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Formation of high viscous inverse lyotropic liquid crystalline phases in situ upon exposure of low viscous drug-loaded lipid preformulations to synovial fluid provides a promising approach for design of depot formulations for intra-articular drug delivery. Rational formulation design relies on a fundamental understanding of the synovial fluid-mediated dynamic structural transitions occurring at the administration site. At conditions mimicking the in vivo situation, we investigated in real-time such transitions at multiple positions by synchrotron small-angle X-ray scattering (SAXS) combined with an injection-cell. An injectable diclofenac-loaded quaternary preformulation consisting of 72/8/10/10% (w/w) glycerol monooleate/1,2-dioleoyl-glycero-3-phospho-rac-(1-glycerol)/ethanol/water was injected into hyaluronic acid solution or synovial fluid. A fast generation of a coherent drug depot of inverse bicontinuous Im3m and Pn3m cubic phases was observed. Through construction of 2D spatial maps from measurements performed 60 min after injection of the preformulation, it was possible to differentiate liquid crystalline rich- and excess hyaluronic acid solution- or synovial fluid-rich regimes. Synchrotron SAXS findings confirmed that the exposure of the preformulation to the media leads to alterations in structural features in position- and time-dependent manners. Effects of biologically relevant medium composition on the structural features, and implications for development of formulations with sustained drug release properties are highlighted.

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.

Pluronic F127-stabilized non-lamellar liquid crystalline aqueous nanodispersions are promising injectable platforms for drug and contrast agent delivery. These nanodispersions, however, trigger complement activation in the human blood, where the extent of complement activation and opsonization processes may compromise their biological performance and safety. Here, we introduce a broad family of nanodispersions from glycerol monooleate (GMO) and oleic acid (OA) in different weight ratios, and stabilized with a plethora of nonionic methoxypoly(ethylene glycol) (mPEG)-lipids of different PEG chain length and variable lipid moiety (monounsaturated or saturated diglycerides or D-α-tocopheryl succinate). Through an integrated biophysical approach involving dynamic light scattering, synchrotron small-angle scattering, and cryo-transmission electron microscopy, we examine the impact of nonionic mPEG-lipid stabilization on size, internal self-assembled architecture, and gross morphological characteristics of nanodispersions. The results show how the nonionic mPEG-lipid type and concentration, and dependent on GMO/OA weight ratio, can variably modulate the internal architectures of nanoparticles. Assessment of complement profiling from selected nanodispersions with diverse structural heterogeneity further suggests a variable modulatory role for the lipid type of the nonionic mPEG-lipid in the extent of complement activation, which span from no activation to moderate to high levels. We comment on plausible mechanisms driving the observed complement activation variability and discuss the potential utility of these nanodispersions for future development of injectable nanopharmaceuticals.

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles, with their tunable structural features and capability of loading a wide range of drugs and reporter probes, are emerging as versatile injectable nanopharmaceuticals. Secondary emulsifiers, such as Pluronic block copolymers, are commonly used for colloidal stabilization of LLC nanoparticles, but their inclusion often compromises the biological safety (e.g., poor hemocompatibility and enhanced cytotoxicity) of the formulation. Here, we introduce a library of colloidally stable, structurally tunable, and pH-responsive lamellar and non-lamellar liquid crystalline nanoparticles from binary mixtures of a phospholipid (phosphatidylglycerol) and three types of omega-3 fatty acids (ω-3 PUFAs), prepared in the absence of a secondary emulsifier and organic solvents. We study formulation size distribution, morphological heterogeneity, and the arrangement of their internal self-assembled architectures by nanoparticle tracking analysis, synchrotron small-angle X-ray scattering, and cryo-transmission electron microscopy. The results show the influence of type and concentration of ω-3 PUFAs in nanoparticle structural transitions spanning from a lamellar (Lα) phase to inverse discontinuous (micellar) cubic Fd3m and hexagonal phase (H2) phases, respectively. We further report on cell-culture medium-dependent dynamic fluctuations in nanoparticle size, number and morphology, and simultaneously monitor uptake kinetics in two human cell lines. We discuss the role of these multiparametric biophysical transformations on nanoparticle-cell interaction kinetics and internalization mechanisms. Collectively, our findings contribute to the understanding of fundamental steps that are imperative for improved engineering of LLC nanoparticles with necessary attributes for pharmaceutical development.

