. 2015), coupled to synchrotron small angle X-ray scattering (SAXS), have begun to shed light on this subject. Such studies of digestion using in situ SAXS measurements are complex and have limitations regarding the resolution of intermediate structures. Using a microfluidic device, the digestion of lipid systems may be monitored with far better control over the mixing of the components and the application of enzyme, thereby elucidating a finer understanding of the structural progression of these lipid systems. This work compares a simple T-junction microcapillary device and a custom-built microfluidic chip featuring hydrodynamic flow focusing, with an equivalent experiment with the full scale pH-stat approach. Both microfluidic devices were found to be suitable for in situ SAXS measurements in tracking the kinetics with improved time and signal sensitivity compared to other microfluidic devices studying similar lipid-based systems, and producing more consistent and controllable structural transformations. Particle sizing of the nanoparticles produced in the microfluidic devices were more consistent than the pH-stat approach.

Phospholipid-based self-assembled mesophase systems for light-activated drug delivery.

The manipulation of the structure of phospholipid-based mesophases to induce a slow to fast drug release profile has potential for use in therapeutic situations where continuous absorption of drug is not desirable and reduce the frequency of injection for short acting or rapidly cleared drugs in treatments for diseases such as macular degeneration. This study had two aims; firstly to confirm the phase behaviour of 20 mol% cholesterol in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), which was previously reported to transition from lamellar (slow release) to bicontinuous cubic (fast release) phase with increasing temperature. Contrary to the literature, no bicontinuous cubic phase was observed but a transition to the inverse hexagonal phase occurred at all POPE : cholesterol ratios investigated. The second aim was to render these mesophases responsive to near-infrared laser (NIR) irradiation by incorporation of gold nanorods (GNR) incorporated into the POPE system to induce photothermal heating. The inclusion of 3 nM GNR in POPE systems induced reversible disruption of lipid packing equivalent to increasing the temperature to 55 °C when irradiated for 30 s. This study confirmed that although the previously published phase behavior was not correct, GNR and NIR can be used to manipulate the self-assembled mesophases in phospholipid-based systems and highlights the potential for a phospholipid-based light-activated drug delivery system.

Magnetically-stimulated transformations in the nanostructure of PEGylated phytantriol-based nanoparticles for on-demand drug release.

Lipid-based liquid crystalline (LLC) systems are formed by the self-assembly of lipid materials in aqueous environments. The internal nanostructures of LLC systems can be manipulated using remote stimuli and have the potential to serve as 'on-demand' drug delivery systems. In this study, a magnetically-responsive system that displayed a transition in nanostructure from liposomes to cubosomes/hexasomes under external alternating magnetic field (AMF) was established by the incorporation of iron oxide nanoparticles (IONPs) into a PEGylated phytantriol (PHYT)-based LLC system. Small angle X-ray scattering (SAXS) was utilized to assess the equilibrium phase behaviour of the systems with different compositions of the lipids to find the optimized formulation. Time-resolved SAXS was then used to determine the dynamic transformation of nanostructures of the IONP-containing systems with the activation of AMF. The formulation containing PHYT and DSPE-PEG2000 at a 95 to 5 molar percent ratio produced a transition from lamellar phase to bicontinuous cubic phase, showing a slow-to-fast drug release profile. Inclusion of either 5 nm or 15 nm IONPs imparted magnetic-responsiveness to the system. The magnetically-responsive system produced an 'on-demand' drug delivery system from which the drug release was able to be triggered externally by AMF-stimulation.

Exposure of liposomes containing nanocrystallised ciprofloxacin to digestive media induces solid-state transformation and altered in vitro drug release.

