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In vitro and in vivo tests for therapeutic agents are typically conducted in sterile environments, but many target areas for drug delivery are home to thousands of microbial species. Here, we examine the behaviour of lipidic nanomaterials after exposure to representative strains of four bacterial species found in the gastrointestinal tract and skin. Small angle X-ray scattering measurements show that the nanostructure of monoolein cubic and inverse hexagonal phases are transformed, respectively, into inverse hexagonal and inverse micellar cubic phases upon exposure to a strain of live Staphylococcus aureus often present on skin and mucosa. Further investigation demonstrates that enzymatic hydrolysis and cell membrane lipid transfer are both likely responsible for this effect. The structural responses to S. aureus are rapid and significantly reduce the rate of drug release from monoolein-based nanomaterials. These findings are the first to demonstrate how a key species in the live human microbiome can trigger changes in the structure and drug release properties of lipidic nanomaterials. The effect appears to be strain specific, varies from patient to patient and body region to body region, and is anticipated to affect the bioapplication of monoglyceride-based formulations., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wye-Khay Fong reports financial support was provided by Noxopharm Limited. Jonathan Caukwell reports financial support was provided by Australian Nuclear Science and Technology Organisation. Salvatore Assenza reports financial support was provided by LaCaixa Foundation. Salvatore Assenza reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. Salvatore Assenza reports financial support was provided by Spanish Scientific Research Council. Livia Salvati Manni reports financial support was provided by Swiss National Science Foundation. Karl Hassan, Brett Neilan reports financial support was provided by Australian Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polymer-homopolypeptide block copolymers are a class of bioinspired materials that combine the processability and stability of synthetic polymers with the biocompatibility and unique secondary structures of peptides, such as α-helices and β-sheets. These properties make them ideal candidates for a wide variety of applications, for example, in the pharmaceutical field, where they are frequently explored as building blocks for polymeric micelle drug delivery systems. While homopolypeptide side chains can be furnished with an array of different moieties to impart the copolymers with desirable properties, such as stimulus responsivity, pyridine derivatives represent an underutilized functional group for this purpose. Additionally, the interplay between polypeptide side chain structure, secondary conformation, and micelle morphology is not yet well understood, particularly in the case of structural regioisomers. Therefore, in this work, a series of polymer-homopolypeptide copolymers were prepared from a poly(ethylene glycol)- b -poly(glutamic acid) (PEG- b -PGA) backbone, where the pendant carboxylic acid groups were covalently conjugated to a series of pyridine regioisomers by carbodiimide coupling. These pyridine regioisomers differed only in the position of the nitrogen heteroatom, ortho, meta or para, relative to the linking group, generating a series of PEG- b -poly(pyridinylmethyl glutamate) (PEG- b -PMG) copolymers. Following self-assembly of the copolymers in aqueous solutions, dynamic light scattering (DLS) revealed differences in micelle hydrodynamic diameter ( D h ) (ranging from ∼60 to 120 nm), while transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) revealed distinctive morphologies ranging from ellipsoidal, to cylindrical, and disc-like, suggesting that subtle changes in positional isomers in the polypeptide block may influence the micelle structure. Analysis of the PEG- b -PMG copolymer micelles by circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that differences in the morphology were associated with changes in polypeptide secondary structure, which in turn was influenced by the position of the pyridine heteroatom. Overall, these findings contribute to the broader understanding of the relationship between polypeptide structure and micelle morphology and serve as useful insight for the rational design of polymer-polypeptide nanoparticles.

This review focuses on the current understanding regarding lipid crystallisation at oil-water interfaces. The main aspects of crystallisation in bulk lipids will be introduced, allowing for a more comprehensive overview of the crystallisation processes within emulsions. Additionally, the properties of an emulsion and the impact of lipid crystallisation on emulsion stability will be discussed. The effect of different emulsifiers on lipid crystallisation at oil-water interfaces will also be reviewed, however, this will be limited to their impact on the interfacial crystallisation of monoglycerides and diglycerides. The final part of the review highlights the recent methodologies used to study crystallisation at oil-water interfaces., Competing Interests: Declaration of competing interest Marta Krasowska reports financial support was provided by Australian Research Council. David A. Beattie reports financial support was provided by Australian Research Council. Graeme Gillies reports financial support was provided by Australian Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Lipid-based lyotropic liquid crystalline nanoparticles (LCNPs) face stability challenges in biological fluids during clinical translation. Ionic Liquids (ILs) have emerged as effective solvent additives for tuning the structure of LCNP's and enhancing their stability. We investigated the effect of a library of 21 choline-based biocompatible ILs with 9 amino acid anions as well as 10 other organic/inorganic anions during the preparation of phytantriol (PHY)-based LCNPs, followed by incubation in human serum and serum proteins. Small angle X-ray scattering (SAXS) results show that the phase behaviour of the LCNPs depends on the IL concentration and anion structure. Incubation with human serum led to a phase transition from the inverse bicontinuous cubic (Q 2 ) to the inverse hexagonal (H 2 ) mesophase, influenced by the specific IL present. Liquid chromatography-mass spectrometry (LC-MS) and proteomics analysis of selected samples, including PHY control and those with choline glutamate, choline hexanoate, and choline geranate, identified abundant proteins in the protein corona, including albumin, apolipoproteins, and serotransferrin. The composition of the protein corona varied among samples, shedding light on the intricate interplay between ILs, internal structure and surface chemistry of LCNPs, and biological fluids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polymeric micelles have been extensively studied as vectors for the delivery of hydrophobic drugs for the treatment of cancers and other diseases. Despite intensive research, few formulations provide significant benefits, and even fewer have been clinically approved. While many traditional non-responsive micelles have excellent safety profiles, they lack the ability to respond to the intracellular environment and release their cargo in a spatiotemporally defined manner to effectively deliver large doses of cytotoxic drugs into the cytosol of cells that overwhelm efflux pumps. As a novel and adaptable strategy, we hypothesized that well-established non-responsive polymeric micelles could be augmented with a pH-trigger via the co-encapsulation of cytocompatible oligoelectrolytes, which would allow rapid cargo release in the endosome, leading to increased cytotoxicity. Herein, we demonstrate how this strategy can be applied to render non-responsive micelles pH-responsive, resulting in abrupt cargo release at specific and tunable pH values compatible with endosomal delivery, which significantly increased their cytotoxicity up to 3-fold in an ovarian adenocarcinoma (SKOV-3) cell line compared to non-responsive micelles. In comparison, the oligoelectrolyte-loaded micelles were significantly less toxic to healthy 3T3 fibroblasts, indicating a selective cargo release in cancer cell lines. Oligoelectrolytes can be co-encapsulated in the micelles along with drugs at high encapsulation efficiency percentages, which are both ejected from the micelle core upon oligoelectrolyte ionization. Mechanistically, the increase in cytotoxicity appears to also result from the accelerated endosomal escape of the cargo caused by disruption of the endosomal membrane by the simultaneous release of the oligoelectrolytes from the micelles. Furthermore, we show how this approach is broadly applicable to non-responsive micelles regardless of their composition and various classes of hydrophobic chemotherapeutics. The preliminary studies presented here reveal the versatility and wide scope of oligoelectrolyte-mediated, pH-triggered drug release as a compelling and powerful strategy to enhance the cytotoxicity of non-responsive polymeric micelles.