Administration of parenteral liquid crystalline phases, forming in-vivo with tunable nanostructural features and sustained release properties, offers an attractive approach for treatment of infections and local drug delivery. It has also a potential use for postoperative pain management after arthroscopic knee surgery. However, the optimal use of this drug delivery principle requires an improved understanding of the involved dynamic structural transitions after administration of low-viscous stimulus-responsive lipid precursors and their fate after direct contact with the biological environment. These precursors (preformulations) are typically based on a single biologically relevant lipid (or a lipid combination) with non-lamellar liquid crystalline phase forming propensity. In relation to liquid crystalline depot design for intra-articular drug delivery, it was our interest in the present study to shed light on such dynamic structural transitions by combining synchrotron SAXS with a remote controlled addition of synovial fluid (or buffer containing 2% (w/v) albumin). This combination allowed for monitoring in real-time the hydration-triggered dynamic structural events on exposure of the lipid precursor (organic stock solution consisting of the binary lipid mixture of monoolein and castor oil) to excess synovial fluid (or excess buffer). The synchrotron SAXS findings indicate a fast generation of inverse bicontinuous cubic phases within few seconds. The effects of (i) the organic solvent N-methyl-2-pyrolidone (NMP), (ii) the lipid composition, and (iii) the albumin content on modulating the structures of the self-assembled lipid aggregates and the implications of the experimental findings in the design of liquid crystalline depots for intra-articular drug delivery are discussed.

This article presents the use of a shearing procedure for the preparation of stable nanostructured dispersions of lipid mesophases. This new application of the shearing technique is compared with the well-established ultrasonication method for the emulsification of these mesophases in water in terms of particle size, particle size distribution and available concentration range. With a laboratory-built shear device based on a Couette cell, it was possible to produce high quantities of internally self-assembled emulsion particles of controlled size at concentrated hydrophobic phase contents ( ϕ o ) of up to 70 wt%. The concentration limit of 70 wt% could be reached however, the maximum attainable concentration depended on the internal structure type of the particles. The limit was thus easily attained for emulsified microemulsions (EME) as well as for the emulsified inverse hexagonal phase ( H 2 ), whereas it was found to be lower for emulsified discontinuous (Fd3m) and bicontinuous (Pn3m) cubic phases. Moreover, by shearing, it was possible to keep the size of the particles relatively constant when increasing ϕ o , whereas the particle size significantly increased with ϕ o when ultrasonication was employed. By means of ultrasonication, the hydrodynamic radius of the particles could be tuned linearly between 85 to 180 nm as a function of ϕ o up to a maximum of 20 to 30 wt%. Below the maximum concentration limit, particles displayed a well-controlled size.

In this paper we present the structural characterization of a five-component food-grade microemulsion containing Tween 80, R(+)-limonene, ethanol, glycerol, and water. Our main approach to investigating the microstructure of dense microemulsions, and how it can be influenced by the various components, was to employ small-angle neutron scattering and the new evaluation technique for dense, interacting systems, the Generalized Indirect Fourier Transformation. We started our investigation with the impact of glycerol and ethanol on Tween 80 micelles in water. We found that glycerol increases the aggregation number and withdraws the hydrating agents from the headgroup region of the surfactant, resulting in a higher packing density of molecules in a micelle at slightly increasing size. The same trend holds when the micelles are oil swollen and/or ethanol is present. Ethanol, on the other hand, redistributes mainly between water and the interface–headgroup region of the surfactant. Part of it replaces surfactant molecules in the micelles, which increases the available interface and results in a higher number of micelles with shrinking size. The same trend holds when the micelles are oil swollen and/or glycerol is present in the aqueous phase. We also investigated samples along the dilution of a mixture of surfactant and oil phase (R(+)-limonene and ethanol), which can be diluted with aqueous phase (mixture of water and glycerol) without the occurrence of phase separation. In some samples of this dilution most probably bicontinuous structures are present. To elucidate this point, we also employed dynamic light scattering, viscosity, and conductivity measurements.