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Hypothesis: pH-responsive nanocarriers have the potential to provide targeted delivery of antimicrobial peptides (AMPs) to sites of bacterial infection with typically abnormal pH levels in the body. However, the local pH of the infected sites varies substantially among different infection-related diseases, calling for the development of delivery systems capable of targeting local pathological conditions in an adjustable pH range. Experiments: In this study, a highly versatile pH-responsive nanocarrier platform, based on dispersions of oleic acid (OA) and glycerol monooleate (GMO) self-assemblies with the human cathelicidin AMP LL-37, was designed and characterized. Findings: A detailed pH-composition phase diagram was constructed from small angle X-ray scattering and cryogenic transmission electron microscopy data. In addition, the protonation state and apparent pKa of OA embedded in these nano-self-assemblies were investigated by electrophoretic mobility measurements at different pHs and found to be strongly dependent on nanocarrier composition. By varying composition of these nanocarriers, the apparent pKa of embedded OA molecules could be tuned from 7.8 to 6.3, shifting the range of nanocarriers' pH-response. The study advances our fundamental understanding of self-assembly and pH-responsiveness in lipid-peptide systems containing monounsaturated long-chain fatty acids. The results may guide the future design of highly adaptable nanocarriers for patient-optimized pH-targeted AMP delivery.

Hypothesis The development of advanced oral delivery systems for bioactive compounds requires the fundamental understanding of the digestion process within the gastrointestinal tract. Towards this goal, dynamic in vitro digestion models, capable of characterising the molecular as well as colloidal aspects of food, together with their biological interactions with relevant in vitro cell culture models, are essential. Experiments In this study, we demonstrate a novel digestion model that combines flow-through time resolved small angle X-ray scattering (SAXS) with an in vitro Caco-2/HT-29 cell co-culture model that also contained a mucus layer. This set-up allows the dynamic in situ characterisation of colloidal structures and their transport across a viable intestinal cell layer during simulated digestion. Findings An integrated online SAXS – in vitro cell co-culture model was developed and applied to study the digestion of nature’s own emulsion, milk. The impact of the in vitro cell culture on the digestion-triggered formation and evolution of highly ordered nanostructures in milk is demonstrated. Reported is also the crucial role of the mucus layer on top of the cell layer, protecting the cells from degradation by digestive juice components such as lipase. The novel model can open unique possibilities for the dynamic investigation of colloidal structure formation during lipid digestion and their effect on the uptake of bioactive molecules by the cells., Journal of Colloid and Interface Science, 574, ISSN:0021-9797, ISSN:1095-7103

pH-responsive aminolipid self-assemblies are promising platforms for the targeted delivery of antimicrobial peptides (AMPs), with the potential to improve their therapeutic efficiency and physico-chemical stability.pH-sensitive nanocarriers based on dispersed self-assemblies of 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) with the human cathelicidin LL-37 in excess water were characterized at different pH values using small-angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering. Fluorescence and electrophoretic mobility measurements were used to probe the encapsulation efficiency of LL-37 and the nanocarriers' surface potential.Upon decreasing pH in the DODAP/water systems, normal oil-in-water emulsions at pH ≥ 5.0 transitioned to emulsions encapsulating inverse hexagonal and cubic structures at pH between 4.5 and 4.0, and mostly positively-charged vesicles at pH 4.0. These colloidal transformations are driven by the protonation of DODAP upon pH decrease. The larger lipid-water interfacial area provided by the DODAP self-assemblies at pH ≤ 4.5 allowed for an adequate encapsulation efficiency of LL-37, favouring the formation of vesicles in a concentration-dependent manner. Contrary, LL-37 was found to dissociate from the emulsion droplets at pH 6.0. The knowledge on the pH-triggered self-assembly of LL-37 and DODAP, combined with the results on peptide release from the structures contribute to the fundamental understanding of lipid/peptide self-assembly. The results can guide the rational design of future pH-responsive AMP delivery systems.

This article introduces a simple microfluidic device that can be combined with synchrotron small-angle X-ray scattering (SAXS) for monitoring dynamic structural transitions. The microfluidic device is a thiol-ene-based system equipped with 125 μm-thick polystyrene windows, which are suitable for X-ray experiments. The device was prepared by soft lithography using elastomeric molds followed by a simple UV-initiated curing step to polymerize the chip material and simultaneously seal the device with the polystyrene windows. The microfluidic device was successfully used to explore the dynamics of the structural transitions of phytantriol/dioleoylphosphatidylglycerol-based cubosomes on exposure to a buffer containing calcium ions. The resulting SAXS data were resolved in the time frame between 0.5 and 5.5 s, and a calcium-triggered structural transition from an internal inverted-type cubic phase of symmetry Im3m to an internal inverted-type cubic phase of symmetry Pn3m was detected. The combination of microfluidics with X-ray techniques opens the door to the investigation of early dynamic structural transitions, which is not possible with conventional techniques such as glass flow cells. The combination of microfluidics with X-ray techniques can be used for investigating protein unfolding, for monitoring the formation of nanoparticles in real time, and for other biomedical and pharmaceutical investigations. A combination of microfluidics with X-ray techniques has been used to perform dynamic structural studies on nanoparticulate formulations. (Less)