Human milk is proposed as a drug delivery vehicle suitable for use in neonatal patients. Clofazimine, traditionally used for the treatment of leprosy and tuberculosis, is emerging as a treatment for cryptosporidiosis in infants, however its poor aqueous solubility has led to its commercial formulation as a waxy lipid formulation in a capsule, a format that is not suitable for infants. In this study, the evaluation of pasteurised human milk for the delivery of clofazimine was investigated using an in vitro lipolysis model to simulate gastric and intestinal digestion. The total lipid composition of the human milk was characterised alongside the liberated fatty acid species following digestion for comparison to alternative lipid-based delivery systems. Small-angle X-ray scattering was used to measure the presence of crystalline clofazimine during digestion and hence the extent of drug solubilisation. High-performance liquid chromatography was used to quantify the mass of clofazimine solubilised per gram of human milk fat (drug-to-fat ratio) in digested and undigested human milk. The digestion process was essential for the solubilisation of clofazimine, with digested human milk solubilising a sufficient dose of clofazimine for treatment of a premature infant. Human milk solubilised the clofazimine to a greater extent than bovine milk and infant formula during digestion, most likely as a result of differing lipid composition and increased long-chain fatty acid concentrations. These findings show that human milk enhances the solubility of clofazimine as a model drug and may be a suitable drug delivery vehicle for infant populations requiring therapeutic treatment., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Colostrum provides bioactive components that are essential for the colonization of microbiota in the infant gut, while preventing infectious diseases such as necrotizing enterocolitis. As colostrum is not always available from the mother, particularly for premature infants, effective and safe substitutes are keenly sought after by neonatologists. The benefits of bioactive factors in colostrum are recognized; however, there have been no accounts of human colostrum being studied during digestion of the lipid components or their self-assembly in gastrointestinal environments. Due to the weaker bile pool in infants than adults, evaluating the lipid composition of human colostrum and linking it to structural self-assembly behavior is important in these settings and thus enabling the formulation of substitutes for colostrum. This study is aimed at the rational design of an appropriate lipid component for a colostrum substitute and determining the ability of this formulation to reduce inflammation in intestinal cells. Gas chromatography was utilized to map lipid composition. The self-assembly of lipid components occurring during digestion of colostrum was monitored using small-angle X-ray scattering for comparison with substitute mixtures containing pure triglyceride lipids based on their abundance in colostrum. The digestion profiles of human colostrum and the substitute mixtures were similar. Subtle differences in lipid self-assembly were evident, with the substitute mixtures exhibiting additional non-lamellar phases, which were not seen for human colostrum. The difference is attributable to the distribution of free fatty acids released during digestion. The biological markers of necrotizing enterocolitis were modulated in cells that were treated with bifidobacteria cultured on colostrum substitute mixtures, compared to those treated with infant formula. These findings provide an insight into a colostrum substitute mixture that resembles human colostrum in terms of composition and structural behavior during digestion and potentially reduces some of the characteristics associated with necrotizing enterocolitis.

Sphingomyelin (SM) and cholesterol complex to form functional liquid-ordered (L o ) domains. It has been suggested that the detergent resistance of these domains plays a key role during gastrointestinal digestion of the milk fat globule membrane (MFGM), which is rich in both SM and cholesterol. Small-angle X-ray scattering was employed to determine the structural alterations that occur when milk sphingomyelin (MSM)/cholesterol, egg sphingomyelin (ESM)/cholesterol, soy phosphatidylcholine (SPC)/cholesterol, and milk fat globule membrane (MFGM) phospholipid/cholesterol model bilayer systems were incubated with bovine bile under physiological conditions. The persistence of diffraction peaks was indicative of multilamellar vesicles of MSM with cholesterol concentrations > 20 % mol, and also for ESM with or without cholesterol. The complexation of ESM with cholesterol is therefore capable of inhibiting the resulting vesicles from disruption by bile at lower cholesterol concentrations than MSM/cholesterol. After subtraction of background scattering by large aggregates in the bile, a Guinier fitting was used to determine changes in the radii of gyration (R g s) over time for the biliary mixed micelles after mixing the vesicle dispersions with bile. Swelling of the micelles by phospholipid solubilization from vesicles was a function of cholesterol concentration, with less swelling of the micelles occurring as the cholesterol concentration was increased. With 40% mol cholesterol, the R g s of the bile micelles mixed with MSM/cholesterol, ESM/cholesterol, and MFGM phospholipid/cholesterol were equal to the control (PIPES buffer + bovine bile), indicating negligible swelling of the biliary mixed micelles., Competing Interests: Competing Interests The authors declare they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polymeric micelles are promising carriers for the delivery of poorly water-soluble drugs, providing enhanced drug solubility, blood circulation times, and bioavailability. Nevertheless, the storage and long-term stability of micelles in solution present challenges requiring the lyophilization and storage of formulations in the solid state, with reconstitution immediately prior to application. Therefore, it is important to understand the effects of lyophilization/reconstitution on micelles, particularly their drug-loaded counterparts. Herein, we investigated the use of β-cyclodextrin (β-CD) as a cryoprotectant for the lyophilization/reconstitution of a library of poly(ethylene glycol- b -ε-caprolactone) (PEG- b -PCL) copolymer micelles and their drug-loaded counterparts, as well as the effect of the physiochemical properties of different drugs (phloretin and gossypol). The critical aggregation concentration (CAC) of the copolymers decreased with increasing weight fraction of the PCL block ( f PCL ), plateauing at ~1 mg/L when the f PCL was >0.45. The blank (empty) and drug-loaded micelles were lyophilized/reconstituted in the absence and presence of β-CD (9% w / w ) and analyzed via dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) to assess for changes in aggregate size (hydrodynamic diameter, D h ) and morphology, respectively. Regardless of the PEG- b -PCL copolymer or the use of β-CD, the blank micelles displayed poor redispersibility (<10% relative to the initial concentration), while the fraction that redispersed displayed similar D h to the as-prepared micelles, increasing in D h as the f PCL of the PEG- b -PCL copolymer increased. While most blank micelles displayed discrete morphologies, the addition of β-CD or lyophilization/reconstitution generally resulted in the formation of poorly defined aggregates. Similar results were also obtained for drug-loaded micelles, with the exception of several that retained their primary morphology following lyophilization/reconstitution, although no obvious trends were noted between the microstructure of the copolymers or the physicochemical properties of the drugs and their successful redispersion.

The development of formulation approaches to coadminister lopinavir and ritonavir antiretroviral drugs to children is necessary to ensure optimal treatment of human immunodeficiency virus (HIV) infection. It was previously shown that milk-based lipid formulations show promise as vehicles to deliver antimalarial drugs by enhancing their solubilization during the digestion of the milk lipids under intestinal conditions. In this study, we investigate the role of digestion of milk and infant formula on the solubilization behavior of lopinavir and ritonavir to understand the fate of drugs in the gastrointestinal (GI) tract after oral administration. Small angle X-ray scattering (SAXS) was used to probe the presence of crystalline drugs in suspension during digestion. In particular, the impact of one drug on the solubilization of the other was elucidated to reveal potential drug-drug interactions in a drug combination therapy. Our results showed that lopinavir and ritonavir affected the solubilization of each other during digestion in lipid-based formulations. While addition of ritonavir to lopinavir improved the overall solubilization of lopinavir, the impact of lopinavir was to reduce ritonavir solubilization as digestion progressed. These findings highlight the importance of assessing the solubilization of individual drugs in a combined matrix in order to dictate the state of drugs available for subsequent absorption and metabolism. Enhancement in the solubilization of lopinavir and ritonavir in a drug combination setting in vitro also supported the potential for food effects on drug exposure.