A recently reported approach to nanocrystallise encapsulated ciprofloxacin within liposomes has generated increased interest in the solid-state properties of drug nanocrystals within liposomal confinement. To explore the potential application of nanocrystallised drug liposomes in oral delivery, a liposomal ciprofloxacin formulation was used as a model system. An in vitro digestion model coupled to small angle X-ray scattering was used to analyse the solid-state properties of the drug nanocrystals during digestion of the liposomal ciprofloxacin nanocrystal formulations. Results showed a complete polymorphic transformation of the ciprofloxacin hydrate nanocrystals to a new salt form at a threshold sodium taurodeoxycholate to ciprofloxacin molar ratio of 0.6. The in vitro drug release from the nanocrystallised drug containing liposomes showed controlled drug release behaviour under non-digestive conditions, while a 3.5-fold increase in the drug release was seen when they were exposed to the simulated digestive environment. In conclusion, the solid state of the drug inside the liposomes is important in dictating the drug release behaviour from the liposomes. The identification of the solid state transformation during digestion in real time and the bile salt-induced polymorphic transformation of ciprofloxacin from nanocrystallised ciprofloxacin liposome are important to understand how the drug is released in vivo, as well as for future formulation design.

Coupling in vitro cell culture with synchrotron SAXS to understand the bio-interaction of lipid-based liquid crystalline nanoparticles with vascular endothelial cells.

Nonlamellar lipid-based liquid crystalline (LLC) nanoparticles possessing different internal nanostructures, specifically the 3D-ordered cubosomes (V2 phase) and the 2D-ordered hexosomes (H2 phase), are of increasing interest as drug delivery systems. To facilitate their development, it is important that we understand their interactions with healthy human umbilical vein endothelial cells (HUVECs). To this end, a 3D cells-in-a-tube model that recapitulates the basic morphology (i.e. tubular lumen) and in vivo microenvironment (i.e. physiological shear stress) of blood vessels was employed as a biomimetic testing platform, and the bio-nanoparticle interactions were compared with that of the conventional 2D planar cell culture. Confocal microscopy imaging revealed internalisation of the nanoparticles into HUVECs within 2 h and that the nanoparticle-cell interactions of cubosomes and hexosomes were not significantly different from one another. Low fluid shear stress conditions (i.e. venous simulation at 0.8 dynes/cm2) were shown to impose subtle effects on the degree of nanoparticle-cell interactions as compared with the static 2D culture. The unexpected similarity of cellular interactions between cubosomes and hexosomes was clarified via a real-time phase behaviour analysis using the synchrotron-based small-angle X-ray scattering (SAXS) technique. When the nanoparticles came into contact with HUVECs under circulating conditions, the cubosomes gradually evolved into hexosomes (within 16 min). In contrast, the hexosomes retained their original internal structure with minimal changes to the lattice parameters. This study highlights the need to couple cellular studies with high-resolution analytics such as time-resolved SAXS analysis to ensure that particle structures are verified in situ, enabling accurate interpretation of the dynamics of cellular interactions and potential bio-induced changes of particles intended for biomedical applications. Graphical abstract.

Probing cell-nanoparticle (cubosome) interactions at the endothelial interface: do tissue dimension and flow matter?

In the research field of nanostructured systems for biomedical applications, increasing attention has been paid to using biomimetic, dynamic cellular models to adequately predict their bio-nano behaviours. This work specifically evaluates the biointeractions of nanostructured lipid-based particles (cubosomes) with human vascular cells from the aspects of tissue dimension (conventional 2D well plate versus 3D dynamic tubular vasculature) and shear flow effect (static, venous and arterial flow-mimicking conditions). A glass capillary-hosted, 3D tubular endothelial construct was coupled with circulating luminal fluid flow to simulate the human vascular systems. In the absence of fluid flow, the degree of cell-cubosome association was not significantly different between the 2D planar and the 3D tubular systems. Under flow conditions simulating venous (0.8 dynes per cm2) and arterial (10 dynes per cm2) shear stresses, the cell-cubosome association notably declined by 50% and 98%, respectively. This highlights the significance of shear-guided biointeractions of non-targeted nanoparticles in the circulation. Across all 2D and 3D cellular models with and without flow, cubosomes had little effect on the cell-cell contact based on the unchanged immunoexpression of the endothelial-specific intercellular junction marker PECAM-1. Interestingly, there were dissimilar nanoparticle distribution patterns between the 2D planar (showing discrete punctate staining) and the 3D tubular endothelium (with a more diffused, patchy fashion). Taken together, these findings highlight the importance of tissue dimension and shear flow in governing the magnitude and feature of cell-nanoparticle interactions.