Hypothesis: Dairy proteins and mono- and diglycerides (MDG) are often used in unison to tailor the properties of dairy-based emulsions. However, there are significant gaps in our understanding of how proteins affect lipid crystallisation at the oil-water interface. We have used a unique combination of interfacially-sensitive techniques to elucidate the impact of dairy proteins on interfacial MDG crystal formation., Experiments: The formation temperature of interfacial MDG crystals was assessed through interfacial tension studies via drop shape analysis. Small and Wide-Angle X-ray Scattering measurements were performed on isolated oil-water interfaces, allowing for in-situ interrogation of MDG crystal structure and concentration at and near the interface., Findings: Dairy proteins are seen to reduce the temperature at which MDG crystals form at the oil-water interface. The displacement of proteins upon interfacial crystal formation was also clearly observed in interfacial tension measurements. For the first time, lipid crystals formed at the oil-water interface have been characterised using X-ray scattering. All scattering studies showed no change to the MDG crystal structures at the oil-water interface in the presence of adsorbed proteins. The results demonstrate that informed selection of emulsifier components is critical to controlling interfacial crystallisation with concomitant impact on emulsion stability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Praziquantel is a poorly water-soluble drug used to treat parasitic infections. Previous studies have suggested that its rate and extent of dissolution in milk and biorelevant media are slow and limited compared to dissolution in the pharmacopoeial-recommended medium, despite being reported as displaying a positive food effect upon administration. This study aimed to revisit the dissolution of praziquantel in biorelevant media and milk to better understand this apparent dichotomy. The context of digestion was introduced to better understand drug solubilisation under more relevant gastrointestinal conditions. The amount of praziquantel solubilised in the various media during digestion was quantified using high performance liquid chromatography (HPLC) and the kinetics of dissolution were confirmed by tracking the disappearance of solid crystalline drug using in situ small angle X-ray scattering (SAXS). For the dissolution media, where sodium lauryl sulfate (SLS) is typically included as a wetting agent, a prominent effect of SLS on drug dissolution was also apparent where >2.5 fold more drug was solubilised in SLS-containing dissolution medium compared to that without (0.1 M HCl only). In milk, significant dissolution of praziquantel was observed only during digestion and not during dispersion, hence suggesting that (1) milk can be potentially administered with praziquantel to improve oral bioavailability and (2) incorporating a digestion step into existing in vitro dissolution testing can better reflect the potential for a positive food effect when lipids are present.

The crystallinity of polymers strongly affects their properties. For block copolymers, whereby two crystallisable blocks are covalently tethered to one another, the molecular weight of the individual blocks and their relative weight fraction are important structural parameters that control their crystallisation. In the case of block copolymer micelles, these parameters can influence the crystallinity of the core, which has implications for drug encapsulation and release. Therefore, in this study, we aimed to determine how the microstructure of poly(ethylene glycol-b-caprolactone) (PEG-b-PCL) copolymers contributes to the crystallinity of their hydrophobic PCL micelle cores. Using a library of PEG-b-PCL copolymers with PEG number-average molecular weight (Mn) values of 2, 5, and 10 kDa and weight fractions of PCL (fPCL) ranging from 0.11 to 0.67, the thermal behaviour and morphology were studied in blends, bulk, and micelles using differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WXRD), and Synchrotron wide-angle X-ray scattering (WAXS). Compared to PEG and PCL homopolymers, the block copolymers displayed reduced crystallinity in the bulk phase and the individual blocks had a large influence on the crystallisation of one another. The fPCL was determined to be the dominant contributor to the extent and order of crystallisation of the two blocks. When fPCL < 0.35, the initial crystallisation of PEG led to an amorphous PCL phase. At fPCL values between 0.35 and 0.65, PEG crystallisation was followed by PCL crystallisation, whereas this behaviour was reversed when fPCL > 0.65. For lyophilised PEG-b-PCL micelles, the crystallinity of the core increased with increasing fPCL, although the core was predominately amorphous for micelles with fPCL < 0.35. These findings contribute to understanding the relationships between copolymer microstructure and micelle core crystallinity that are important for the design and performance of micellar drug delivery systems, and the broader application of polymer micelles.

Hydrogen sulfide (H 2 S) is an important signalling molecule with potential pharmaceutical applications. In pursuit of a suitable delivery system for H 2 S, herein we apply an amphiphilic trisulfide to concomitantly alter the mesophase behaviour of dispersed lipid particles and enable triggered H 2 S release. Amperometric release studies indicate the trisulfide acts as a sustained H 2 S donor, with inclusion into the mesophase attenuating release vs neat dispersed trisulfide. Taken together the results highlight the potential for including trisulfide-based additives in stimuli-responsive drug delivery vehicles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human milk is critical for the survival and development of infants. This source of nutrition contains components that protect against infections while stimulating immune maturation. In cases where the mother's own milk is unavailable, pasteurized donor milk is the preferred option. Although pasteurization has been shown to have minimal impact on the lipid and FA composition before digestion, no correlation has been made between the impact of pasteurization on the FFA composition and the self-assembly of lipids during digestion, which could act as delivery mechanisms for poorly water-soluble components. Pooled nonpasteurized and pasteurized human milk from a single donor was used in this study. The evolving FFA composition during digestion was determined using GC coupled to a flame ionization detector. In vitro digestion coupled to small-angle X-ray scattering was utilized to investigate the influence of different calcium levels, fat content, and the presence of bile salts on the extent of digestion and structural behavior of human milk lipids. Almost complete digestion was achieved when bile salts were added to the systems containing high calcium to milk fat ratio, with similar structural behavior of lipids during digestion of both types of human milk being apparent. In contrast, differences in the colloidal structures were formed during digestion in the absence of bile salt because of a greater amount of FFAs being released from the nonpasteurized than pasteurized milks. This difference in FFAs released from both types of human milk could result in varying nutritional implications for infants., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022. Published by Elsevier Inc.)

Mesoporous silica particles (MSPs) are emerging as an interesting option to reduce calorific uptake as a treatment for obesity and other metabolic conditions. However, their further development under the pharmaceutical regulatory framework is hindered by poor understanding of the mechanisms by which they exert their effects. In the current study the interaction of MSPs with the lipid digestion process is investigated, specifically interactions with lipase enzymes and lipid digestion products as a key contributing factor to lipid absorption and calorific intake. The impact of exposing lipase to MSPs on the enzyme activity was assessed directly using the tributyrin digestion test. The extent of interaction of digestion products with MSPs was studied using selectively radiolabeled bile components and lipids, while the impact on in vivo absorption of lipids was studied by incorporation of radiolabelled lipid (triolein) into milk and administration with and without particles. The studies showed that particles that inhibited lipase activity also tended to interact more extensively with lipid digestion products. In vitro X-ray scattering studies revealed the interaction of some MSPs with lipid digestion products through changes in lipid self-assembly during digestion. The MSPs led to reduced lipid absorption in vivo compared to the control particles and MSP-free milk. While the specific properties of the MSPs that drive the differences between the behavior of MSPs during lipid digestion remain elusive, the studies highlight that interactions with the lipid digestion and absorption pathways are a likely mechanism for reducing calorific uptake., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Dairy emulsions contain an intrinsically heterogeneous lipid phase, whose components undergo crystallisation in a manner that is critical to dairy product formulation, storage, and sensory perception. Further complexity is engendered by the diverse array of interfacially-active molecules naturally present within the serum of dairy systems, and those that are added for specific formulation purposes, all of which interact at the lipid-serum interface and modify the impact of lipid crystals on dairy emulsion stability. The work described in this article addresses this complexity, with a specific focus on the impact of temperature cycling and the effect of emulsifier type on the formation and persistence of lipid crystals at lipid-solution interfaces. Profile analysis tensiometry experiments were performed using single droplets of the low melting fraction of dairy lipids, in the presence and absence of emulsifiers (Tween 80 and whey protein isolate, WPI) and during the temperature cycling, to study the formation of monoacylglycerol (MAG) crystals at the lipid-solution interface. Companion experiments on the same lipid systems, and at the same cooling and heating rates, were undertaken with synchrotron small angle X-ray scattering, to specifically analyse the effect of emulsifier type on the formation of triacylglycerol (TAG) crystals at the lipid-solution interface of a model dairy emulsion. These two complementary techniques have revealed that Tween 80 molecules delay MAG and TAG crystal formation by lowering the temperature at which the crystallisation occurs during two cooling cycles. WPI molecules delay the crystallisation of MAGs and TAGs during the first cooling cycle, while MAG crystals form without delay during the second cooling cycle at the same temperature as MAG crystals in an emulsifier free system. The crystallisation of TAGs is inhibited during the second cooling cycle. The observed differences in crystallisation behaviour at the interface upon temperature cycling can provide further insight into the impact of emulsifiers on the long-term stability of emulsion-based dairy systems during storage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lipid-based formulations improve the absorption capacity of poorly-water-soluble drugs and digestion of the formulation is a critical step in that absorption process. A recent approach to understanding the propensity for drug to dissolve in digesting lipid-based formulations couples an in vitro pH-stat lipolysis model to small-angle X-ray scattering (SAXS) by means of a flow-through capillary. However, the conventional pH-stat apparatus used to measure the extent of lipid digestion during such experiments requires digest volumes of 15-30 mL and drug doses of 50-200 mg, which is problematic for scarce compounds and can require excessive amounts of formulation reagents. This manuscript describes an approach to reduce the amount of material required for in vitro lipolysis experiments coupled to SAXS, for use in instances where the amount of drug or formulation medium is limited. Importantly, this was achieved while maintaining the pH stat conditions, which is critical for maintaining biorelevance and driving digestion to completion. The digestibility of infant formula with the poorly-water-soluble drugs halofantrine and clofazimine dispersed into it was measured as an exemplar paediatric-friendly lipid formulation. Halofantrine was incorporated in its powdered free base form and clofazimine was incorporated both as unformulated drug powder and as drug in nanoparticulate form prepared using Flash NanoPrecipitation. The fraction of triglyceride digested was found to be independent of vessel size and the incorporation of drug. The dissolution of the two forms of clofazimine during the digestion of infant formula were then measured using synchrotron SAXS, which revealed complete and partial solubilisation over 30 min of digestion for the powdered drug and nanoparticle formulations, respectively. The main challenge in reducing the volume of the measurements was in ensuring that thorough mixing was occurring in the smaller digestion vessel to provide uniform sampling of the dispersion medium., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Andrew Clulow reports a relationship with Australian Research Council that includes: funding grants., (© 2022 The Authors.)

Understanding the microstructural parameters of amphiphilic copolymers that control the formation and structure of aggregated colloids (e.g., micelles) is essential for the rational design of hierarchically structured systems for applications in nanomedicine, personal care and food formulations. Although many analytical techniques have been employed to study such systems, in this investigation we adopted an integrated approach using non-interfering techniques - diffusion nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) - to probe the relationship between the microstructure of poly(ethylene glycol-b-caprolactone) (PEG-b-PCL) copolymers [e.g., block molecular weight (MW) and the mass fraction of PCL (f PCL )] and the structure of their aggregates. Systematic trends in the self-assembly behaviour were determined using a large family of well-defined block copolymers with variable PEG and PCL block lengths (number-average molecular weights (M n ) between 2 and 10 and 0.5-15 kDa, respectively) and narrow dispersity (Ð < 1.12). For all of the copolymers, a clear transition in the aggregate structure was observed when the hydrophobic f PCL was increased at a constant PEG block M n , although the nature of this transition is also dependent on the PEG block M n . Copolymers with low M n PEG blocks (2 kDa) were observed to transition from unimers and loosely associated unimers to metastable aggregates and finally, to cylindrical micelles as the f PCL was increased. In comparison, copolymers with PEG block M n of between 5 and 10 kDa transitioned from heterogenous metastable aggregates to cylindrical micelles and finally, well-defined ellipsoidal micelles (of decreasing aspect ratios) as the f PCL was increased. In all cases, the diffusion NMR spectroscopy, DLS and synchrotron SAXS results provided complementary information and the grounds for a phase diagram relating copolymer microstructure to aggregation behaviour and structure. Importantly, the absence of commonly depicted spherical micelles has implications for applications where properties may be governed by shape, such as, cellular uptake of nanomedicine formulations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This review will focus on orally administered lipid-based drug delivery vehicles and specifically the influence of lipid digestion on the structure of the carrier lipids and their entrained drug cargoes. Digestion of the formulation lipids, which are typically apolar triglycerides, generates amphiphilic monoglycerides and fatty acids that can self-assemble into a diverse array of liquid crystalline structures. Tracking the dynamic changes in self-assembly of the lipid digestion products during digestion has recently been made possible using synchrotron-based small angle X-ray scattering. The influence of lipid chain length and degree of unsaturation on the resulting lipid structuring will be described in the context of the critical packing parameter theory. The chemical and structural transformation of the formulation lipids can also have a dramatic impact on the physical state of drugs co-administered with the formulation. It is often assumed that the best strategy for drug development is to maximise drug solubility in the undigested formulation lipids and to incorporate additives to maintain drug solubility during digestion. However, it is possible to improve drug absorption using lipid digestion in cases where the solubility of the dosed drug or one of its polymorphic forms is greater in the digested lipids. Three different fates for drugs administered with digestible lipid-based formulations will be discussed: (1) where the drug is more soluble in the undigested formulation lipids; (2) where the drug undergoes a polymorphic transformation during lipid digestion; and (3) where the drug is more soluble in the digested formulation lipids., (© 2021 The Author(s).)

Efficient organic light-emitting diodes (OLEDs) consist of an emissive layer comprising a blend of a light-emitting and host material in contact with one or more charge transporting layers. The distribution of the active material in the guest-host emissive layer blend and the changes that may occur upon thermal annealing are two important factors in determining the stability and efficiency of OLEDs. We have combined neutron reflectometry and photoluminescence measurements to investigate the structures of films comprising an emissive layer containing a phosphorescent poly(dendrimer) material blended with 4,4'- N , N '-di(carbazolyl)biphenyl. This combination has been shown to give rise to highly efficient OLEDs. Here, we show that the emissive poly(dendrimer) material was not uniformly distributed in the host, but formed a concentration gradient within the emissive layer. Upon heating, the adjacent electron transport layer was found to intermix with the emissive layer, accompanied by changes in the material distribution in the emissive layer. The intermixing of the materials led to a decrease in the photoluminescence from the poly(dendrimer) within the film. The decrease in the photoluminescence was ascribed to an increase in interchromophore interactions that could arise from a conformational change of the poly(dendrimer) or phase separation leading to aggregation. The results indicate that, while uniform mixing of the guest and host is not essential for efficiency, the thermal stabilities of both host and charge transport materials are important for device durability.

Lipids play an important role in regulating bodily functions and providing a source of energy. Lipids enter the body primarily in the form of triglycerides in our diet. The gastrointestinal digestion of certain types of lipids has been shown to promote the self-assembly of lipid digestion products into highly ordered colloidal structures. The formation of these ordered colloidal structures, which often possess well-recognized liquid crystalline morphologies (or "mesophases"), is currently understood to impact the way nutrients are transported in the gut and absorbed. The formation of these liquid crystalline structures has also been of interest within the field of drug delivery, as it enables the encapsulation or solubilization of poorly water-soluble drugs in the aqueous environment of the gut enabling a means of absorption. This review summarizes the evidence for structure formation during the digestion of different lipid systems associated with foods, the techniques used to characterize them and provides areas of focus for advancing our understanding of this emerging field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pham, Clulow and Boyd.)