Controlling the size and shape of liposomal ciprofloxacin nanocrystals by varying the lipid bilayer composition and drug to lipid ratio.

Drug nanocrystals precipitated inside liposomes are of increasing interest in liposomal drug delivery. For liposomal nanocrystal formulations, the size and shape of the drug nanocrystals can influence the apparent drug release properties, providing opportunities for developing tailored liposomal drug release systems. Small angle X-ray scattering (SAXS) and quantitative transmission electron microscopy (TEM) can be used to analyse the size distributions of the nanoparticles. In this study, by changing the fluidity of the membrane through the use of different membrane phospholipids with varying cholesterol content, the impact of lipid phase, fluidity and permeability on the size distribution of ciprofloxacin nanocrystals were investigated using standard TEM and SAXS as orthogonal techniques. The results show that the phospholipid phase behaviour has a direct effect on the nanocrystal size distribution, where shorter and thinner nanocrystals were formed in liposomes made from hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids with higher phase transition temperatures than 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with lower transition temperatures. This is mainly due to the phase behaviour of the liposome during nanocrystal formation. The addition of cholesterol that reduces fluidity and permeability of the DOPC liposomes was also shown to restrict the growth of the ciprofloxacin nanocrystals. Moreover, increasing the drug loading of the liposomes made from HSPC and DPPC produced longer and wider nanocrystals. The findings open new opportunities to tailor nanocrystal size distributions, as well as the aspect ratio of the enclosing liposomes with potential to alter drug release and in vivo behaviour.

PEGylation and surface functionalization of liposomes containing drug nanocrystals for cell-targeted delivery.

Liposomal formulations have important therapeutic applications in anti-cancer treatments but current formulations suffer from serious side effects, high dosage requirements and prolonged treatment. In this study, PEGylated azide-functionalized liposomes containing drug nanocrystals were investigated with the aim of increasing the drug payload and achieving functionalization for targeted delivery. Liposomes were characterized using cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small and ultra-small angle neutron scattering (SANS/USANS) and small and wide angle X-ray scattering (SAXS/WAXS). Cryo-TEM experiments revealed the dimensions of the nanocrystal-loaded liposomes and the change of shape from spherical to elongated after the formation of nanocrystals. Results from SANS/USANS experiments confirmed the asymmetric particle shape. SAXS/WAXS experiments confirmed that the crystalline drug only occurred in freeze-thawed samples and correlated with a new unidentified polymorphic form of ciprofloxacin. Using a small molecule dye, dibenzocyclooctyne (DBCO)-cy5, specific conjugation between DBCO groups and surface azide groups on the liposomes was confirmed; this indicates the promise of this system for tumour-targeted delivery.

Bulk and dispersed aqueous behaviour of an endogenous lipid, selachyl alcohol: Effect of Tween 80 and Pluronic F127 on nanostructure.

Tween 80 has been reported to provide a means of targeting drug nanocarriers to the blood- brain barrier. This study investigated the influence of addition of Tween 80 on the formation of different bulk and dispersed lyotropic liquid crystalline phases in selachyl alcohol-based systems. The effect of increasing concentrations of Tween 80 and Pluronic F127 (as a control) (0-25% w/w relative to SA) on the bulk phase behaviour and dispersions of selachyl alcohol (SA) were investigated using small angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy. The addition of Tween 80 to SA bulk phase samples triggered concentration-dependent phase changes with the structure sequentially evolving from a reverse hexagonal phase (H2) to a mixed H2 and inverse bicontinuous cubic (V2) then a V2 phase alone. In contrast, the addition of Pluronic F127 resulted in a phase change from H2 phase to a mixed lamellar and H2 phase system. The mean particle size of internally structured particles was 125-190 nm with low polydispersity indices (0.1-0.2). Nanoparticles retained the bulk phase internal structure in the presence of Tween 80, whereas in the presence of Pluronic F127, the additional lamellar phase that formed in bulk phase systems was not observed. Cryo-TEM revealed the formation of cubosomes and hexosomes by SA in excess water in the presence of Tween 80 and Pluronic F127 respectively. In summary, it was shown that stabilisation of SA dispersions using Tween 80 resulted in a decrease in negative curvature leading to a change in internal structure from H2 to V2 phase. The studies provide the core understanding of particle structure to progress these structured lipid nanocarriers into delivery studies with Tween 80 as a mechanism to target the blood-brain barrier.

Solubilisation behaviour of poorly water-soluble drugs during digestion of solid SMEDDS.

Lipid based-formulations can enhance the bioavailability of poorly water-soluble lipophilic drugs through enhanced solubilisation of drugs in the gastrointestinal (GI) tract during digestion. This study investigates the solubilisation behaviour of poorly water-soluble drugs upon digestion of solid self-microemulsifying drug delivery system (S-SMEDDS). The S-SMEDDS were prepared using two different core lipids, Gelucire® 44/14 (GEL) or glyceryl monooleate (GMO), and were loaded with two model drugs, fenofibrate (FEN) and cinnarizine (CINN). S-SMEDDS formulations were characterized using wide-angle X-ray scattering (WAXS) and Raman spectroscopy, and their structural behaviour and drug solubilisation behaviour were monitored using drug-related diffraction peaks during digestion under fasted and fed simulated intestinal conditions using time-resolved small and wide-angle X-ray scattering (SAXS/WAXS). The concentrations of FEN and CINN released into the aqueous phase (AP) during digestion were quantified using high-performance liquid chromatography (HPLC). Both model drugs, FEN and CINN, had greater solubility in the GMO-based S-SMEDDS formulations and were partially solubilised into lipid matrix and uniformly distributed in solid formulations. The extent of digestion was greater for the GEL-based systems (92-94%) than GMO-based systems (65-75%) as was the rate of digestion. GEL-based S-SMEDDS formulations formed a lamellar phase during digestion in the fasted state and formed mixed micelles in the fed state. In contrast, the GMO-based system formed the mixed micelles in both intestinal conditions. The time-resolved SAXS profiles revealed solubilisation of crystalline drugs into the lipolysis products. Synchrotron SAXS results were in correlation with the HPLC measurements, confirming the ability of the SAXS technique to monitor drug behaviour and showing that the digestion of S-SMEDDS can enhance drug solubilisation.

Immobilised lipase for in vitro lipolysis experiments.

In vitro lipolysis experiments are used to assess digestion of lipid-based formulations, and probe solubilisation by colloidal phases during digestion. However, proteins and other biological components in the pancreatin often used as the lipase result in high-background scattering when interrogating structures using scattering approaches, complicating the resolution of colloidal structures. In this study, to circumvent this problem, a modified in vitro digestion model employing lipase immobilised on polymer beads, which allows for separation of the lipid digestion components during lipolysis, was investigated. Titration of the fatty acids released during digestion of medium chain triglycerides using pancreatin compared with immobilised lipase, combined with HPLC was used to follow the digestion, and small-angle X-ray scattering was used to determine colloidal structure formation. Digestion of medium chain triglycerides at the same nominal activity revealed that for the immobilised lipase, a longer digestion time was required to achieve the same extent of digestion. However, the same structural endpoint was observed, indicating that structure formation was not affected by the choice of lipase used. Lipolysis with immobilised lipase led to the reduction of parasitic scattering, resulting in clearer and more defined scattering from the structures generated by the lipolysis products.