Delamanid is a poorly water-soluble drug currently being used for the treatment of tuberculosis. The high frequency of dosing leads to poor adherence for patients who live in lower economic and nomadic populations. Non-digestible self-assembling lipids as a formulation approach for poorly water-soluble drugs have previously been shown to extend the window of absorption through gastric retention. We hypothesise that this approach could lead to the reduction of dosing frequency for delamanid and thereby has potential to improve adherence. Formulations of delamanid were prepared in selachyl alcohol and phytantriol as non-digestible self-assembling lipid vehicles, and their behaviour was compared with reconstituted milk powder, as a digestible lipid-based formulation, and an aqueous suspension. The self-assembly of selachyl alcohol and phytantriol in aqueous media in the presence of delamanid was studied using small angle X-ray scattering and produced the inverse hexagonal (H 2 ) and inverse bicontinuous cubic (V 2 ) liquid crystal structures, respectively. The times at which maximum delamanid levels in plasma were observed (T max ) after oral administration of the phytantriol, selachyl alcohol and reconstituted milk powder formulations of delamanid to rats were 27 ± 3, 20 ± 4 and 6.5 ± 1.0 h, respectively, compared with the aqueous suspension formulation with a T max of 3.4 ± 1 h, which confirms the hypothesis of an extended duration of absorption after administration in non-digestible self-assembling lipids. The digestion products of the triglycerides in the milk formulation increased the solubilisation of delamanid in the gastrointestinal tract, leading to an increase in exposure compared with the aqueous suspension formulation but did not significantly extend T max . Overall, the non-digestible nanostructured lipid formulations extended the duration of absorption of delamanid well beyond that from milk or suspension formulations. Graphical abstract.

Hypothesis: The digestion of different milks and milk substitutes leads to the formation of a variety of self-assembled lipid structures, with the structuring of human milk being paramount for infant nutrition. It was hypothesised that mixing cow milk fat rich in medium/long-chain lipids with canola oil rich in long-chain unsaturated lipids would replicate the structuring of human milk by balancing lipid chain lengths and saturation levels., Experiments: Emulsions of cow milk fat/canola oil mixtures were prepared in two ways - by pre-mixing ghee and canola oil before dispersing them and by dispersing canola oil directly into commercial cow milk. Small angle X-ray scattering combined with titration of the fatty acids produced during digestion allowed for the correlation of dynamic lipid self-assembly with the extent of lipid digestion. Laser light scattering was used to show that the particle sizes in the digesting mixtures were similar and coherent anti-Stokes Raman spectroscopy (CARS) microscopy was used to confirm the mixing of canola oil into cow milk fat globules., Findings: As the amount of long-chain unsaturated canola oil lipids in the mixtures increased, the lipid self-assembly tended towards colloidal structures of greater interfacial curvature. When the ratio of cow milk fat to canola oil lipids was 1:1 (w/w), the digesting lipids assembled themselves into the same liquid crystalline structures as human breast milk. This observation was independent of the method used to mix the lipids, with CARS microscopy indicating uniform mixing of the canola oil into cow milk upon ultrasonication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The combination of Flash NanoPrecipitation and hydrophobic ion pairing (HIP) is a valuable approach for generating nanocarrier formulations of ionic water-soluble drugs with controllable release properties dictated by liquid crystalline structuring of the ion pairs. However, there are few examples of this in practice in the literature. This work aims to decipher the influence of the nature of the hydrophobic counterion used in HIP and its consequent impact on liquid crystalline structuring and drug release. The hypothesis of this study was that hydrophobic counterions with different head and tail groups used for FNP with HIP would give rise to different liquid crystalline structures, which in turn would result in different drug release behavior. A cationic, water-soluble antibiotic, polymixin B, was complexed with eight different hydrophobic counterions with varying head and tail groups and encapsulated into nanocarriers 100-400 nm in size prepared using FNP. Sixteen formulations were assessed for internal structure by synchrotron small-angle X-ray scattering, and drug release was measured in vitro in physiological conditions. The liquid crystalline phases formed depended on the counterion head group and tail geometry, drug:counterion charge ratio, and the ionic strength and pH of the release medium. Drug release occurred more rapidly when no liquid crystalline phases were present and more slowly when higher-ordered phases existed. Specific findings include that phosphonic acid counterions lead to the formation of lamellar structures that persisted at pH 2.0 but were not present at pH 7.3. In contrast, sulfonic acids lead to lamellar or hexagonal phases that persisted at both pH 7.3 and 2.0, while hydrophobic counterions without alkyl tails did not form internal structures. It was also clear that the lipophilicity of the counterion does not dictate drug release. These findings confirm that the liquid crystalline phase behavior of the drug:counterion complex dictates drug release and significantly improves our understanding of the types of controlled release formulations that are possible using FNP with HIP.

Hypothesis: Hydrophobic ion pairing (HIP), a solubility engineering technique in which ionic hydrophilic molecules are paired with a hydrophobic counterion, is an attractive strategy for encapsulating ionic water-soluble species into nanocarriers (NCs). Drug release from NCs containing HIP complexes is sensitive to ionic strength, pH, and drug:counterion charge ratio, but the exact mechanism for this was unknown, as was the underlying microstructure inside the NCs. We hypothesize that HIP complexes arrange into liquid crystalline structures in NC cores and that these structures are responsible for salt- and pH-dependent release., Experiment: A model hydrophobic ion pair from the cationic antimicrobial peptide polymyxin B sulfate and the anionic counterion sodium oleate is encapsulated into ~100 nm NCs formed using Flash NanoPrecipitation (FNP) and stabilized with an amphiphilic diblock copolymer, poly(caprolactone)-b-poly(ethylene glycol). Internal structures are observed using synchrotron small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) following NC formulation and are found to vary with polymyxin:oleate charge ratio. In vitro drug release is also measured with changes in pH and two charge ratio., Findings: For a formulation containing a four-fold charge excess of oleate relative to polymyxin, internal structures rearranged from a lamellar phase into an inverse hexagonal phase. The hexagonal phase formation corresponds to a greatly reduced rate of polymyxin release, suggesting that the polymyxin was incorporated into the center of hexagonally-packed rods. When release tests were repeated using phosphate-buffered saline (PBS) at pH 2.0 to ensure protonation of the oleic acid, all internal structures were eliminated and release occurs much faster than at neutral pH, regardless of charge ratio. These findings shed light on the mechanism behind stimulus-responsive drug release from systems containing hydrophobic ion pairs and enable the rational design of controlled-release formulations by manipulating the formation and dynamics of liquid crystalline phases inside NCs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

Spontaneous formation of vesicles from the self-assembly of two specific surfactants, one zwitterionic (oleyl amidopropyl betaine, OAPB) and the other anionic (Aerosol-OT, AOT), is explored in water using small-angle scattering techniques. Two factors were found to be critical in the formation of vesicles: surfactant ratio, as AOT concentrations less than equimolar with OAPB result in cylindrical micelles or mixtures of micellar structures, and salt concentration, whereby increasing the amount of NaCl promotes vesicle formation by reducing headgroup repulsions. Small-angle neutron scattering measurements reveal that the vesicles are approximately 30-40 nm in diameter, depending on sample composition. Small-angle X-ray scattering measurements suggest preferential partitioning of OAPB molecules on the vesicle inner layer to support vesicular packing. Heating the vesicles to physiological temperature (37 °C) causes them to collapse into smaller ellipsoidal micelles (2-3 nm), with higher salt concentrations (≥10 mM) inhibiting this transition. These aggregates could serve as responsive carriers for loading or unloading of aqueous cargoes such as drugs and pharmaceuticals, with temperature changes serving as a simple release/uptake mechanism.

The nature of the interaction of bile salt micelles with exogenous surfactants used in formulations and the consequent impact on drug solubilisation is not well understood. It is often assumed that addition of any surfactant will lead to an enhanced solubility of drug, which is often true in water alone. In this study we have investigated the interaction of a range of typical non-ionic formulation surfactants (Kolliphor EL, Vitamin E TPGS and a range of Pluronics) with bile salt + phospholipid (BS + PL) mixed micelles using small angle X-ray scattering. The solubility of the model poorly water-soluble drug fenofibrate was determined in the mixed micelles and compared to solubility in the presence of increasing exogenous surfactant alone. It was found that while Pluronic F68 did not appear to interact with bile salt micelles and did not impact on the solubility of the drug in the BS + PL micellar system, addition of hydrophobic surfactants led to a synergistic boost in drug solubility, while addition of more hydrophilic surfactants led to a net reduction in drug solubility. With the exception of Pluronic F68, both hydrophobic and hydrophilic surfactants swelled the bile salt mixed micelles leading to the conclusion that although the micelle size was increased, the solubilising environment was less favourable than in bile salt micelles alone. The results serve as a warning to formulators using these surfactants as solubilising agents to consider their likely interactions with endogenous colloidal structures., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recent studies have shown that the solubilization of two antimalarial drug candidates, artefenomel (OZ439) and ferroquine (FQ), designed to provide a single-dose combination therapy for uncomplicated malaria can be enhanced using milk as a lipid-based formulation. However, milk as an excipient faces significant quality and regulatory hurdles. We therefore have investigated infant formula as a potential alternative formulation approach. The significance of the lipid species present in a formula with different lipid compositions upon the solubilization of OZ439 and FQ during digestion has been investigated. Synchrotron small-angle X-ray scattering was used to measure the diffraction from a dispersed drug during digestion and thereby determine the extent of drug solubilization. High-performance liquid chromatography was used to quantify the amount of drug partitioned into the digested lipid phases. Our results show that both the lipid species and the amount of lipids administered were key determinants for the solubilization of OZ439, while the solubilization of FQ was independent of the lipid composition. Infant formulas could therefore be designed and used as milk substitutes to tailor the desired level of drug solubilization while circumventing the variability of components in naturally derived milk. The enhanced solubilization of OZ439 was achieved during the digestion of medium-chain triacylglycerols (MCT), indicating the potential applicability of MCT-fortified infant formula powder as a lipid-based formulation for the oral delivery of OZ439 and FQ.

Lipids in mammalian milks such as bovine milk and human breast milk have been shown to self-assemble into various liquid crystalline materials during digestion. In this study, the direct correlation between the composition of the lipids from three types of mammalian milk, three brands of infant formulas (IFs), and soy milk and the liquid crystalline structures that form during their digestion was investigated to link the material properties to the composition. The self-assembly behavior was assessed using in vitro digestion coupled with in situ small-angle X-ray scattering (SAXS). Lipid composition was determined during in vitro digestion using ex situ liquid chromatography-mass spectrometry. All tested milks self-assembled into ordered structures during digestion, with the majority of milks displaying nonlamellar phases. Milks that released mostly long-chain fatty acids (>95 mol % of the top 10 fatty acids released) with more than 47 mol % unsaturation predominantly formed a micellar cubic phase during digestion. Other milks released relatively more medium-chain fatty acids and medium-chain monoglycerides and produced a range of ordered liquid crystalline structures including the micellar cubic phase, the hexagonal phase, and the bicontinuous cubic phase. One infant formula did not form liquid crystalline structures at all as a consequence of differences in fatty acid distributions. The self-assembly phenomenon provides a powerful discriminator between different classes of nutrition and a roadmap for the design of human milklike systems and is anticipated to have important implications for nutrient transport and the delivery of bioactives., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

In the advent of the post-antibiotic era, new strategies are urgently required to improve the efficacy of antimicrobials and outsmart multi-drug resistant bacteria. Exploiting a basic survival mechanism of bacteria, lipase production, monoolein liquid crystal nanoparticles (MO-LCNPs) were investigated as a bacterial-triggered drug delivery system for three different antimicrobial compounds and compared with model sn-1/3 regiospecific and non-regiospecific lipases via pH-stat titration, proton nuclear magnetic resonance and in situ synchrotron small-angle X-ray scattering. The release of model hydrophobic (rifampicin) and macromolecular (alginate lyase) antimicrobials were triggered from MO-LCNPs at 82-fold and 7-fold higher rates (respectively) due to bacterial lipase digestion of MO-LCNPs, which could not be stimulated with a small hydrophilic antibiotic (ciprofloxacin HCl) or non-digestible, phytantriol-LCNPs. While sn-1/3 regiospecific lipase rapidly digested MO-LCNPs in a two-phase process, the single-phase digestion kinetics of the non-regiospecific lipase steadily digested the cubic Im3m structure and gave rise to lamellar structures that ultimately stimulated the triggered antibiotic release. Accordingly, MO-LCNPs have an application for localised Pseudomonas aeruginosa and Staphylococcus aureus infections that produce non-regiospecific lipases and for concentration-dependent antibiotics that have macromolecular (MW ~ 30 kDa) or hydrophobic (logP ~ 4) chemistries, as a triggered bolus release would be clinically efficacious for improved bacterial eradication., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Techniques enabling in situ monitoring of drug solubilization and changes in the solid-state of the drug during the digestion of milk and milk-based formulations are valuable for predicting the effectiveness of such formulations in improving the oral bioavailability of poorly water-soluble drugs. We have recently reported the use of low-frequency Raman scattering spectroscopy (region of analysis <200 cm -1 ) as an analytical approach to probe solubilization of drugs during digestion in milk using ferroquine (SSR97193) as the model compound. This study investigates the wider utilization of this technique to probe the solubilization behavior of other poorly water-soluble drugs (halofantrine, lumefantrine, and clofazimine) in not only milk but also infant formula in the absence or presence of bile salts during in vitro digestion. Multivariate analysis was used to interpret changes to the spectra related to the drug as a function of digestion time, through tracking changes in the principal component (PC) values characteristic to the drug signals. Characteristic low-frequency Raman bands for all of the drugs were evident after dispersing the solid drugs in suspension form in milk and infant formula. The drugs were generally solubilized during the digestion of the formulations as observed previously for ferroquine and correlated with behavior determined using small-angle X-ray scattering (SAXS). A greater extent of drug solubilization was also generally observed in the infant formula compared to milk. However, in the case of the drug clofazimine, the correlation between low-frequency Raman scattering and SAXS was not clear, which may arise due to background interference from clofazimine being an intense red dye, which highlights a potential limitation of this new approach. Overall, the in situ monitoring of drug solubilization in milk and milk-based formulations during digestion can be achieved using low-frequency Raman scattering spectroscopy, and the information obtained from studying this spectral region can provide better insights into drug solubilization compared to the mid-frequency Raman region.

Hypothesis: The distribution of three cell-penetrating peptides (CPPs) with different architectures (short, long linear and branched) on poly(lactic-co-glycolic) acid (PLGA) nanoparticles depends on the conjugation approach. Here, we explore the utilization of a zero-length crosslinking reaction for the covalent attachment of CPPs to PLGA nanoparticles and the translation of the reaction into a microfluidic platform., Experiments: A microfluidic device with a staggered herringbone mixer was used for the formulation of CPP-tagged PLGA nanoparticles. CPP-tagged PLGA nanoparticles were labeled with gold nanoparticles (AuNPs) and transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) were used to elucidate the distribution of CPPs., Findings: The SAXS scattering profiles for the CPP-tagged PLGA nanoparticles prepared with the in situ microfluidics conjugation approach indicated a distribution of the Au-labeled CPPs throughout the PLGA nanoparticles. For the post-microfluidics conjugation approach, the SAXS scattering profiles did not show the feature of the Au-labeled CPPs distributed throughout the PLGA nanoparticles and an arrangement of the Au-labeled CPP on the surface was support by TEM micrographs. The distribution of the CPPs was highly dependent on the conjugation approach and was not influenced by the architecture of the CPPs. The results provided insight for the rational design of CPP-tagged PLGA nanoparticles using microfluidics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Clofazimine, a drug previously used to treat leprosy, has recently been identified as a potential new drug for the treatment for cryptosporidiosis: a diarrheal disease that contributes to 500 000 infant deaths a year in developing countries. Rapid dissolution and local availability of the drug in the small intestine is considered key to the treatment of the infection. However, the commercially available clofazimine formulation (Lamprene) is not well-suited to pediatric use, and therefore reformulation of clofazimine is desirable. Development of clofazimine nanoparticles through the process of flash nanoprecipitation (FNP) has been previously shown to provide fast and improved drug dissolution rates compared to clofazimine crystals and Lamprene. In this study, we investigate the effects of milk-based formulations (as possible pediatric-friendly vehicles) on the in vitro solubilization of clofazimine formulated as either lecithin- or zein/casein-stabilized nanoparticles. Milk and infant formula were used as the lipid vehicles, and time-resolved synchrotron X-ray scattering was used to monitor the presence of crystalline clofazimine in suspension during in vitro lipolysis under intestinal conditions. The study confirmed faster dissolution of clofazimine from all the FNP formulations after the digestion of infant formula was initiated, and a reduced quantity of fat was required to achieve similar levels of drug solubilization compared to the reference drug material and the commercial formulation. These attributes highlight not only the potential benefits of the FNP approach to prepare drug particles but also the fact that enhanced dissolution rates can be complemented by considering the amount of co-administered fat in lipid-based formulations to drive the solubilization of poorly soluble drugs.

We have recently shown that real-time monitoring of drug solubilization and changes to solid state of the drug during digestion of milk can be achieved using synchrotron small-angle X-ray scattering. A complementary laboratory-based method to explore such changes is low-frequency Raman spectroscopy, which has been increasingly used to characterize crystalline drugs and their polymorphs in powders and suspensions. This study investigates the use of this technique to monitor in situ drug solubilization in milk during the process of digestion, using a lipolysis model/flow-through configuration identical to that used previously for in situ synchrotron small-angle X-ray scattering studies. An antimalarial drug, ferroquine (SSR97193), was used as the model drug for this study. The Raman spectra were processed using multivariate analysis to extract the drug signals from the milk digestion background. The results showed disappearance of the ferroquine peaks in the low-frequency Raman region (<200 cm -1 ) after approximately 15-20 min of digestion when milk fat was present in the system, which indicated drug solubilization and was in good agreement with the in situ small-angle X-ray scattering measurements. This proof-of-concept study therefore suggests that low-frequency Raman spectroscopy can be used to monitor drug solubilization in a complex digesting milk medium because of the unique vibrational modes of the drug crystal lattices.

Milk is an attractive lipid-based formulation for the delivery of poorly water-soluble drugs to pediatric populations. We recently observed that solubilization of artefenomel (OZ439) during in vitro intestinal lipolysis was driven by digestion of triglycerides in full-cream bovine milk, reflecting the ability of milk to act as an enabling formulation in the clinic. However, when OZ439 was co-administered with a second antimalarial drug, ferroquine (FQ) the exposure of OZ439 was reduced. The current study therefore aimed to understand the impact of the presence of FQ on the solubilization of OZ439 in milk during in vitro intestinal digestion. Synchrotron small-angle X-ray scattering was used for in situ monitoring of drug solubilization (inferred via decreases in the intensity of drug diffraction peaks) and polymorphic transformations that occurred during the course of digestion. Quantification of the amount of each drug solubilized over time and analysis of their distributions across the separated phases of digested milk were determined using high-performance liquid chromatography. The results show that FQ reduced the solubilization of OZ439 during milk digestion, which may be due to competitive binding of FQ to the digested milk products. Interactions between the protonated FQ-H + and ionized liberated free fatty acids resulted in the formation of amorphous salts, which removes the low-energy crystalline state as a barrier to dissolution of FQ, while inhibiting the solubilization of OZ439. We conclude that although milk could enhance the solubilization of poorly water-soluble OZ439 during in vitro digestion principally due to the formation of fatty acids, the solubilization efficiency was reduced by the presence of FQ by competition for the available fatty acids. Assessment of the solubilization of both drugs during digestion of fixed-dose combination lipid formulations (such as milk) is important and may rationalize changes in bioavailability when compared to that of the individual drugs in the same formulation.

Phytantriol is an interfacially-active lipid that is chemically robust, non-digestible and forms particles with internal bicontinuous cubic phase structures (cubosomes) when dispersed with non-ionic surfactants at ambient and physiological temperatures. The liquid crystalline internal structure of phytantriol-based cubosomes can be changed to alter the interfacial contact area/topology with the aqueous dispersant to trigger bioactive payload release or to alter the local membrane curvature around bound or embedded proteins. To enable the study of payload distribution, structure and transformation kinetics within phytantriol particles by neutron scattering techniques it is desirable to have access to a deuterated version of this molecule but to date a synthetic route has not been available. The first successful synthesis of phytantriol-d 39 is presented here alongside a preliminary physical characterisation of related particle structures when phytantriol-d 39 is dispersed using two non-ionic surfactants, Tween® 80 and Pluronic® F127. Synchrotron small angle X-ray scattering (SAXS) was used to confirm that phytantriol-d 39 -based nanoparticles in D 2 O form similar liquid crystalline structures to those of their natural isotopic abundance (phytantriol/H 2 O) counterparts as a function of temperature. Finally, small angle neutron scattering (SANS) with solvent contrast to match out the phytantriol-d 39 structuring was used to show that the spatial correlations between the Tween® and Pluronic® non-ionic surfactant molecules are different within dispersed phytantriol-d 39 particles with different liquid crystalline structures in D 2 O. The surfactant molecules in phytantriol-d 39 /Tween® 80 particles with Im3m cubic structures were found to follow a self-avoiding walk, whereas in phytantriol-d 39 /Pluronic® F127 particles with Pn3m cubic structures they were found to follow a more rod-like packing arrangement., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oral delivery of dispersible tablets is a preferred route of administration for paediatrics due to ease of administration and dose control. Milk has gained interest as a drug delivery system due to its ability to dissolve poorly water-soluble drugs. There are no reports of milk tablet formulations being assessed in the context of lipid digestion, which is critical in influencing orally administered drug solubility and bioavailability. Milk-drug tablets were formulated by blending freeze-dried bovine milk or infant formula with the poorly water-soluble drug cinnarizine, which were directly compressed. Tablet strength, friability and dispersibility were quantified and synchrotron X-ray scattering was used to determine the lipid liquid crystalline phases formed during in vitro digestion of dispersed tablets and their effects on drug solubilisation. Tableting had a significant impact on the self-assembly of lipids in redispersed milk tablets whereas no effect was seen for infant formula tablets. Incorporation of the disintegrant poly(vinylpolypyrrolidone) to reduce tablet dispersion times promoted the formation of hexagonal liquid crystalline phases upon digestion but had minimal effect on drug solubilisation. These findings show that similar to the use of liquid milk, the formulation of milk-drug tablets can be used to improve solubilisation of poorly water-soluble drugs., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6 DBW and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Milk has previously been considered as a potential lipid-based drug delivery system for poorly water soluble drugs but it has never gained significant attention. This is in part because relying on solubility in lipid-based formulations (in this case milk) does not provide a complete picture of the behavior of such systems upon digestion. Herein, we demonstrate using time resolved X-ray scattering that the digestion of milk is actually crucial to the solubilisation of a poorly water-soluble drug, halofantrine. Halofantrine was chosen because its behaviour in lipid-based formulations has been widely investigated and because of its close structural relationship to lumefantrine, an antimalarial drug of current interest for the treatment of paediatric malaria. The transformation of the drug from a crystalline solid form in suspension in milk, to a solubilised form as a direct consequence of lipolysis highlights that consideration of digestion of the milk lipids as a critical process that influences drug solubilisation and availability for absorption is vital., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Milk has been used as a vehicle for the delivery of antimalarial drugs during clinical trials to test for a food effect and artefenomel (OZ439) showed enhanced oral bioavailability with milk. However, the nature of the interaction between milk and OZ439 in the gastrointestinal tract remains poorly understood. To understand the role of milk digestion on the solubilization of OZ439 and polymorphism, we conducted real-time monitoring of crystalline drug in suspension during in vitro intestinal lipolysis of milk containing OZ439 using synchrotron X-ray scattering. OZ439 formed an unstable solid-state intermediate free base form (OZ439-FB form 1) at intestinal pH and was partially solubilized by milk fat globules prior to lipolysis. Dissolution of the free base form 1 and recrystallization of OZ439 in a more stable polymorphic form (OZ439-FB form 2) occurred during in vitro lipolysis in milk. Simply stirring the milk/drug suspension in the absence of lipase or addition of lipase to OZ439 in a lipid-free buffer did not induce this polymorphic transformation. The formation of OZ439-FB form 2 was therefore accelerated by the solubilization of OZ439-FB form 1 during the digestion of milk. Our findings confirmed that although crystalline precipitates of OZ439-FB form 2 could still be detected after in vitro digestion, milk-based lipid formulations provided a significant reduction in crystalline OZ439 compared to lipid-free formulations, which we attribute to the formation of colloidal structures by the digested milk lipids. Milk may therefore be particularly suited as a form of lipid-based formulation (LBF) for coadministration with OZ439, from which both an enhancement in OZ439 oral bioavailability and the delivery of essential nutrients should result.

Efforts to develop orally administered drugs tend to place an exceptional focus on aqueous solubility as this is an essential criterion for their absorption in the gastrointestinal tract. In this work we examine the solid state behavior and solubility of OZ439, a promising single-dose cure for malaria being developed as the highly water-soluble mesylate salt. The aqueous phase behavior of the OZ439 mesylate salt was determined using a combination of small angle neutron and X-ray scattering (SANS and SAXS, respectively). It was found that this salt has low solubility at low concentrations with the drug largely precipitated in free base aggregates. However, with increasing concentration these crystalline aggregates were observed to dissociate into cationic micelles and lamellar phases, effectively increasing the dissolved drug concentration. It was also found that the dissolved OZ439 spontaneously precipitated in the presence of biologically relevant anions, which we attribute to the high lattice energies of most of the salt forms of the drug. These findings show that aqueous solubility is not always what it seems in the context of amphiphilic drug molecules and highlights that its use as the principal metric in selecting drug candidates for development can be perilous.

Milk has recently been reported to form complex self-assembled liquid crystalline structures during digestion by lipolytic enzymes. The formation of cubic phases at the endpoint of digestion was of particular interest as this requires a fine balance in self-assembly. This manuscript probes the robustness of the kinetic structural behaviour when milk is subjected to a range of processes that are encountered by milk and/or are relevant to the use of milk in pharmaceutical applications (homogenisation, lyophilisation, freeze-thawing and freeze-drying) using time-resolved small angle X-ray scattering (TR-SAXS). The nature of the persistent lamellar phase that occurs during digestion is elucidated using SAXS and X-ray photoelectron spectroscopy, and the interplay between the formation of structured mesophases and the evolution of particle size during digestion is determined using laser light scattering studies. This closer look at milk lipids during digestion establishes the dependence of the structural behaviour of milk on lipid composition and not processing, and clarifies the phase behaviour and kinetic effects on particle size distribution under lipolytic conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Organic light-emitting devices containing solution-processed emissive dendrimers can be highly efficient. The most efficient devices contain a blend of the light-emitting dendrimer in a host and one or more charge-transporting layers. Using neutron reflectometry measurements with in situ photoluminescence, we have investigated the structure of the as-formed film as well as the changes in film structure and dendrimer emission under thermal stress. It was found that the as-formed film stacks comprising poly(3,4-ethylenedioxythiophene):polystyrene sulfonate/host:dendrimer/1,3,5-tris(N-phenylbenzimidazol-2-yl)benzene (where the host was deuterated 4,4'-N,N'-di(carbazolyl)biphenyl or tris(4-carbazol-9-ylphenyl)amine, the host:dendrimer layer was solution-processed, and the 1,3,5-tris(N-phenylbenzimidazol-2-yl)benzene evaporated) had well-defined interfaces, indicating good wetting of each of the layers by the subsequently deposited layer. Upon thermal annealing, there was no change in the poly(3,4-ethylenedioxythiophene):polystyrene sulfonate/host:dendrimer interface, but once the temperature reached above the T g of the host:dendrimer layer, it became a supercooled liquid into which 1,3,5-tris(N-phenylbenzimidazol-2-yl)benzene dissolved. When the film stacks were held at a temperature just above the onset of the diffusion process, they underwent an initial relatively fast diffusion process before reaching a quasi-stable state at that temperature.

Nanoparticle dispersions open up an ecofriendly route toward printable organic solar cells. They can be formed from a variety of organic semiconductors by using miniemulsions that employ surfactants to stabilize the nanoparticles in dispersion and to prevent aggregation. However, whenever surfactant-based nanoparticle dispersions have been used to fabricate solar cells, the reported performances remain moderate. In contrast, solar cells from nanoparticle dispersions formed by precipitation (without surfactants) can exhibit power conversion efficiencies close to those of state-of-the-art solar cells processed from blend solutions using chlorinated solvents. In this work, we use small-angle neutron scattering measurements and transient absorption spectroscopy to investigate why surfactant-free nanoparticles give rise to efficient organic solar cells. We show that surfactant-free nanoparticles comprise a uniform distribution of small semiconductor domains, similar to that of bulk-heterojunction films formed using traditional solvent processing. This observation differs from surfactant-based miniemulsion nanoparticles that typically exhibit core-shell structures. Hence, the surfactant-free nanoparticles already possess the optimum morphology for efficient energy conversion before they are assembled into the photoactive layer of a solar cell. This structural property underpins the superior performance of the solar cells containing surfactant-free nanoparticles and is an important design criterion for future nanoparticle inks.

The absorption of hydrophobic drugs and nutrients from the intestine is principally determined by the amount that can be dissolved by the endogenous fluids present in the gut. Human intestinal fluids (HIFs) comprise a complex mixture of bile salts, phospholipids, steroids and glycerides that vary in composition in the fed and fasted state and between subjects. A number of simulated intestinal fluid (SIF) compositions have been developed to mimic fasted and fed state intestinal conditions and allow the in vitro determination of drug solubility as a proxy for the maximum dissolved concentration it is possible to reach. In particular these solvents are used during the development of lipophilic and poorly water-soluble drugs but questions remain around the differences that may arise from the source and methods of preparation of these fluids. In this work, a range of SIFs were studied using small-angle X-ray scattering (SAXS), cryogenic-transmission electron microscopy (cryo-TEM) and molecular dynamics (MD) simulations in order to analyze their structures. In-house prepared SIFs based on sodium taurodeoxycholate (NaTDC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) formed oblate ellipsoidal micelles irrespective of lipid concentration and preparation conditions. In contrast, commercially available SIFs based on sodium taurocholate and lecithin formed prolate ellipsoidal micelles in the fed state and vesicles in the fasted state. These structural variations are the likely reason for the dramatic differences sometimes observed in the solubility enhancements for hydrophobic drugs, nutrients and digestion products when using different SIFs. However, the structural homogeneity of the NaTDC/DOPC micelles makes them ideal candidates for standardizing SIF formulations as the structures of the solubilizing nanoaggregates therein are not sensitive to the preparation method.

The effect of varying the emitter concentration on the structural properties of an archetypal phosphorescent blend consisting of 4,4'-bis(N-carbazolyl)biphenyl and tris(2-phenylpyridyl)iridium(III) has been investigated using non-equilibrium molecular dynamics (MD) simulations that mimic the process of vacuum deposition. By comparison with reflectometry measurements, we show that the simulations provide an accurate model of the average density of such films. The emitter molecules were found not to be evenly distributed throughout film, but rather they can form networks that provide charge and/or energy migration pathways, even at emitter concentrations as low as ≈5 weight percent. At slightly higher concentrations, percolated networks form that span the entire system. While such networks would give improved charge transport, they could also lead to more non-radiative pathways for the emissive state and a resultant loss of efficiency., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)